Subsystem Technology Research Articles

Comparison of Mycobacterium tuberculosis Strains with H37Rv Using Whole Genome Sequence Analysis to Identify Virulence Factors and Phylogenetic Relationships

Mycobacterium tuberculosis (Mtb) poses a significant disease burden in developing countries, largely due to the rising issue of drug resistance. In Pakistan, the genetic and molecular characteristics of resistant strains have not been thoroughly analyzed or compared with the reference Mtb strain, H37Rv, to understand the molecular and genetic epidemiology. Mtb employs various strategies to resist anti-tuberculosis drugs, and antimicrobial resistance, including single and multidrug resistance, has increased extensively in developing nations like Pakistan. In this study, the whole genomes of three Mtb strains (335, 335-2, and 311) were uploaded to the Rapid Annotation Subsystems Technology (RAST) server to retrieve coding DNA sequences (CDS). The RAST server identified 4,454, 4,445, and 4,396 CDS for Mtb strains 335, 335-2, and 311, respectively. Mutation analysis was performed using NCBI nucleotide BLAST and CLUSTAL Omega, comparing the sequences with the H37Rv reference strain. The analysis revealed 909, 832, and 1,135 mutations in the 335, 335-2, and 311 strains, respectively. Phylogenetic analysis based on the most virulent protein (EccC3) indicated a close resemblance between all three strains and the H37Rv strain. Furthermore, Mycobacteriumcanettii (accession number WP_203037448.1) was identified as the closest related bacterium to the studied strains and H37Rv.

Detection of Genes Related to Antibiotic Resistance in Leptospira.

Leptospirosis is a disease caused by the bacteria of the Leptospira genus, which can usually be acquired by humans through contact with urine from infected animals; it is also possible for this urine to contaminate soils and bodies of water. The disease can have deadly consequences in some extreme cases. Fortunately, until now, patients with leptospirosis have responded adequately to treatment with doxycycline and azithromycin, and no cases of antibiotic resistance have been reported. However, with the extensive use of such medications, more bacteria, such as Staphylococci and Enterococci, are becoming resistant. The purpose of this study is to determine the presence of genes related to antibiotic resistance in the Leptospira genus using bioinformatic tools, which have not been undertaken in the past. Whole genomes from the 69 described Leptospira species were downloaded from NCBI's GeneBank and analyzed using CARD (The Comprehensive Antibiotic Resistant Database) and RAST (Rapid Annotations using Subsystem Technology). After a detailed genomic search, 12 genes associated with four mechanisms were found: resistance to beta-lactamases, vancomycin, aminoglycoside adenylyltransferases, as well as multiple drug efflux pumps. Some of these genes are highly polymorphic among different species, and some of them are present in multiple copies in the same species. In conclusion, this study provides evidence of the presence of genes related to antibiotic resistance in the genomes of some species of the genus Leptospira, and it is the starting point for future experimental evaluation to determine whether these genes are transcriptionally active in some species and serovars.

Indigenous alkaliphiles as an effective tool for bioremediation of bauxite residue (red mud)

The microorganisms thriving in ageing Bauxite residue, or red mud, have captured scientific interest for their adaptability to extreme conditions. This study investigates extremophilic microbial communities present in Indian red mud for their potential to neutralize the residue and extracting metals. These communities thrive in the highly alkaline, sodic, and metal-rich conditions of this challenging environment. The research specifically highlights alkali-halophilic species and their ability to withstand pH fluctuations (7–11) and varying NaCl levels (0–3 M). Out of the 13 isolates analyzed, all preferred a pH range of 9–10 and tolerated NaCl up to 1.5–2 M. Notably, Evansella cellulosilytica and Halalkalibacterium halodurans, showed superior tolerance index for Al3+ and Cr6+ at 2000 ppm, as well as Co2+ at 1000 ppm, followed by Sutcliffiella cohnii. However, the tolerance index for Cu2+, Te4+, and Hg2+ was relatively low for all tested strains. Additionally, Alkalihalobacillus sp. demonstrated remarkable tolerance to 10% red mud, facilitated by the production of mixed acids, neutralizing the pH within 24 h. The study proposes a potential mechanism for metal and red mud tolerance through genomic analysis using Rapid Annotation Subsystem Technology (RAST), revealing stress tolerance mechanisms, metal resistance genes, ion transporters, hydrolytic enzymes, siderophore production, and organic acid synthesis. Indigenous species like E. cellulosilytica, H. halodurans, S. cohnii, and Alkalihalobacillus sp. emerge as promising candidates for red mud bioremediation, providing insights into sustainable strategies for red mud disposal.

First Report of Pantoea ananatis causing leaf streak disease on wheat (Triticum aestivum) in the United States of America.

Wheat (Triticum aestivum) loses 21.5% yield to pests and diseases annually (Savary et al. 2019). Among the wheat diseases, bacterial leaf streak (BLS) is a growing problem, costing $78.5 million in losses (https://cropprotectionnetwork.org/). In July 2022, we sampled winter wheat leaf samples at Volga (44.30, -96.92), South Dakota, USA with an estimated disease incidence of 40% (n=100). The typical symptoms were water-soaking with large necrotic and chlorotic streaks extending the length of the leaves and were strikingly similar to BLS. To isolate the pathogen, leaves were cut lengthwise into 1 cm pieces and surface-sterilized using a 10% NaOCl solution for 3 min, followed by 70% ethanol for 3 min, and then rinsed with sterile distilled water and placed in 500 ul of sterile distilled water for 5 min and using a sterile loop the water was streaked over a plate of Nutrient Agar (NA). Following Duveiller et al. (1997), the streaked plate was incubated in the dark at 28℃ for 48 h. Observed single colonies were sub-cultured thrice onto fresh NA plates to obtain a pure culture. We named the culture SD101. Bacteria were found to be gram-negative with a colony morphology initially raised, smooth, and white that later turned yellow. DNA was extracted using the Wizard HMW DNA Extraction Kit (Promega, Madison, WI) following the manufacturer's protocol, and sequenced using Nanopore MinION R9.4 (Oxford Nanopore Technology). We used the Rapid Annotation Using Subsystems Technology approach (Aziz eal. 2008) to extract the 16S rDNA, DNA gyrase subunit B (gyrB), and translation initiation factor IF-2 (infB) gene sequences that were deposited in GenBank under accession numbers PP329908.1 for 16S rDNA, PP496481 for infB, and PP328920.1 for gyrB. Homology analysis using CLC Genomics Workbench 22.0.2 (QIAGEN) and BLASTn against the GenBank nucleotide database resulted in a 99.74% match (1543/ 1547 bp) of the 16S sequence, 99.59% match (2674/ 2685 bp) of the infB sequence, and 99.42% match (2396/ 2410 bp) of the gyrB sequence with Pantoea ananatis strain AJ13355 (AP012032). To test pathogenicity, seeds of spring wheat breeding line SD4892 were planted in 30 cm × 30 cm pots in a greenhouse under a 16 h light photoperiod. The inoculum was prepared from 48-h-old NA plates of SD101 rinsed with 1X Phosphate Buffer Saline (PBS buffer), adjusted to an OD600 = 1.0, and amended with two drops of Tween 20 (polyoxyethylene sorbitol ester, Millipore Sigma). PBS with Tween 20 was used as a negative control. The inoculum was sprayed on 15 replicates of 15-day-old seedlings, kept at 95% relative humidity for 48 h, then moved to the greenhouse at 23 to 25°C. The symptoms appeared as water soaking that later turned to necrotic streaks with surrounding chlorosis on all 15 inoculated plants while control plants remained healthy. The pathogen was re-isolated from the leaves as described above. The 16S rDNA, infB, and gyrB sequences amplified and sequenced were identical to the gene sequences from the whole genome sequencing. The experiment was repeated with the same results, completing Koch's postulates. Although P. ananatis is pathogenic on corn, rice, and other plant species in the United States (Coutinho et al. 2009), and was reported pathogenic on wheat in Poland (Krawczyk et al. 2020), this is the first report of its pathogenicity on wheat in the United States. The prevalence, and incidence of BLS disease caused by P. ananatis in wheat is needed to estimate its threat to wheat production and to develop management strategies.

Characteristics of Genomes of Opportunistic Bacteria in Zoogenic Ecosystems of the Russian Western Arctic Islands

Introduction: Arctic ecosystems are the most important object of microbiological surveillance that helps monitor a rapidly changing natural environment affected by climate change and industrial development of the northern territories. Objective: To assess the epidemic potential of opportunistic bacteria associated with animals from the Arctic islands of the Barents and Kara Seas. Materials and methods: We have studied four strains of gram-negative opportunistic bacteria (Serratia fonticola, Aeromonas salmonicida, Yersinia kristensenii, and Yersinia rochesterensis) isolated in 2021–2022 during microbiological testing of 46 samples of zoogenic biologic materials from the natural ecosystems of Vaigach Island, Novaya Zemlya and Franz Josef Land archipelagos. Genomes of the isolated microbial strains were sequenced using the Illumina MiSeq sequencing system and, after de novo assembly using the SPAdes 13.0 genomic assembler, annotated with RAST (Rapid Annotation using Subsystem Technology). Results: The strains were shown to possess a number of virulence factors and antibiotic resistance genes, which allows them to be considered as potential pathogens. Classes A, B, and C beta-lactamases were found in the genomes of all the bacteria under study, and operons for siderophores and type IV secretion systems were typical of virulence factors. In addition, the pertussis-like YtxA enterotoxin was identified in Yersinia genomes. Conclusions: Our findings indicate the possibility of emergence of natural foci of notifiable infections on the islands in the high-latitude Arctic promising in terms of tourism development and economic growth.

Exploring the biotechnological potential of Acinetobacter soli ANG344B: A novel bacterium for 2-phenylethanol production

A bacterium, Acinetobacter soli ANG344B, isolated from river water, exhibited an exceptional capacity to produce 2-phenylethanol (2-PE) using L-phenylalanine (L-Phe) as a precursor—a capability typically observed in yeasts rather than bacteria. Bioreactor experiments were conducted to evaluate the production performance, using glucose as the carbon source for cellular growth and L-Phe as the precursor for 2-PE production. Remarkably, A. soli ANG344B achieved a 2-PE concentration of 2.35 ± 0.26 g/L in just 24.5 h of cultivation, exhibiting a global volumetric productivity of 0.10 ± 0.01 g/L.h and a production yield of 0.51 ± 0.01 g2-PE/gL-Phe, a result hitherto reported only for yeasts. These findings position A. soli ANG344B as a highly promising microorganism for 2-PE production.Whole-genome sequencing of A. soli strain ANG344 revealed a genome size of 3.52 Mb with a GC content of 42.7 %. Utilizing the Rapid Annotation using Subsystem Technology (RAST) server, 3418 coding genes were predicted, including genes coding for enzymes previously associated with the metabolic pathway of 2-PE production in other microorganisms, yet unreported in Acinetobacter species. Through gene mapping, 299 subsystems were identified, exhibiting 30 % subsystem coverage. The whole genome sequence data was submitted to NCBI GeneBank with the BioProject ID PRJNA982713. These draft genome data offer significant potential for exploiting the biotechnological capabilities of A. soli strain ANG344 and for conducting further comparative genomic studies.

Biogas upgrading towards acetic acid production using Clostridium thailandense supplemented with granular activated carbon (GAC) and L-arginine: A genomic analysis approach

This study explores the impact of granular activated carbon (GAC) and L-arginine supplementation on biogas upgrading and acetic acid production employing Clostridium thailandense. GAC and L-arginine concentrations ranged from 0 to 20 g/L and 0 to 5 g/L, respectively, with H2 acting as the electron donor at an H2 to CO2 ratio of 2:1 (v/v). Experiments were conducted at 30 °C with an agitation speed of 150 rpm. Additionally, gene annotation of the C. thailandense genome using Rapid Annotations using Subsystems Technology (RAST) identified genes involved in CO2 to acetic acid conversion. Results indicate that adding 7.5 g/L GAC boosts CH4 purity in biogas, elevating CO2 and H2 consumption efficiencies to 88.3 % and 98.7 %, respectively. This enhancement leads to a CH4 content increase to 93.3 %, accompanied by 0.90 g/L acetic acid production. Conversely, L-arginine demonstrates no significant impact on CO2 conversion. Leveraging RAST, the study identifies hydrogenase genes and NADH-dependent ferredoxin-NADP+ oxidoreductase (Nfn), as crucial for heightened H2 consumption efficiencies and cell growth facilitated by GAC, thus enhancing biogas upgrading efficiency in C. thailandense. This research provides vital insights into optimizing sustainable biogas production through strategic GAC utilization and elucidates the roles of hydrogenase genes and Nfn.

Levels of Cytokines in Leptospirosis Patients with Different Serovars and rfb Locus.

Leptospirosis has a wide spectrum of clinical manifestations ranging from mild to severe disease. The cytokine response is considered one of the key drivers for this varying manifestation. The different cytokine response observed in patients with leptospirosis could be due to the variation of infecting serovars. Since the rfb locus codes for the lipopolysaccharide synthesis of the bacterial cell wall, which also determines the serovar, this locus may play a role in driving a specific cytokine response in the host. We investigated 12 commonly used cytokine profiles in serum samples of culture, microscopic agglutination test (MAT), or polymerase chain reaction (PCR)-positive patients with leptospirosis. The sequences of the rfb locus in culture-positive samples were generated from whole genome sequencing and serovar status was drawn from original data published. Isolated cultures were subjected to whole genome sequencing using the PacBio RS II system, and the resulting data were used to determine the species. The recovered genomic data were annotated with the Rapid Annotation using Subsystem Technology (RAST) subsystem, and the rfb locus was extracted. The cytokine analysis was carried out using the Qiagen human ELISA kit. Eighteen samples were found to be positive by culture, while the other 7 samples were positive by PCR or MAT. Infections from Leptospira interrogans serovar Autumnalis (5), Pyrogens (3), Icterohaemorrhagiae (1) Leptospira borgpetersenii (all 7 samples clustered in same clonal group with serovar status not determined), Leptospira weilii (1 with serovar status not determined), and Leptospira kirschneri serovar Grippotyphosa (1) were included in the analysis. Three patients [infected with Leptospira interrogansserovar Autumnalis (2) and Pyrogens (1)] and 2 MAT-positive patients (highest titer against serovar Bratislava of L.interrognas) were reported to have severe clinical manifestations, while the rest had mild to moderate symptoms. Although the serum cytokine concentration of patients with severe clinical manifestation was comparatively higher, a statistically significant difference was observed only for interleukin (IL)-1β (P < 0.05). IL-10/tumor necrosis factor-alpha (TNF-α) ratio was high in patients with severe complications. In general, patients infected with L. interrogans showed higher concentration of cytokines compared to L. borgpetersenii.

Determinação da diversidade microbiana na conjuntiva ocular de cães saudáveis pelo sequenciamento completo do DNA

Abstract The conjunctiva plays an essential role in eye health and immunity and acts as a barrier to the entry of microorganisms. Conjunctival infections are common in dogs and result from both the invasion of pathogenic microorganisms and the uncontrolled growth of the existing microbiota. Most of the existing data come from studies based on traditional culture methods. These reports indicate the predominance of gram-positive bacteria, especially Staphylococcus spp. In the present study, we analyzed the microbiota present on the conjunctival surface from a heterogeneous dog population without ophthalmological disorders using DNA sequencing. After a thorough ophthalmological examination, conjunctival swabs were collected from both eyes of 30 dogs. After processing and nucleic acid extraction, the sample pool was subjected to shotgun DNA sequencing through the Illumina platform and analyzed via the Metagenomic Rapid Annotations using Subsystems Technology (MGRAST) server. A predominance of the phylum Proteobacteria and the genera Ralstonia and Burkholderia were identified along with a minority of fungi, whereas viruses were not found. Microbial DNA sequencing has provided new data on this subject, revealing the presence of noncultivable organisms that were previously unknown as part of the ocular microbiome.

Genomic analysis of Oceanotoga teriensis strain UFV_LIMV02, a multidrug-resistant thermophilic bacterium isolated from an offshore oil reservoir.

Bacteria of the species Oceanotoga teriensis belong to the family Petrotogaceae, are Gram-negative bacilli, are moderately thermophilic and are included in the group of thiosulfate-reducing bacteria, being capable of significantly accelerating corrosion in metallic structures. However, no in-depth study on the genome, antibiotic resistance and mobile elements has been carried out so far. In this work, the isolation, phenotypic and genotypic characterization of the multi-resistant O. teriensis UFV_LIMV02 strain was carried out, from water samples from an offshore oil extraction platform in Rio de Janeiro (Brazil). We determined that the isolate has a genome of 2 812 778 bp in size, with 26 % GC content, organized into 34 contigs. Genomic annotation using Rapid Annotation using Subsystem Technology revealed the presence of genes related to resistance to antibiotics and heavy metals. By evaluating the antimicrobial resistance of the isolate using the disc diffusion technique, resistance was verified for the classes of antibiotics, beta-lactams, fluoroquinolones, aminoglycosides, sulfonamides, lincosamides and rifamycins, a total of 14 antibiotics. The search for genomic islands, prophages and defence systems against phage infection revealed the presence of five genomic islands in its genome, containing genes related to resistance to heavy metals and antibiotics, most of which are efflux pumps and several transposases. No prophage was found in its genome; however, nine different defence systems against phage infection were detected. When analysing the clustered regularly interspaced short palindromic repeat (CRISPR) systems, four CRISPR arrays, classified as types I-B and III-B, with 272 spacers, can provide the strain with immunity to different mobile genetic elements and bacteriophage infection. The results found in this study show that the isolate UFV_LIVM02 is an environmental bacterium, resistant to different classes of antibiotics, and that the proteins encoded by the predicted genomic islands may be associated with the development of greater resistance to antibiotics and heavy metals. They provide evidence that environmental bacteria found in offshore oil exploration residues may pose a risk for the spread of antibiotic resistance genes. More comprehensive studies on the microbial community present in oil waste are needed to assess the risks of horizontal gene transfer.

Safety assessment of five candidate probiotic lactobacilli using comparative genome analysis.

Micro-organisms belonging to the Lactobacillus genus complex are often used for oral consumption and are generally considered safe but can exhibit pathogenicity in rare and specific cases. Therefore, screening and understanding genetic factors that may contribute to pathogenicity can yield valuable insights regarding probiotic safety. Limosilactobacillus mucosae LM1, Lactiplantibacillus plantarum SK151, Lactiplantibacillus plantarum BS25, Limosilactobacillus fermentum SK152 and Lactobacillus johnsonii PF01 are current probiotics of interest; however, their safety profiles have not been explored. The genome sequences of LM1, SK151, SK152 and PF01 were downloaded from the NCBI GenBank, while that of L. plantarum BS25 was newly sequenced. These genomes were then annotated using the Rapid Annotation using Subsystem Technology tool kit pipeline. Subsequently, a command line blast was performed against the Virulence Factor Database (VFDB) and the Comprehensive Antibiotic Resistance Database (CARD) to identify potential virulence factors and antibiotic resistance (AR) genes. Furthermore, ResFinder was used to detect acquired AR genes. The query against the VFDB identified genes that have a role in bacterial survivability, platelet aggregation, surface adhesion, biofilm formation and immunoregulation; and no acquired AR genes were detected using CARD and ResFinder. The study shows that the query strains exhibit genes identical to those present in pathogenic bacteria with the genes matched primarily having roles related to survival and surface adherence. Our results contribute to the overall strategies that can be employed in pre-clinical safety assessments of potential probiotics. Gene mining using whole-genome data, coupled with experimental validation, can be implemented in future probiotic safety assessment strategies.

Whole genome sequence and comparative genomics analysis of multidrug-resistant Staphylococcus xylosus NM36 isolated from a cow with mastitis in Basrah city

BackgroundStaphylococcus xylosus is a coagulase-negative, gram-positive coccus that is found in the environment and as a commensal organism on the skin and mucosal surfaces of animals. Despite the fact that S. xylosus is considered a nonpathogenic bacterium, several studies have linked S. xylosus to opportunistic infections in both animals and humans. During an investigation of mastitis-causing agents in the governorate of Basrah, Iraq, we identified an antibiotic-resistant strain of S. xylosus NM36 from a milk sample from a cow with chronic mastitis. In addition to robust biofilm formation, multiple antibiotic resistance phenotypes were found. To further understand the genetic background for these phenotypes, the full genome of S. xylosus NM36 was analyzed. ResultsThe genome consisted of a single circular 2,668,086 base pairs chromosome containing 32.8% G + C. There were 2454 protein-coding sequences, 4 ribosomal RNA (rRNA) genes, and 50 transfer RNA (tRNA) genes in the genome. In addition, genetic variation was studied by searching sequence data against a representative reference genome. Consequently, single-nucleotide polymorphism analysis was conducted and showed that there were 46,610 single-nucleotide polymorphisms (SNPs), 523 insertions, and 551 deletions. In order to overcome antibiotics, S. xylosus NM36 had been armed with several antibiotic resistance genes from several groups and families. The genome annotation service in PathoSystems Resource Integration Center (PATRIC) and Rapid Annotation using Subsystem Technology (RAST) annotation servers showed that there are multiple antimicrobial resistance elements, including antibiotic inactivation enzymes (BlaZ family, FosB), antibiotic resistance gene clusters (TcaB, TcaB2, TcaR), proteins involved in methicillin resistance (LytH, FmtA, FemC, HmrB, HmrA), TetR family transcriptional regulators, and efflux pumps conferring antibiotic resistance (NorA). In addition, we investigated and categorized the biofilm and quorum-sensing elements of the NM36 strain and found that it has multiple subsets of biofilm regulators, confirming its pathogenic nature. ConclusionsThese findings necessitate a reevaluation of microbial and clinical interventions when dealing with coagulase-negative staphylococci, particularly in the context of studies pertaining to public health. This is the first time, to our knowledge, that the entire genome of S. xylosus has been sequenced in Iraq.

331 Investigating the Ruminal Metagenome of Grass and Forage-Fed Bison to Uncover Metabolic Activities That Impact the Efficiency of Plant Fiber Utilization

Abstract The ability of bison to thrive on grass and forage-based diets is of great interest for the bison industry. However, raising bison on pasture presents challenges, including longer finishing times. While some producers overcome this by feeding grain or concentrate to bison, this practice conflicts with the concept of natural production. Considering that bison maintain better on feed of poor quality compared with domesticated grazers, it can be inferred that there is great potential for improving their productivity on pastures. Because ruminants rely on symbiotic microorganisms to break down and ferment plant polysaccharides into nutrients they can absorb and assimilate, the metabolic capabilities of rumen microbial communities in bison would likely have an important role in contributing to the efficiency of their host. To gain further insight, we investigated the ruminal metagenome of six grass-fed bison. Raw sequence reads generated using an Illumina Miseq (2x250) platform from ruminal microbial genomic DNA were assembled into genomic contigs using custom written Perl scripts. A total of 6,297 contigs, representing a combined total length of 23,822,104 nucleotides, were obtained for all samples. Coding sequences were identified and annotated using RAST (Rapid Annotation using Subsystem Technology), which was followed by assignment to metabolic pathways using references from KEGG (Kyoto Encyclopedia of Genes and Genomes), with a particular focus on carbohydrate metabolism and amino acid synthesis. This analysis resulted in the identification of nine of the ten enzymes necessary for glycolysis, indicating the capacity to metabolize glucose into pyruvate. Two outcomes were predicted for pyruvate: conversion to lactate through lactate dehydrogenases (EC 1.1.1.27, EC 1.1.1.28), as well as the production of acetate through the sequential activity of pyruvate-flavodoxin oxidoreductase (EC 1.2.7.1), phosphate acetyltransferase (EC 2.3.1.8), and acetate kinase (EC 2.7.2.1). Propionate and butyrate were also predicted as end-products being produced in bison rumen, as well as synthesis of precursors for aromatic amino synthesis through the Chorismate-Shikimate pathway. In addition, enzymes involved in the fermentation of five-carbon sugars, such as arabinose (EC 5.1.3.4, EC 2.7.1.16, and EC 5.3.1.4) and xylose (EC 2.7.1.17 and EC 5.3.1.5), were also identified. Characterization of the metabolic potential of ruminal bacteria from pasture-fed bison, such as the utilization of glucose and pentose sugars that form the backbone of structural polysaccharides to produce short chain fatty acids and essential amino acids, will provide further insight on the use of forage and grass-based diets by these animals.

Whole genome sequence of Streptococcus pluranimalium SP21–2, a porcine strain harbouring optrA and lsa(E) with chromosomal location

The aim of this study was to characterise the whole genome sequence of multidrug-resistant Streptococcus pluranimalium strain SP21-2 of swine origin in China. Illumina Miseq (200X coverage) and Nanopore PromethION platform (100X coverage) were used for genome sequencing. Rapid Annotation using Subsystem Technology (RAST) was used to annotate the genome of SP21-2. The antimicrobial resistance genes (ARGs) were identified using ResFinder-4.1. The assembled circular genome of S. pluranimalium SP21-2 was 1,987,058 bp in length with a GC content of 39.54%, and no plasmid sequence was detected. A total of 2086 coding sequences were predicted by RAST. Oxazolidinone-phenicol resistance gene, optrA, and pleuromutilin-lincosamide-streptogramin A resistance gene, lsa(E), are both located on chromosomes, associated with IS1216 and ISS1S, respectively. In addition, SP21-2 harbours lnu(B) (lincosamide), ant (6)-Ia and aac(6')-aph(2") (aminoglycoside), erm(B) (macrolide), and tet(O) (tetracycline). We firstly report the oxazolidinone-phenicol gene, optrA, and pleuromutilin-lincosamide-streptogramin A resistance gene, lsa(E), in S. pluranimalium. In this strain, we firstly identified ISS1S and IS1216 carrying ARGs in S. pluranimalium, which will provide a valuable reference to understanding potential transfer mechanisms of ARGs in S. pluranimalium.

Detailed genomic and biochemical characterization and plant growth promoting properties of an arsenic-tolerant isolate of Bacillus pacificus from contaminated groundwater of West Bengal, India

In this study, arsenic tolerating bacteria Bacillus pacificus (AKS1a) was isolated from arsenic contaminated groundwater of Purbasthali, Purba Bardhaman, West Bengal, India and its bioremediation potential was preliminary screened. This multimetal resistant strain was able to grow against more than 20 mM arsenate and 10 mM arsenite salts. The genome was more than 5.16 Mb in length, with an average of around 35.2% GC content, bearing 5403 protein coding genes. Arsenic resistant genes like arsC, arsB, arsR, etc. were also identified. Rapid Annotation using Subsystem Technology (RAST) identified 328 subsystems within the genome. Presence of six Genomic Islands (GIs) and five phage virus genomic parts indicated its ecological adaptations to overcome environmental stresses. The production of about 415 μg mL−1 indole acetic acid (IAA), 258.0 μg mL−1 gibberellic acid (GA), and 183 μg mL−1 proline by the bacterium, along with nitrogen fixation ability under in-vitro conditions, indicate its plant growth promoting potential. This was further confirmed through rice seedling growth enhancement under arsenic stress. Beside arsenite oxidation to arsenate, its arsenic adsorption property was confirmed through X-ray Fluorescence spectroscopy (XRF), Fourier Transform Infrared spectroscopy (FTIR), and Energy Dispersive X-ray spectroscopic (EDS) analysis. Genomic comparisons among 25 different strains of B. pacificus showed that there are tremendous genetic differences in respect to their accessory genome content. In future, this strain can be applied as biofertilizer or biostimulant for improving rice plant growth.

Draft genome sequence of Staphylococcus aureus sequence type 5 SA01 isolated from bloodstream infection and comparative analysis with reference strains.

A Staphylococcus aureus isolate (SA01) obtained from bloodstream infection exhibited a remarkable drug resistance profile. In this study, we report the draft genome sequence of S. aureus ST 5 SA01, a multidrug-resistant isolate, and analyzed the genes associated with drug resistance and virulence. The genome sketch of S. aureus ST5 SA01 was sequenced with Illumina and annotated using the Prokka software. Rapid Annotation Subsystem Technology (RAST) was used to verify the gene functions in the genome subsystems. The Comprehensive Antibiotic Resistance Database (CARD) and Virulence Factor Database (VFDB) were used in the analysis. The RAST indicated a contribution of 25 proteins to host adenine, fibronectin-binding protein A (FnbA), and biofilm formation as an intercellular polysaccharide adhesive system (PIA). The MLST indicated that S. aureus ST 5 SA01 belongs to ST5 (CC5). In silico analyses also showed an extensive repertoire of genes associated with toxins, such as LukGH leukocidin, enterotoxins, and superantigen staphylococcal classes (SSL). The 11 genes for antimicrobial resistance in S. aureus ST 5 SA01 showed similarity and identity above ≥ 99% with nucleotide sequences deposited in GenBank. Although studies on ST5 clones in Brazil are scarce, monitoring the clone of S. aureus ST 5 SA01 is essential, as it has become a problem in pediatrics in several countries.

Unveiling the draft genome sequence of diesel-degrading Paenibacillus sp. strain d9, a surfactant producer isolated from diesel-contaminated soil

Introduction: Gram-positive bacteria, particularly Bacillus and Paenibacillus spp., have gained significant attention for their potential in environmental bioremediation (biosurfactant production) and diverse biotechnological applications. Among these, Paenibacillus sp. D9, isolated from oil-contaminated soil, has shown diesel and engine oil degradation capabilities and biosurfactant production. However, its role in alkane degradation remains unexplored. Methodology and Result: To shed light on its unique attributes, we conducted whole-genome sequencing of Paenibacillus sp. D9 using the Illumina HiSeq 2000 platform. The draft genome comprised 56 contigs and 7 scaffolds, with a size of 5,645,302 bp at 157.94× coverage and a G + C content of 58.13%. A total of 9,950 Coding Sequences (CDSs) were predicted, and functional annotation revealed 3,283 (43.19%) and 3,155 (58.8%) putative genes based on Bacterial Annotation System (BASys) and Rapid Annotation using the Subsystems Technology (RAST) subsystem categorization, respectively. Furthermore, 93 tRNA and 23 rRNA genes were identified. Conclusion: This genome announcement provides valuable insights into the genetic potential of Paenibacillus sp. D9 and paves the way for future research in its biotechnological applications.

Characterization and genomic analysis of a broad-spectrum lytic phage HZ2201 and its antibiofilm efficacy against Pseudomonas aeruginosa

Pseudomonas aeruginosa is a clinically common conditionally pathogenic bacterium, and the abuse of antibiotics has exacerbated its drug resistance in recent years. This has resulted in extensive reports about the usage of Pseudomonas aeruginosa phage as a novel antibacterial drug. In this study, we isolated a novel phage HZ2201 with a broad lytic spectrum. The lytic rate of this phage against Pseudomonas aeruginosa reached 78.38% (29/37), including 25 multi-drug- and carbapenem-resistant Pseudomonas aeruginosa strains. Transmission electron microscopy revealed that phage HZ2201 belongs to the class Caudoviricetes. Biological characterization showed that phage HZ2201 had an latent period of 40 min, a lytic period of 20 min, and a burst size of 440 PFU/cell, with improved tolerance to temperature and pH. Considering genomic analysis, the HZ2201 genome was a circular double-stranded DNA with a size of 45,431 bp and a guanine-cytosine (G + C) content of 52.16%, and contained 3 tRNAs. 27 of the 74 open reading frames (ORFs) annotated by the Rapid Annotation using Subsystem Technology (RAST) tool could be matched to the genomes of known functions, and no genes related to virulence and antibiotic resistance were found. The phylogenetic tree suggests that phage HZ2201 is highly related to the phage ZCPS1 and PaP3, and ORF57 and ORF17 are predicted to encode a holin and an endolysin, respectively. Cell lysis by HZ2201 proceeds through the holin-endolysin system, suggesting that it is a novel phage. Additionally, we demonstrated that phage HZ2201 has a high inhibitory capacity against Pseudomonas aeruginosa biofilms. The results of our study suggest that phage HZ2201 is a novel potential antimicrobial agent for treating drug-resistant Pseudomonas aeruginosa infection.

Whole-genome sequencing of Alcaligenes sp. strain MMA: insight into the antibiotic and heavy metal resistant genes.

Introduction: A wide range of pollutants, including the likes of xenobiotics, heavy metals, and antibiotics, are characteristic of marine ecosystems. The ability of the bacteria to flourish under high metal stress favors the selection of antibiotic resistance in aquatic environments. Increased use and misuse of antibiotics in medicine, agriculture, and veterinary have posed a grave concern over antimicrobial resistance. The exposure to these heavy metals and antibiotics in the bacteria drives the evolution of antibiotic and heavy metal resistance genes. In the earlier study by the author Alcaligenes sp. MMA was involved in the removal of heavy metals and antibiotics. Alcaligenes display diverse bioremediation capabilities but remain unexplored at the level of the genome. Methods: To shed light on its genome, the Alcaligenes sp. strain MMA, was sequenced using Illumina Nova Seq sequencer, which resulted in a draft genome of 3.9Mb. The genome annotation was done using Rapid annotation using subsystem technology (RAST). Given the spread of antimicrobial resistance and the generation of multi-drug resistant pathogens (MDR), the strain MMA was checked for potential antibiotic and heavy metal resistance genes Further, we checked for the presence of biosynthetic gene clusters in the draft genome. Results: Alcaligenes sp. strain MMA, was sequenced using Illumina Nova Seq sequencer, which resulted in a draft genome of 3.9Mb. The RAST analysis revealed the presence of 3685 protein-coding genes, involved in the removal of antibiotics and heavy metals. Multiple metal-resistant genes and genes conferring resistance to tetracycline, beta-lactams, and fluoroquinolones were present in the draft genome. Many types of BGCs were predicted, such as siderophore. The secondary metabolites of fungi and bacteria are a rich source of novel bioactive compounds which have the potential to in new drug candidates. Discussion: The results of this study provide information on the strain MMA genome and are valuable for the researcher in further exploitation of the strain MMA for bioremediation. Moreover, whole-genome sequencing has become a useful tool to monitor the spread of antibiotic resistance, a global threat to healthcare.

Genomic analysis of Paenibacillus macerans strain I6, which can effectively saccharify oil palm empty fruit bunches under nutrient-free conditions

The improper disposal of palm oil industrial waste has led to serious environmental pollution. In this study, we isolated Paenibacillus macerans strain I6, which can degrade oil palm empty fruit bunches generated by the palm oil industry in nutrient-free water, from bovine manure biocompost and sequenced its genome on PacBio RSII and Illumina NovaSeq 6000 platforms. We obtained 7.11 Mbp of genomic sequences with 52.9% GC content from strain I6. Strain I6 was phylogenetically closely related to P.macerans strains DSM24746 and DSM24 and was positioned close to the head of the branch containing strains I6, DSM24746, and DSM24 in the phylogenetic tree. We used the RAST (rapid annotation using subsystem technology) server to annotate the strain I6 genome and discovered genes related to biological saccharification; 496 genes were related to carbohydrate metabolism and 306 genes were related to amino acids and derivatives. Among them were carbohydrate-active enzymes (CAZymes), including 212 glycoside hydrolases. Up to 23.6% of the oil palm empty fruit bunches was degraded by strain I6 under anaerobic and nutrient-free conditions. Evaluation of the enzymatic activity of extracellular fractions of strain I6 showed that amylase and xylanase activity was highest when xylan was the carbon source. The high enzyme activity and the diversity in the associated genes may contribute to the efficient degradation of oil palm empty fruit bunches by strain I6. Our results indicate the potential utility of P.macerans strain I6 for lignocellulosic biomass degradation.