Rapid Annotation Using Subsystem Technology Research Articles

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.

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.

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.

Characterization of a Potential Probiotic Lactiplantibacillus plantarum LRCC5310 by Comparative Genomic Analysis and its Vitamin B6 Production Ability.

Safety assessment and functional analysis of probiotic candidates are important for their industrial applications. Lactiplantibacillus plantarum is one of the most widely recognized probiotic strains. In this study we aimed to determine the functional genes of L. plantarum LRCC5310, isolated from kimchi, using next-generation, whole-genome sequencing analysis. Genes were annotated using the Rapid Annotations using Subsystems Technology (RAST) server and the National Center for Biotechnology Information (NCBI) pipelines to establish the strain's probiotic potential. Phylogenetic analysis of L. plantarum LRCC5310 and related strains showed that LRCC5310 belonged to L. plantarum. However, comparative analysis revealed genetic differences between L. plantarum strains. Carbon metabolic pathway analysis based on the Kyoto Encyclopedia of Genes and Genomes database showed that L. plantarum LRCC5310 is a homofermentative bacterium. Furthermore, gene annotation results indicated that the L. plantarum LRCC5310 genome encodes an almost complete vitamin B6 biosynthetic pathway. Among five L. plantarum strains, including L. plantarum ATCC 14917T, L. plantarum LRCC5310 detected the highest concentration of pyridoxal 5'-phosphate with 88.08 ± 0.67 nM in MRS broth. These results indicated that L. plantarum LRCC5310 could be used as a functional probiotic for vitamin B6 supplementation.

Genomic Diversity among Actinomyces naeslundii Strains and Closely Related Species

The aim of this study was to investigate and clarify the ambiguous taxonomy of Actinomyces naeslundii and its closely related species using state-of-the-art high-throughput sequencing techniques, and, furthermore, to determine whether sub-clusters identified within Actinomyces oris and Actinomyces naeslundii in a previous study by multi locus sequence typing (MLST) using concatenation of seven housekeeping genes should either be classified as subspecies or distinct species. The strains in this study were broadly classified under Actinomyces naeslundii group as A. naeslundii genospecies I and genospecies II. Based on MLST data analysis, these were further classified as A. oris and A. naeslundii. The whole genome sequencing of selected strains of A. oris (n = 17) and A. naeslundii (n = 19) was carried out using Illumina Genome Analyzer IIxe and Roche 454 allowing paired-end and single-reads sequencing, respectively. The sequences obtained were aligned using CLC Genomic workbench version 5.1 and annotated using RAST (Rapid Annotation using Subsystem Technology) release version 59 accessible online. Additionally, genomes of seven publicly available strains of Actinomyces (k20, MG1, c505, OT175, OT171, OT170, and A. johnsonii) were also included. Comparative genomic analysis (CGA) using Mauve, Progressive Mauve, gene-by-gene, Core, and Pan Genome, and finally Digital DNA-DNA homology (DDH) analysis was carried out. DDH values were obtained using in silico genome-genome comparison. Evolutionary analysis using ClonalFrame was also undertaken. The mutation and recombination events were compared using chi-square test among A. oris and A. naeslundii isolates (analysis methods are not included in the study). CGA results were consistent with previous traditional classification using MLST. It was found that strains of Actinomyces k20, MG1, c505, and OT175 clustered in A. oris group of isolates, while OT171, OT170, and A. johnsonii appeared as separate branches. Similar clustering to MLST was observed for other isolates. The mutation and recombination events were significantly higher in A. oris than A. naeslundii, highlighting the diversity of A. oris strains in the oral cavity. These findings suggest that A. oris forms six distinct groups, whereas A. naeslundii forms three. The correct designation of isolates will help in the identification of clinical Actinomyces isolates found in dental plaque. Easily accessible online genomic sequence data will also accelerate the investigation of the biochemical characterisation and pathogenesis of this important group of micro-organisms.

Draft genome sequence of a multidrug-resistant Campylobacter coli ST825 carrying several acquired antibiotic resistance genes isolated from poultry in China

Campylobacter coli is a typical food-borne pathogen worldwide known to cause bacterial gastroenteritis in humans. This study reported a draft whole genome sequence of C. coli isolate obtained from the caecal contents of poultry in Jinhua, China. Whole genomic DNA was sequenced using an Illumina Novaseq 6000 platform in 150 bp paired-end mode. The generated reads were de novo assembled by SPAdes v.3.12.0. All probable coding sequences were annotated using the RAST (Rapid Annotation using Subsystem Technology), and antibiotic resistance-related genes were also further identified by ResFinder 4.1 and rgi 5.1.1. The draft genome contained 1 794 608 bp, a total of 69 contigs, belonging to sequence type (ST) ST825, comprising 1972 coding genes, 42 transfer RNAs, 2 ribosomal RNA, and with a GC content of 31.2%. The RAST analysis revealed a total of 698 subsystems in the genome of C. coli WL32 strain, with most of the genes associated with amino acids and derivatives (21.35%) and protein metabolism (17.05%). The genes related to antibiotic resistance, including erm(B) gene associated with macrolide resistance, blaOXA-61 gene associated with resistance to β-lactams, aac (6')-aph(2'), ant(6)-Ia, aph(2')-If, aph(3')-III gene associated with resistance to aminoglycosides, tetO gene associated with resistance to tetracycline, cat gene associated with amphenicol, and gyrA with fluoroquinolone Thr-86-Ile substitution, were identified. Also, the virulence genes, including motA, motB, flaG, fliE, fliF, fliG, flhB, and flhF genes, were identified by WGS analysis. We report the draft genome sequence of C. coli ST825 isolate obtained from a poultry in China, which could provide potential information for tracking the potential spread of such a multidrug-resistant clone from poultry product processing to human beings.

Comparative Genomic Analysis Reveals Intestinal Habitat Adaptation of Ligilactobacillus equi Rich in Prophage and Degrading Cellulase

Ligilactobacillus equi is common in the horse intestine, alleviates the infection of Salmonella, and regulates intestinal flora. Despite this, there have been no genomic studies on this species. Here, we provide the genomic basis for adaptation to the intestinal habitat of this species. We sequenced the genome of L. equi IMAU81196, compared this with published genome information from three strains in NCBI, and analyzed genome characteristics, phylogenetic relationships, and functional genes. The mean genome size of L. equi strains was 2.08 ± 0.09 Mbp, and the mean GC content was 39.17% ± 0.19%. The genome size of L. equi IMAU81196 was 1.95 Mbp, and the GC content was 39.48%. The phylogenetic tree for L. equi based on 1454 core genes showed that the independent branch of strain IMAU81196 was far from the other three strains. In terms of genomic characteristics, single-nucleotide polymorphism (SNP) sites, rapid annotation using subsystem technology (RAST), carbohydrate activity enzymes (CAZy), and predictions of prophage, we showed that strain L. equi JCM 10991T and strain DSM 15833T are not equivalent strains.It is worth mentioning thatthestrain of L. equi has numerous enzymes related to cellulose degradation, and each L. equi strain investigated contained at least one protophage. We speculate that this is the reason why these strains are adapted to the intestinal environment of horses. These results provide new research directions for the future.

Announcement of the 2022 winners of the Spychala and Women in Science Awards

Announcement of the 2022 winners of the Spychala and Women in Science Awards

IProbiotics: a machine learning platform for rapid identification of probiotic properties from whole-genome primary sequences.

Lactic acid bacteria consortia are commonly present in food, and some of these bacteria possess probiotic properties. However, discovery and experimental validation of probiotics require extensive time and effort. Therefore, it is of great interest to develop effective screening methods for identifying probiotics. Advances in sequencing technology have generated massive genomic data, enabling us to create a machine learning-based platform for such purpose in this work. This study first selected a comprehensive probiotics genome dataset from the probiotic database (PROBIO) and literature surveys. Then, k-mer (from 2 to 8) compositional analysis was performed, revealing diverse oligonucleotide composition in strain genomes and apparently more probiotic (P-) features in probiotic genomes than non-probiotic genomes. To reduce noise and improve computational efficiency, 87376k-mers were refined by an incremental feature selection (IFS) method, and the model achieved the maximum accuracy level at 184 core features, with a high prediction accuracy (97.77%) and area under the curve (98.00%). Functional genomic analysis using annotations from gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Rapid Annotation using Subsystem Technology (RAST) databases, as well as analysis of genes associated with host gastrointestinal survival/settlement, carbohydrate utilization, drug resistance and virulence factors, revealed that the distribution of P-features was biased toward genes/pathways related to probiotic function. Our results suggest that the role of probiotics is not determined by a single gene, but by a combination of k-mer genomic components, providing new insights into the identification and underlying mechanisms of probiotics. This work created a novel and free online bioinformatic tool, iProbiotics, which would facilitate rapid screening for probiotics.

Genomic-based characterization of Enterococcus spp.: an emerging pathogen isolated from human gut.

Enterococci are ubiquitous microorganisms having diverse ecological niches but most prominently in gastrointestinal tract of humans and animals. Production of enterocins makes them a good probiotic candidate. However, their role as probiotics has become ambiguous in the last few years because of the presence of virulence factors and antibiotic resistance genes. These virulence traits are known to be transferred genetically, which makes them opportunistic pathogens in the gastrointestinal tract leading to serious concerns about their being used as probiotics. In the present study, Enterococcusspp. isolated from the human gut were subjected to Whole-Genome Sequencing (WGS) to determine the presence of resistance and virulence genes. Four human origins Enterococcus spp. including Enterococcus faecalis, Enterococcus casseliflavus, and two Enterococcus gallinarum were isolated from human fecal samples and further cultured on blood agar. Sanger sequencing was done using Applied Biosystems 3730xl DNA Analyzer. These strains were further subjected to WGS using oxford nanopore technology MinION. Raw data were analyzed using the free online tool epi2me. The Comprehensive Antibiotic Resistance Database (CARD) and RAST (Rapid Annotation using Subsystem Technology) software were used to look for the presence of antibiotic resistance genes in these strains. Resistance determinants for clinically important antibiotics (vancomycin) and functional virulence factor genes were detected. G-view server was used for comparative genomics of all strains. The genomic sequencing of Enterococcus suggested that E. faecalis, E. casseliflavus, and E. gallinarum strains are opportunistic pathogens, having antibiotic resistance genes. All isolates had vancomycin resistance genes, which were expressed phenotypically. Genes related to bacteriocin resistance were also present in E. casseliflavus and E. gallinarum.

Deciphering the Potential of Rhizobium pusense MB-17a, a Plant Growth-Promoting Root Endophyte, and Functional Annotation of the Genes Involved in the Metabolic Pathway.

Plant growth-promoting rhizobacteria (PGPR) are root endophytic bacteria used for growth promotion, and they have broader applications in enhancing specific crop yield as a whole. In the present study, we have explored the potential of Rhizobium pusense MB-17a as an endophytic bacterium isolated from the roots of the mung bean (Vigna radiata) plant. Furthermore, this bacterium was sequenced and assembled to reveal its genomic potential associated with plant growth-promoting traits. Interestingly, the root endophyte R. pusense MB-17a showed all essential PGPR traits which were determined by biochemical and PGPR tests. It was noted that this root endophytic bacterium significantly produced siderophores, indole acetic acid (IAA), ammonia, and ACC deaminase and efficiently solubilized phosphate. The maximum IAA and ammonia produced were observed to be 110.5 and 81 μg/ml, respectively. Moreover, the PGPR potential of this endophytic bacterium was also confirmed by a pot experiment for mung bean (V. radiata), whose results show a substantial increase in the plant's fresh weight by 76.1% and dry weight by 76.5% on the 60th day after inoculation of R. pusense MB-17a. Also, there is a significant enhancement in the nodule number by 66.1% and nodule fresh weight by 162% at 45th day after inoculation with 100% field capacity after the inoculation of R. pusense MB-17a. Besides this, the functional genomic annotation of R. pusense MB-17a determined the presence of different proteins and transporters that are responsible for its stress tolerance and its plant growth-promoting properties. It was concluded that the unique presence of genes like rpoH, otsAB, and clpB enhances the symbiosis process during adverse conditions in this endophyte. Through Rapid Annotation using Subsystem Technology (RAST) analysis, the key genes involved in the production of siderophores, volatile compounds, indoles, nitrogenases, and amino acids were also predicted. In conclusion, the strain described in this study gives a novel idea of using such type of endophytes for improving plant growth-promoting traits under different stress conditions for sustainable agriculture.

Distribution of Important Probiotic Genes and Identification of the Biogenic Amines Produced by Lactobacillus acidophilus PNW3.

The genome of Lactobacillus acidophilus PNW3 was assessed for probiotic and safety potentials. The genome was completely sequenced, assembled using SPAdes, and thereafter annotated with NCBI prokaryotic genome annotation pipeline (PGAP) and rapid annotation using subsystem technology (RAST). Further downstream assessment was determined using appropriate bioinformatics tools. The production of biogenic amines was confirmed through HPLC analysis and the evolutionary trend of the strain was determined through the Codon Tree pipeline. The strain was predicted as a non-human pathogen. A plethora of encoding genes for lactic acids and bioactive peptides production, adhesion molecules, resistance to the harsh gut environmental conditions, and improvement of the host metabolism, which are putative for important probiotic functionalities, were located at different loci within the genome. A bacteriocin predicted to be helveticin J was identified as one of the secondary metabolites. The maximum zone of inhibition exhibited by the crude bacteriocin against STEC E. coli O177 was 21.7 ± 0.58 mm and 24.3 ± 1.15 mm after partial purification (250 µg/mL). Three coding sequences were identified for insertion sequences and one for the CRISPR-Cas fragment. The protein-encoding sequence for Ornithine decarboxylase was found within the genome. L. acidophilus PNW3 presents important features categorizing it as a viable and safe probiotic candidate, though further safety investigations are necessary. The application of probiotics in livestock-keeping would ensure improved public health and food security.