Average Nucleotide Identity Analysis Research Articles

Sphingomonas Immobilis sp. nov., and Sphingomonas natans sp. nov. bacteria isolated from soil.

Two novel strains of bacteria, CA1-15T and BIUV-7T, were isolated from soil samples gathered in Cheonan-si, Republic of Korea, and Inje-gun, Republic of Korea, respectively. These bacteria are Gram-negative, aerobic, and non-motile. Phylogenetic evaluations, using the sequence of the 16S rRNA gene, showed that strains CA1-15T and BIUV-7T belong to a distinctive clade within the family Sphingomonadaceae (order Sphingomonadales, class Alphaproteobacteria). The strains exhibited the highest similarity in their genetic makeup with representatives of the genus Sphingomonas. Strain CA1-15T was closely related to Sphingomonas echinoides NRRL B-3126T (97.8% similarity in 16S rRNA gene sequence), Sphingomonas oligophenolica JCM 12,082T (97.8%), Sphingomonas glacialis C16yT (97.6%) and Sphingomonas psychrolutea MDB1-AT (97.3%). Strain BIUV-7T was closely related to Sphingomonas nostoxanthinifaciens AK-PDB1-5T (97.0%), Sphingomonas vulcanisoli SN6-13T (96.3%), Sphingomonas naphthae DKC-5-1T (96.2%), and Sphingomonas prati W18RDT (95.7%). The optimal growth conditions for strains CA1-15T and BIUV-7T were determined to be at pH 7.0 and a temperature of 25°C. Analysis of the cellular fatty acids of strain CA1-15T and BIUV-7T revealed that summed feature 8 (C18:1ω7c/C18:1ω6c) (60.4%), summed feature 8 (C18:1ω7c/C18:1ω6c) (62.9%) were the major component, respectively. Additionally, both strains exhibited ubiquinone Q-10 as their major respiratory quinone, and diphosphatidylglycerol (DPG), glycosphingolipid (SGL), and phosphatidylethanolamine (PE) as the major polar lipid. The genome of strain CA1-15T measures 4,133,944bp, comprising 4,026 coding sequences (CDSs) and 46 tRNA genes. Similarly, the genome of strain BIUV-7T is 4,563,252bp, characterized by 4,226 CDSs and 44 tRNA genes. The average nucleotide identity (ANI) analysis and digital DNA-DNA hybridization (dDDH) values between strain CA1-15T and other Sphingomonas species range from 73.2 to 79.9% and 19.4-22.9%, respectively. Comparatively, ANI and dDDH values between strain BIUV-7T and other Sphingomonas species are in the range of 72.9-76.5% and 19.3-20.9%, respectively. Based on the biochemical, chemotaxonomic, and phylogenetic analyses, it is evident that strains CA1-15T and BIUV-7T represent two novel bacterial species within the genus Sphingomonas. Accordingly, the names Sphingomonas immobilis sp. nov. and Sphingomonas natans sp. nov. are proposed. also, CA1-15T(= KCTC 92960T = NBRC 116547T) is the type strain of Sphingomonas immobilis and BIUV-7T(= KCTC 92961T = NBRC 116546T) is the type strain of Sphingomonas natans.

Discovery and characterization of a novel pathogen Erwinia pyri sp. nov. associated with pear dieback: taxonomic insights and genomic analysis.

In 2022, a novel disease similar to pear fire blight was found in a pear orchard in Zhangye City, Gansu Province, China. The disease mainly damages the branches, leaves, and fruits of the plant. To identify the pathogen, tissue isolation and pathogenicity testing (inoculating the potential pathogen on healthy plant tissues) were conducted. Furthermore, a comprehensive analysis encompassing the pathogen's morphological, physiological, and biochemical characteristics and whole-genome sequencing was conducted. The results showed that among the eight isolates, the symptoms on the detached leaves and fruits inoculated with isolate DE2 were identical to those observed in the field. Verifying Koch's postulates confirmed that DE2 was the pathogenic bacterium that causes the disease. Based on a 16S rRNA phylogenetic tree, isolate DE2 belongs to the genus Erwinia. Biolog and API 20E results also indicated that isolate DE2 is an undescribed species of Erwinia. Isolate DE2 was negative for oxidase. Subsequently, the complete genome sequence of isolate DE2 was determined and compared to the complete genome sequences of 29 other Erwinia species based on digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) analyses. The ANI and dDDH values between strain DE2 and Erwinia species were both below the species thresholds (ANI < 95-96%, dDDH<70%), suggesting that isolate DE2 is a new species of Erwinia. We will temporarily name strain DE2 as Erwinia pyri sp. nov. There were 548 predicted virulence factors in the genome of strain DE2, comprising 534 on the chromosome and 5 in the plasmids. The whole genome sequence of strain DE2 has been submitted to the NCBI database (ASM3075845v1) with accession number GCA_030758455.1. The strain DE2 has been preserved at the China Center for Type Culture Collection (CCTCC) under the deposit number CCTCC AB 2024080. This study represents the initial report of a potentially new bacterial species in the genus Erwinia that causes a novel pear dieback disease. The findings provide a valuable strain resource for the study of the genus Erwinia and establish a robust theoretical foundation for the prevention and control of emerging pear dieback diseases.

Rhodoferax potami sp. nov. and Rhodoferax mekongensis sp. nov., isolated from the Mekong River in Thailand.

Four newly discovered Gram-stain-negative bacteria, designated as BL00010T, BL00058, D8-11T and BL00200, were isolated from water samples collected at three hydrological monitoring stations (namely Chiang Saen, Chiang Khan and Nong Khai) located along the Mekong River in Thailand. An investigation encompassing phenotypic, chemotaxonomic and genomic traits was conducted. The results of phylogenetic analysis based on 16S rRNA gene sequences indicated that all four isolates represented members of the genus Rhodoferax. These isolates were closely related to Rhodoferax bucti KCTC 62564T with a similarity of 99.59%. The major fatty acids of the four novel isolates included C16:0 and C16:1ω7c and/or C16 : 1ω6c, whereas the major respiratory quinone was identified as ubiquinone Q-8. In addition, phosphatidylethanolamine was identified as a major polar lipid in these bacteria. The genomes of BL00010T, BL00058, D8-11T and BL00200 were similar in size (3.88-4.01 Mbp) and DNA G+C contents (59.5, 59.3, 59.5 and 59.3 mol%, respectively). In contrast to R. bucti KCTC 62564T and Rhodoferax aquaticus KCTC 32394T, the newly discovered species possessed several genes involved in nitrite and nitrile metabolism, which may be related to their unique adaptation to nitrile-rich environments. From the results of the pairwise analysis of average nucleotide identity of the whole genome and digital DNA-DNA hybridisation, it was evident that BL00010T and D8-11T represented two novel species, for which we propose the nomenclature Rhodoferax potami sp. nov., with the type strain BL00010T (TBRC 17198T = NBRC 116413T), and Rhodoferax mekongensis sp. nov., with the type strain D8-11T (TBRC 17307T = NBRC 116415T).

Kitasatospora cathayae sp. nov., a novel endophytic actinomycete isolated from the leaves of Cathaya argyrophylla.

Strain HUAS 3-15T was isolated from the leaves of Cathaya argyrophylla collected from Chenzhou, Hunan Province, PR China. The main fatty acids (>5.0 %) of the strain were anteiso-C15 : 0, C16 : 0, C18 : 1 ω9c, iso-C16 : 0, summed feature 5 (C18 : 2 ω6,9c/C18 : 0 ante), iso-C15 : 0 and anteiso-C17 : 0. MK-9(H6), MK-9(H8) and MK-9(H4) were detected as respiratory quinones. The diagnostic cell-wall diamino acid was meso-diaminopimelic acid. Galactose, glucose and ribose were also present in the cell wall. The major polar lipids consisted of diphosphatidylglycerol, phosphatidyl ethanolamine, phosphatidylinositol mannosides and unidentified phospholipids. The DNA G+C content of the genome sequence, consisting of 8 860 963 bp, is 72.4 mol%. blast analysis based on 16S rRNA gene sequences revealed that the strain belongs to the genus Kitasatospora, with 99.37, 99.03, 98.95, 98.68 and 98.67 % sequence similarity to Kitasatospora aureofaciens ATCC 10762T, Kitasatospora viridis DSM 44826T, Kitasatospora xanthocidica NBRC 13469T, Kitasatospora aburaviensis NRRL B-2218T and Kitasatospora kifunensis IFO 15206T, respectively. Phylogenetic trees based on 16S rRNA gene and whole-genome sequences demonstrated that strain HUAS 3-15T formed a well-supported cluster with K. aureofaciens ATCC 10762T. Further genomic characterization through average nucleotide identity (ANIb/m) and digital DNA-DNA hybridization analysis between strain HUAS 3-15T and K. aureofaciens ATCC 10762T showed values of 90.62/92.55 % and 45.3 %, respectively, lower than the 95-96 % ANI threshold and 70.0 % cutoff used as guideline values for species delineation in bacteria. Furthermore, the differences between the strain and its phylogenomic neighbour in terms of physiological (e.g. sole carbon source growth) and chemotaxonomic (e.g. cellular fatty composition) characteristics further supported this conclusion. Consequently, we concluded that strain HUAS 3-15T represents a novel species of the genus Kitasatospora, for which the name Kitasatospora cathayae sp. nov. is proposed. The type strain is HUAS 3-15T (=MCCC 1K08542T=JCM 36274T).

First Report of Dickeya dadantii Causing Potato Blackleg in Fujian Province China.

Blackleg and soft rot are harmful diseases in potato (Solanum tuberosum) caused by Pectobacterium spp. and Dickeya spp. (Czajkowski et al. 2015). The occurrence of potato blackleg was serious in potato-producing areas around Xiapu County in Fujian Province, China, in 2021 (6 ha) and 2022 (7 ha), with an incidence of approximately 5%, which reached nearly 23%. Three diseased plants were collected to isolate the pathogen. Single colonies from each sampled plant were isolated and streaked onto fresh plates. DNA from three colonies from different plants was PCR amplified with primer pair 27F/1492R (Lane 1991) for the 16S rRNA gene. Since the sequences were identical, we selected strain M2-3 for further analysis. The strain M2-3 was gram-negative, pectolytic on CVP, grew at 37°C and 5% NaCl. The bacterium was positive for phosphatase activity, erythromycin sensitivity, indole production, gelatin liquefaction, malonic utilization, and acid production from, melibiose, raffinose, and arabinose. The bacterium was negative for sucrose, α-methyl glucoside, sorbitol, trehalose, lactose, and sodium citrate (Fujimoto et al. 2018;),although sucrose and lactose did not provide the expected results, there are exception in all species. The genome of strain M2-3 was sequenced and deposited in the NCBI database under accession numbers: CP077422. An Average Nucleotide Identity (ANI) analysis showed that M2-3 clustered with other D. dadantii strains and has a 98.39% identity with D. dadantii strain DSM 18020 (CP023467). The housekeeping genes (recA, dnaX, acnA, gapA, icd, mdh, mtlD and pgi) were amplified with primer pairs designed previously(Fujimoto et al. 2018; Ma et al. 2007) and sequenced. A multilocus sequence analysis (MLSA) was performed by concatenating the 8 gene sequences and constructing a maximum likelihood phylogenetic tree using PhyloSuite version 1.2.1 (Zhang et al. 2020) and IQ-tree version 1.6.8 (Nguyen et al. 2015) software. Strain M2-3 was clustered together with Dickeya dadantii. For the pathogenicity test, three plants per treatment, totaling nine plants, were used. Bacterial suspensions (1×10^8 CFU/mL) were made in a 10mM PBS buffer. 10 μL of M2-3, D. dadantii type strain 18020 (positive control), and buffer (negative control) were injected into the plant stems near the base. Water stains appeared at the site of inoculation after 2 days and they gradually became black and rotten. The leaves became yellow and wilted, and the petiole base rotted within 5 days of inoculation completing the Koch postulate. According to average nucleotide identity and housekeeping gene sequence analysis, strain M2-3 was identified as Dickeya dadantii. Previous studies have reported several pathogens that cause potato blackleg in China, including P. atrosepticum, P. carotovorum, P. brasiliense, P. parmentieri, P. polaris, and P. punjabense (Li-ping et al. 2020; Wang et al. 2021). To the best of our knowledge, this study is the first to report potato blackleg disease caused by Dickeya dadantii in Fujian Province, China. This finding suggests that this pathogen may cause a threat to potato production in Fujian Province.

Pan-Genome Analysis of Wolbachia, Endosymbiont of Diaphorina citri, Reveals Independent Origin in Asia and North America.

Wolbachia, a group of Gram-negative symbiotic bacteria, infects nematodes and a wide range of arthropods. Diaphorina citri Kuwayama, the vector of Candidatus Liberibacter asiaticus (CLas) that causes citrus greening disease, is naturally infected with Wolbachia (wDi). However, the interaction between wDi and D. citri remains poorly understood. In this study, we performed a pan-genome analysis using 65 wDi genomes to gain a comprehensive understanding of wDi. Based on average nucleotide identity (ANI) analysis, we classified the wDi strains into Asia and North America strains. The ANI analysis, principal coordinates analysis (PCoA), and phylogenetic tree analysis supported that the D. citri in Florida did not originate from China. Furthermore, we found that a significant number of core genes were associated with metabolic pathways. Pathways such as thiamine metabolism, type I secretion system, biotin transport, and phospholipid transport were highly conserved across all analyzed wDi genomes. The variation analysis between Asia and North America wDi showed that there were 39,625 single-nucleotide polymorphisms (SNPs), 2153 indels, 10 inversions, 29 translocations, 65 duplications, 10 SV-based insertions, and 4 SV-based deletions. The SV-based insertions and deletions involved genes encoding transposase, phage tail tube protein, ankyrin repeat (ANK) protein, and group II intron-encoded protein. Pan-genome analysis of wDi contributes to our understanding of the geographical population of wDi, the origin of hosts of D. citri, and the interaction between wDi and its host, thus facilitating the development of strategies to control the insects and huanglongbing (HLB).

First report on in-depth genome and comparative genome analysis of a metal-resistant bacterium Acinetobacter pittii S-30, isolated from environmental sample.

A newly isolated bacterium Acinetobacter pittii S-30 was recovered from waste-contaminated soil in Ranchi, India. The isolated bacterium belongs to the ESKAPE organisms which represent the major nosocomial pathogens that exhibit high antibiotic resistance. Furthermore, average nucleotide identity (ANI) analysis also showed its closest match (>95%) to other A. pittii genomes. The isolate showed metal-resistant behavior and was able to survive up to 5 mM of ZnSO4. Whole genome sequencing and annotations revealed the occurrence of various genes involved in stress protection, motility, and metabolism of aromatic compounds. Moreover, genome annotation identified the gene clusters involved in secondary metabolite production (biosynthetic gene clusters) such as arylpolyene, acinetobactin like NRP-metallophore, betalactone, and hserlactone-NRPS cluster. The metabolic potential of A. pittii S-30 based on cluster of orthologous, and Kyoto Encyclopedia of Genes and Genomes indicated a high number of genes related to stress protection, metal resistance, and multiple drug-efflux systems etc., which is relatively rare in A. pittii strains. Additionally, the presence of various carbohydrate-active enzymes such as glycoside hydrolases (GHs), glycosyltransferases (GTs), and other genes associated with lignocellulose breakdown suggests that strain S-30 has strong biomass degradation potential. Furthermore, an analysis of genetic diversity and recombination in A. pittii strains was performed to understand the population expansion hypothesis of A. pittii strains. To our knowledge, this is the first report demonstrating the detailed genomic characterization of a heavy metal-resistant bacterium belonging to A. pittii. Therefore, the A. pittii S-30 could be a good candidate for the promotion of plant growth and other biotechnological applications.

Hybrid sequence-based analysis reveals the distribution of bacterial species and genes in the oral microbiome at a high resolution

Bacteria in the oral microbiome are poorly identified owing to the lack of established culture methods for them. Thus, this study aimed to use culture-free analysis techniques, including bacterial single-cell genome sequencing, to identify bacterial species and investigate gene distribution in saliva. Saliva samples from the same individual were classified as inactivated or viable and then analyzed using 16S rRNA sequencing, metagenomic shotgun sequencing, and bacterial single-cell sequencing. The results of 16S rRNA sequencing revealed similar microbiota structures in both samples, with Streptococcus being the predominant genus. Metagenomic shotgun sequencing showed that approximately 80 % of the DNA in the samples was of non-bacterial origin, whereas single-cell sequencing showed an average contamination rate of 10.4 % per genome. Single-cell sequencing also yielded genome sequences for 43 out of 48 wells for the inactivated samples and 45 out of 48 wells for the viable samples. With respect to resistance genes, four out of 88 isolates carried cfxA, which encodes a β-lactamase, and four isolates carried erythromycin resistance genes. Tetracycline resistance genes were found in nine bacteria. Metagenomic shotgun sequencing provided complete sequences of cfxA, ermF, and ermX, whereas other resistance genes, such as tetQ and tetM, were detected as fragments. In addition, virulence factors from Streptococcus pneumoniae were the most common, with 13 genes detected. Our average nucleotide identity analysis also suggested five single-cell-isolated bacteria as potential novel species. These data would contribute to expanding the oral microbiome data resource.

First Report of Bacterial Blight of Pennycress Caused by Xanthomonas campestris in Wisconsin.

In May 2023, pennycress (Thlaspi arvense, L.) lines undergoing seed production in the Walnut Street Greenhouse at the University of Wisconsin-Madison displayed symptoms of chlorosis and black necrotic leaf spots (Fig. S1-A). Lesions eventually enlarged to 1-2 cm in diameter, became necrotic, and coalesced to cover a substantial portion of leaves. Symptoms were observed in ~30% of the pennycress lines adversely affecting overall growth and reproduction. Symptomatic leaves were surface sterilized for 30 seconds in 0.75% sodium hypochlorite, rinsed in sterile deionized water, and bacteria were isolated using three-phase streaking of symptomatic tissue onto KB medium (King et al., 1954). Single colonies of three isolates (creamy white to yellow) from this initial isolation were streaked onto KB medium to obtain pure cultures. Individual colonies were transferred for growth overnight in nutrient broth (Difco) and an equal amount of the broth was added to 30% glycerol in deionized (di) water and stored at -80 °C. To validate Koch's Postulates, bacteria were grown from these stocks on Yeast Dextrose Calcium Carbonate medium (Wilson et al., 1967) and were used to inoculate 5-week-old pennycress plants in the greenhouse. The bacteria were grown for 48 hours at 26°C, suspended in 300 ml of 0.05 M PBS buffer (pH=7.2) for inoculum preparation. Plants were inoculated with three bacterial isolates (approx. 108 CFU/ml) by piercing the mid veins or hydathodes with a sterilized toothpick dipped in the suspension. Inoculated plants were then enclosed in clear plastic bags for 24-48 hours and maintained in the greenhouse at a constant temperature of 26°C with a 16-hour photoperiod. After seven days, water-soaked lesions appeared on the inoculated leaves, eventually developing into the characteristic black spots (Fig. S1-B). DNA from the original isolates was extracted, and 16S PCR and sequencing of the positive bands was done. The negative control only produced brown spots at the site of inoculation (Fig. S1-C). The primer sequences were as follows: 27F: AGAGTTTGATCMTGGCTCAG; 1492R: GGTTACCTTGTTACGACTT (Eden et al., 1991; Weisburg et al., 1991). A BLAST analysis showed that the isolates had an E value of 0.0 to the genus Xanthomonas as well as 100% identity. Amplification and sequencing of the bacterium using gyrB amplicons revealed a 99-100% pairwise match with Xc. To enhance taxonomy resolution and confirm the identity of these isolates, the complete genomes of three samples were sequenced using NextSeq2000 Illumina platform (NCBI bioproject ID PRJNA1040293). Average Nucleotide Identity (ANI) analysis was conducted with representative strains from the Xc species (Dubrow et al., 2022), using PanExplorer (Dereeper et al., 2020) featuring integrated FastANI module (Jain et al., 2018). The isolates genomes exhibited over 98% identity and clustered with that of Xc pv. incanae and Xc pv. barbarae (Fig S2). Further work will be required to identify the pathovar of Xc identified in this study through phenotypic host range assay. This marks the first documented case of Xc in pennycress in the Midwestern US. Given the potential use of pennycress as a cover crop in the region, further investigations are warranted to assess its economic impact on production and develop management strategies.

Functional Genome Analysis of a Conditionally Pathogenic Rhizobacterial Strain, Pseudomonas putida AKMP7.

This manuscript reports the whole genome sequence of a conditionally pathogenic rhizobacterial strain, Pseudomonas putida AKMP7, which has been previously reported by us to be beneficial to Arabidopsis thaliana under well-watered conditions and pathogenic to the plant under water stress. As part of a study to understand this unique behavior, the whole genome sequence of this strain was analyzed. Based on the results, it was identified that the total length of the AKMP7 genome is 5,764,016 base pairs, and the total GC content of the genome is 62.93% (typical of P. putida). Using RAST annotation pipeline, it was identified that the genome has 5605 coding sequences, 80 repeat regions, 71 tRNA genes, and 22 rRNA genes. A total of 4487 functional proteins and 1118 hypothetical proteins were identified. Phylogenetic analysis has classified it as P. putida species, with a P value of 0.03. In order to identify close relatives of this strain, comparative genomics was performed with 30 other P. putida strains, taken from publicly available genome databases, using Average Nucleotide Identity (ANI) analysis. Whole genome comparison with these strains reveals that AKMP7 possesses Type-IV Secretion System (T4SS) with conjugative transfer functionality. Interestingly, the T4SS feature is absent in all the beneficial/harmless strains of P. putida that we analyzed. All the plant pathogenic bacteria that were analyzed had the T4SS feature in their genome, indicating its role in pathogenesis. This study aims to address important gaps in understanding the molecular mechanisms involved in the conditional/opportunistic pathogenesis of plant-associated, beneficial soil bacteria, using genomics approaches.

Prevalence of CTX-M types among ESBL-producing pathogenic Escherichia coli isolates from foodborne diarrheal patients in Gyeonggi-do, South Korea

AbstractPrevalence and characteristics of extended-spectrum β-lactamase (ESBL)-producing pathogenic Escherichia coli from foodborne diarrheal patients were studied. Analysis of 495 E. coli isolates revealed that 80 isolates were ESBL-producing pathogenic E. coli, and enteroaggregative E. coli and enterotoxigenic E. coli were two of the most prevalent pathotypes. In silico Clermont phylo-typing of the 80 ESBL-producing E. coli showed that phylogroup A (49/80) and D (22/80) were the predominant phylogroups. The average nucleotide identity analysis of ESBL-producing E. coli disclosed that they could be grouped into two phylogenetic groups; 25 A and 55 B groups. All strains, except one, harbored the blaCTX-M gene. All CTX-M-15 type ESBL-producing strains also carried qnrS, a plasmid-mediated quinolone resistance gene (PMQR). These results suggest that the diversity of ESBL-producing E. coli is high and that co-existence of blaCTX-M-15 and qnrS genes is widespread, highlighting their high risk of antibiotic-resistance spreading in infectious disease outbreaks.

Whole genome sequencing analysis of Komagataeibacter nataicola reveals its potential in food waste valorisation for cellulose production.

Komagataeibacter nataicola (K. nataicola) is a gram-negative acetic acid bacterium that produces natural bacterial cellulose (BC) as a fermentation product under acidic conditions. The goal of this work was to study the complete genome of K. nataicola and gain insight into the functional genes in K. nataicola that are responsible for BC synthesis in acidic environments. The pure culture of K. nataicola was obtained from yeast-glucose-calcium carbonate (YGC) agar, followed by genomic DNA extraction, and subjected to whole genome sequencing on a Nanopore flongle flow cell. The genome of K. nataicola consists of a 3,767,936bp chromosome with six contigs and 4,557 protein coding sequences. The maximum likelihood phylogenetic tree and average nucleotide identity analysis confirmed that the bacterial isolate was K. nataicola. The gene annotation via RAST server discovered the presence of cellulose synthase, along with three genes associated with lactate utilization and eight genes involved in lactate fermentation that could potentially contribute to the increase in acid concentration during BC synthesis. A more comprehensive genome study of K. nataicola may shed light into biological pathway in BC productivity as well as benefit the analysis of metabolites generated and understanding of biological and chemical interactions in BC production later.

Complete genome sequences and multidrug resistance genotypes of nontuberculous mycobacteria isolates from the Central Tuberculosis Reference Laboratory Muhimbili Tanzania

Background: Nontuberculous mycobacteria (NTM) usually comprise a group of environmental bacteria, with emerging but elusive coinfection with tuberculous mycobacteria, causing pulmonary tuberculosis. Whole genome sequencing may give insight into potential antimicrobial resistance genotypes, giving clinicians and policymakers proper directions in clinical applications and management regimens. Methods: WGS was performed on twenty-four gDNA isolates from archival samples at the Central Tuberculosis Reference Laboratory using the MinION Oxford Nanopore Sequencing approach. Out of twenty-four, two were confirmed to belong to the NTM group. Further analysis was done to resolve the complete genomes of two nontuberculous mycobacteria strains isolated from tuberculosis patients. We then combined phylogenomics, reference-based scaffolding and average nucleotide identity (ANI) analysis to delineate each strain's taxonomic position and corresponding features. Results: Our findings reveal that the two strains fit into the genus Mycolicibacterium, and the closest relative is Mycolicibacterium novocastrense. Coupling BacAnt and CARD-based antibiotic resistance analyses revealed multidrug-resistant genotypes of diverse spectra and mechanisms. While the BC02 strain is genetically resistant to beta-lactams, macrolides and rifamycins, the BC05 strain portrays an extended drug resistance genotype encompassing beta-lactams, macrolides, polyamines, and aminoglycosides. Both strains possess a single nucleotide polymorphism (SNP) of the RNA polymerase beta-subunit (rpoB), representing resistance to the first-line rifampicin. Additionally, the BC05 strain genetically portrays resistance to ethambutol, isoniazid and fosfomycin through mechanisms involving target alteration through SNPs, drug inactivation and efflux. Conclusion: Our findings strongly suggest the potential implication of multidrug-resistant NTM clinical isolates in the pathogenesis of pulmonary tuberculosis.

Genome-based approach to evaluate the metabolic potentials and exopolysaccharides production of Bacillus paralicheniformis CamBx3 isolated from a Chilean hot spring.

In the present study, a thermophilic strain designated CamBx3 was isolated from the Campanario hot spring, Chile. Based on 16S rRNA gene sequence, phylogenomic, and average nucleotide identity analysis the strain CamBx3 was identified as Bacillus paralicheniformis. Genome analysis of B. paralicheniformis CamBx3 revealed the presence of genes related to heat tolerance, exopolysaccharides (EPS), dissimilatory nitrate reduction, and assimilatory sulfate reduction. The pangenome analysis of strain CamBx3 with eight Bacillus spp. resulted in 26,562 gene clusters, 7,002 shell genes, and 19,484 cloud genes. The EPS produced by B. paralicheniformis CamBx3 was extracted, partially purified, and evaluated for its functional activities. B. paralicheniformis CamBx3 EPS with concentration 5 mg mL-1 showed an optimum 92 mM ferrous equivalent FRAP activity, while the same concentration showed a maximum 91% of Fe2+ chelating activity. B. paralicheniformis CamBx3 EPS (0.2 mg mL-1) demonstrated β-glucosidase inhibition. The EPS formed a viscoelastic gel at 45°C with a maximum instantaneous viscosity of 315 Pa.s at acidic pH 5. The present study suggests that B. paralicheniformis CamBx3 could be a valuable resource for biopolymers and bioactive molecules for industrial applications.

Whole-genome sequence and resistance determinants of four Elizabethkingia anophelis clinical isolates collected in Hanoi, Vietnam

Four isolates of the opportunistic pathogen Elizabethkingia anophelis were identified for the first time in a Vietnamese hospital and underwent antimicrobial susceptibility testing and genomic characterization by whole-genome sequencing. Complete, fully circularized genome sequences were obtained for all four isolates. Average Nucleotide Identity analysis and single nucleotide polymorphism phylogenetic analysis on the core genome showed that three of the four isolates were genetically distinct, ruling out the hypothesis of a single strain emergence. Antibiotic susceptibility testing highlighted multi-resistant phenotypes against most antimicrobial families, including beta-lactams, carbapenems, aminoglycosides, quinolones, macrolides, amphenicols, rifamycins and glycopeptides. Additionally, in silico genomic analysis was used to correlate the phenotypic susceptibility to putative resistance determinants, including resistance genes, point mutations and multidrug efflux pumps. Nine different resistance genes were located inside a single resistance pocket predicted to be a putative Integrative and Conjugative Element (ICE). This novel ICE was shared by three isolates from two different lineages and displayed similarity with ICEs previously reported in various Elizabethkingia and Chryseobacterium species. The role of such ICEs in pathogenicity, genome plasticity and antimicrobial resistance gene spread within the Flavobacteriaceae family needs to be further elucidated.

Isolation, characterization and application of noble bacteriophages targeting potato common scab pathogen Streptomyces stelliscabiei

Bacteriophages have emerged as promising alternatives to pesticides for controlling bacterial pathogens in crops. Among these pathogens, Streptomyces stelliscabiei (syn. S. stelliscabiei) is a primary causative agent of potato common scab (PCS), resulting in substantial global economic losses. The traditional management methods for PCS face numerous challenges, highlighting the need for effective and environmentally friendly control strategies. In this study, we successfully isolated three novel bacteriophages, namely Psst1, Psst2, and Psst4, which exhibited a broad host range encompassing seven S. stelliscabiei strains. Morphological analysis revealed their distinct features, including an icosahedral head and a non-contractile tail. These phages demonstrated stability across a broad range of temperatures (20–50°C), pH (pH 3–11), and UV exposure time (80 min). Genome sequencing revealed double-stranded DNA phage with open reading frames encoding genes for phage structure, DNA packaging and replication, host lysis and other essential functions. These phages lacked genes for antibiotic resistance, virulence, and toxicity. Average nucleotide identity, phylogenetic, and comparative genomic analyses classified the three phages as members of the Rimavirus genus, with Psst1 and Psst2 representing novel species. All three phages efficiently lysed S. stelliscabiei in the liquid medium and alleviated scab symptom development and reduced pathogen abundance on potato slices. Furthermore, phage treatments of radish seedlings alleviated the growth inhibition caused by S. stelliscabiei with no disease symptoms. In soil potted experiments, phages significantly reduced disease incidence by 40%. This decrease is attributed to a reduction in pathogen density and the selection of S. stelliscabiei strains with reduced virulence and slower growth rates in natural environments. Our study is the first to report the isolation of three novel phages that infect S. stelliscabiei as a host bacterium. These phages exhibit a broad host range, and demonstrate stability under a variety of environmental conditions. Additionally, they demonstrate biocontrol efficacy against bacterial infections in potato slices, radish seedlings, and potted experiments, underscoring their significant potential as biocontrol agents for the effective management of PCS.

Comprehensive genomic analysis of Bacillus subtilis and Bacillus paralicheniformis associated with the pearl millet panicle reveals their antimicrobial potential against important plant pathogens

BackgroundPlant microbiome confers versatile functional roles to enhance survival fitness as well as productivity. In the present study two pearl millet panicle microbiome member species Bacillus subtilis PBs 12 and Bacillus paralicheniformis PBl 36 found to have beneficial traits including plant growth promotion and broad-spectrum antifungal activity towards taxonomically diverse plant pathogens. Understanding the genomes will assist in devising a bioformulation for crop protection while exploiting their beneficial functional roles.ResultsTwo potential firmicute species were isolated from pearl millet panicles. Morphological, biochemical, and molecular characterization revealed their identities as Bacillus subtilis PBs 12 and Bacillus paralicheniformis PBl 36. The seed priming assays revealed the ability of both species to enhance plant growth promotion and seedling vigour index. Invitro assays with PBs 12 and PBl 36 showed the antibiosis effect against taxonomically diverse plant pathogens (Magnaporthe grisea; Sclerotium rolfsii; Fusarium solani; Alternaria alternata; Ganoderma sp.) of crops and multipurpose tree species. The whole genome sequence analysis was performed to unveil the genetic potential of these bacteria for plant protection. The complete genomes of PBs 12 and PBl 36 consist of a single circular chromosome with a size of 4.02 and 4.33 Mb and 4,171 and 4,606 genes, with a G + C content of 43.68 and 45.83%, respectively. Comparative Average Nucleotide Identity (ANI) analysis revealed a close similarity of PBs 12 and PBl 36 with other beneficial strains of B. subtilis and B. paralicheniformis and found distant from B. altitudinis, B. amyloliquefaciens, and B. thuringiensis. Functional annotation revealed a majority of pathway classes of PBs 12 (30) and PBl 36 (29) involved in the biosynthesis of secondary metabolites, polyketides, and non-ribosomal peptides, followed by xenobiotic biodegradation and metabolism (21). Furthermore, 14 genomic regions of PBs 12 and 15 of PBl 36 associated with the synthesis of RiPP (Ribosomally synthesized and post-translationally modified peptides), terpenes, cyclic dipeptides (CDPs), type III polyketide synthases (T3PKSs), sactipeptides, lanthipeptides, siderophores, NRPS (Non-Ribosomal Peptide Synthetase), NRP-metallophone, etc. It was discovered that these areas contain between 25,458 and 33,000 secondary metabolite-coding MiBiG clusters which code for a wide range of products, such as antibiotics. The PCR-based screening for the presence of antimicrobial peptide (cyclic lipopeptide) genes in PBs 12 and 36 confirmed their broad-spectrum antifungal potential with the presence of spoVG, bacA, and srfAA AMP genes, which encode antimicrobial compounds such as subtilin, bacylisin, and surfactin.ConclusionThe combined in vitro studies and genome analysis highlighted the antifungal potential of pearl millet panicle-associated Bacillus subtilis PBs12 and Bacillus paralicheniformis PBl36. The genetic ability to synthesize several antimicrobial compounds indicated the industrial value of PBs 12 and PBl 36, which shed light on further studies to establish their action as a biostimulant for crop protection.

Complete genome sequence of a novel Prescottella sp. R16 isolate from deep-sea sediments in the western Pacific.

Prescottella, a distinct genus separate from Rhodococcus, has garnered attention for its adaptability and ecological versatility. In this study, a Gram-stain positive and ovoid-rod shaped the actinobacterium strain R16 was isolated from deep-sea sediment (with a depth of 6,310m) in the Western Pacific. On the basis of 16S rRNA gene sequence analysis, average nucleotide identity and phylogenomic analysis, strain R16 clearly represents a novel species within the genus Prescottella. Genomic analyses indicate Prescottella sp. R16 contains a circular chromosome of 4,531,251 bp with an average GC content of 68.9%, 4,208 protein-coding genes, 51 tRNA genes, and 12 rRNA operons. Additionally, four CRISPRs and 24 genomic islands are also identified. The presence of rich categories related to catalytic activity, membrane part and metabolic process highlights their involvement in cellular component, biological process, and molecular function. The genome sequence of strain R16 also revealed the presence of 13 putative biosynthetic gene clusters for secondary metabolites, including those for ε-Poly-L-lysine, ectoine, heterobactin, isorenieratene and corynecin, suggesting its potential for antibiotic production and warranting further exploration.

Clostridium strain FAM25158, a unique endospore-forming bacterium related to Clostridium tyrobutyricum and isolated from Emmental cheese shows low tolerance to salt.

The genus Clostridium is a large and diverse group of species that can cause food spoilage, including late blowing defect (LBD) in cheese. In this study, we investigated the taxonomic status of strain FAM25158 isolated from Emmental cheese with LBD using a polyphasic taxonomic and comparative genomic approach. A 16S rRNA gene sequence phylogeny suggested affiliation to the Clostridium sensu stricto cluster, with Clostridium tyrobutyricum DSM 2637T being the closest related type strain (99.16% sequence similarity). Average Nucleotide Identity (ANI) analysis revealed that strain FAM25158 is at the species threshold with C. tyrobutyricum, with ANI values ranging from 94.70 to 95.26%, while the digital DNA-DNA hybridization values were below the recommended threshold, suggesting that FAM25158 is significantly different from C. tyrobutyricum at the genomic level. Moreover, comparative genomic analysis between FAM25158 and its four closest C. tyrobutyricum relatives revealed a diversity of metabolic pathways, with FAM25158 differing from other C. tyrobutyricum strains by the presence of genes such as scrA, srcB, and scrK, responsible for sucrose utilization, and the absence of many important functional genes associated with cold and osmolality adaptation, which was further supported by phenotypic analyses. Surprisingly, strain FAM25158 exhibited unique physiologic traits, such as an optimal growth temperature of 30°C, in contrast to its closest relatives, C. tyrobutyricum species with an optimal growth temperature of 37°C. Additionally, the growth of FAM25158 was inhibited at NaCl concentrations higher than 0.5%, a remarkable observation considering its origin from cheese. While the results of this study provide novel information on the genetic content of strain FAM25158, the relationship between its genetic content and the observed phenotype remains a topic requiring further investigation.

Prevalence and molecular characteristics of Shewanella infection in diarrhea patients in Beijing, China 2017-2019.

Shewanella is an important opportunistic pathogen distributed in marine environments that has caused an increasing number of clinical infections. However, there are few reports on the distribution and characteristics of Shewanella in the diarrheal pathogen spectrum. In this study, we have systematically described the prevalence of Shewanella infections in diarrhea patients in Beijing, China 2017-2019, and genome characteristics and antimicrobial susceptibility of Shewanella isolates. Stool samples were collected from diarrhea patients in a surveillance project from 2017 to 2019. Shewanella strains were isolated, and identified using VITEKR 2 COMPACT and MALDI-TOF MS. Average nucleotide identity (ANI) analysis, multi-locus sequence typing (MLST), phylogenetic analysis, virulence-associated genes and antimicrobial resistance genes analysis were used for genome characteristics description. The antibiotic susceptibility test was performed with microbroth dilution method. 1104 fecal samples were collected, and the Shewanella detection rate was 2.36% (26/1104). The main manifestations of infection caused by Shewanella spp. were diarrhea (100%, 26/26), abdominal pain (65.38%, 17/26), and vomiting (38.46%, 10/26). The 26 isolates were classified into 3 species (S. algae (n = 18), S. indica (n = 5), and S. chilikensis (n = 3)) and 22 sequence types. Core genome single nucleotide polymorphism-based evolutionary tree identified three clone groups corresponding to three infection events in the same months in 2017 and 2019. The putative virulence-associated gene pool consisted of 56 potential virulence genes, including 19 virulence gene factors. The resistance rates of the 26 isolates to 17 antibiotics from high to low were as follows: polymyxin E (76.92%), cefotaxime (57.69%), ampicillin (50%), ampicillin-sulbactam (34.62%), nalidixic acid (15.38%), ciprofloxacin (11.54%), selectrin (3.846%,1/26), and tetracycline (3.846%, 1/26). The rate of multidrug resistance was 38.46% (10/26). Monitoring for Shewanella spp. should be added to the routine surveillance of infectious diarrhea during the epidemic season.