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616100.9910Unraveling radiation resistance strategies in two bacterial strains from the high background radiation area of Chavara-Neendakara: A comprehensive whole genome analysis. This paper reports the results of gamma irradiation experiments and whole genome sequencing (WGS) performed on vegetative cells of two radiation resistant bacterial strains, Metabacillus halosaccharovorans (VITHBRA001) and Bacillus paralicheniformis (VITHBRA024) (D10 values 2.32 kGy and 1.42 kGy, respectively), inhabiting the top-ranking high background radiation area (HBRA) of Chavara-Neendakara placer deposit (Kerala, India). The present investigation has been carried out in the context that information on strategies of bacteria having mid-range resistance for gamma radiation is inadequate. WGS, annotation, COG and KEGG analyses and manual curation of genes helped us address the possible pathways involved in the major domains of radiation resistance, involving recombination repair, base excision repair, nucleotide excision repair and mismatch repair, and the antioxidant genes, which the candidate could activate to survive under ionizing radiation. Additionally, with the help of these data, we could compare the candidate strains with that of the extremely radiation resistant model bacterium Deinococccus radiodurans, so as to find the commonalities existing in their strategies of resistance on the one hand, and also the rationale behind the difference in D10, on the other. Genomic analysis of VITHBRA001 and VITHBRA024 has further helped us ascertain the difference in capability of radiation resistance between the two strains. Significantly, the genes such as uvsE (NER), frnE (protein protection), ppk1 and ppx (non-enzymatic metabolite production) and those for carotenoid biosynthesis, are endogenous to VITHBRA001, but absent in VITHBRA024, which could explain the former's better radiation resistance. Further, this is the first-time study performed on any bacterial population inhabiting an HBRA. This study also brings forward the two species whose radiation resistance has not been reported thus far, and add to the knowledge on radiation resistant capabilities of the phylum Firmicutes which are abundantly observed in extreme environment.202438857267
866910.9909The ins and outs of metal homeostasis by the root nodule actinobacterium Frankia. BACKGROUND: Frankia are actinobacteria that form a symbiotic nitrogen-fixing association with actinorhizal plants, and play a significant role in actinorhizal plant colonization of metal contaminated areas. Many Frankia strains are known to be resistant to several toxic metals and metalloids including Pb(2+), Al(+3), SeO2, Cu(2+), AsO4, and Zn(2+). With the availability of eight Frankia genome databases, comparative genomics approaches employing phylogeny, amino acid composition analysis, and synteny were used to identify metal homeostasis mechanisms in eight Frankia strains. Characterized genes from the literature and a meta-analysis of 18 heavy metal gene microarray studies were used for comparison. RESULTS: Unlike most bacteria, Frankia utilize all of the essential trace elements (Ni, Co, Cu, Se, Mo, B, Zn, Fe, and Mn) and have a comparatively high percentage of metalloproteins, particularly in the more metal resistant strains. Cation diffusion facilitators, being one of the few known metal resistance mechanisms found in the Frankia genomes, were strong candidates for general divalent metal resistance in all of the Frankia strains. Gene duplication and amino acid substitutions that enhanced the metal affinity of CopA and CopCD proteins may be responsible for the copper resistance found in some Frankia strains. CopA and a new potential metal transporter, DUF347, may be involved in the particularly high lead tolerance in Frankia. Selenite resistance involved an alternate sulfur importer (CysPUWA) that prevents sulfur starvation, and reductases to produce elemental selenium. The pattern of arsenate, but not arsenite, resistance was achieved by Frankia using the novel arsenite exporter (AqpS) previously identified in the nitrogen-fixing plant symbiont Sinorhizobium meliloti. Based on the presence of multiple tellurite resistance factors, a new metal resistance (tellurite) was identified and confirmed in Frankia. CONCLUSIONS: Each strain had a unique combination of metal import, binding, modification, and export genes that explain differences in patterns of metal resistance between strains. Frankia has achieved similar levels of metal and metalloid resistance as bacteria from highly metal-contaminated sites. From a bioremediation standpoint, it is important to understand mechanisms that allow the endosymbiont to survive and infect actinorhizal plants in metal contaminated soils.201425495525
908320.9908ARGNet: using deep neural networks for robust identification and classification of antibiotic resistance genes from sequences. BACKGROUND: Emergence of antibiotic resistance in bacteria is an important threat to global health. Antibiotic resistance genes (ARGs) are some of the key components to define bacterial resistance and their spread in different environments. Identification of ARGs, particularly from high-throughput sequencing data of the specimens, is the state-of-the-art method for comprehensively monitoring their spread and evolution. Current computational methods to identify ARGs mainly rely on alignment-based sequence similarities with known ARGs. Such approaches are limited by choice of reference databases and may potentially miss novel ARGs. The similarity thresholds are usually simple and could not accommodate variations across different gene families and regions. It is also difficult to scale up when sequence data are increasing. RESULTS: In this study, we developed ARGNet, a deep neural network that incorporates an unsupervised learning autoencoder model to identify ARGs and a multiclass classification convolutional neural network to classify ARGs that do not depend on sequence alignment. This approach enables a more efficient discovery of both known and novel ARGs. ARGNet accepts both amino acid and nucleotide sequences of variable lengths, from partial (30-50 aa; 100-150 nt) sequences to full-length protein or genes, allowing its application in both target sequencing and metagenomic sequencing. Our performance evaluation showed that ARGNet outperformed other deep learning models including DeepARG and HMD-ARG in most of the application scenarios especially quasi-negative test and the analysis of prediction consistency with phylogenetic tree. ARGNet has a reduced inference runtime by up to 57% relative to DeepARG. CONCLUSIONS: ARGNet is flexible, efficient, and accurate at predicting a broad range of ARGs from the sequencing data. ARGNet is freely available at https://github.com/id-bioinfo/ARGNet , with an online service provided at https://ARGNet.hku.hk . Video Abstract.202438725076
514130.9908Flavobacterium flabelliforme sp. nov. and Flavobacterium geliluteum sp. nov., Two Multidrug-Resistant Psychrotrophic Species Isolated From Antarctica. Despite unfavorable Antarctic conditions, such as cold temperatures, freeze-thaw cycles, high ultraviolet radiation, dryness and lack of nutrients, microorganisms were able to adapt and surprisingly thrive in this environment. In this study, eight cold-adapted Flavobacterium strains isolated from a remote Antarctic island, James Ross Island, were studied using a polyphasic taxonomic approach to determine their taxonomic position. Phylogenetic analyses based on the 16S rRNA gene and 92 core genes clearly showed that these strains formed two distinct phylogenetic clusters comprising three and five strains, with average nucleotide identities significantly below 90% between both proposed species as well as between their closest phylogenetic relatives. Phenotyping revealed a unique pattern of biochemical and physiological characteristics enabling differentiation from the closest phylogenetically related Flavobacterium spp. Chemotaxonomic analyses showed that type strains P4023(T) and P7388(T) were characterized by the major polyamine sym-homospermidine and a quinone system containing predominantly menaquinone MK-6. In the polar lipid profile phosphatidylethanolamine, an ornithine lipid and two unidentified lipids lacking a functional group were detected as major lipids. These characteristics along with fatty acid profiles confirmed that these species belong to the genus Flavobacterium. Thorough genomic analysis revealed the presence of numerous cold-inducible or cold-adaptation associated genes, such as cold-shock proteins, proteorhodopsin, carotenoid biosynthetic genes or oxidative-stress response genes. Genomes of type strains surprisingly harbored multiple prophages, with many of them predicted to be active. Genome-mining identified biosynthetic gene clusters in type strain genomes with a majority not matching any known clusters which supports further exploratory research possibilities involving these psychrotrophic bacteria. Antibiotic susceptibility testing revealed a pattern of multidrug-resistant phenotypes that were correlated with in silico antibiotic resistance prediction. Interestingly, while typical resistance finder tools failed to detect genes responsible for antibiotic resistance, genomic prediction confirmed a multidrug-resistant profile and suggested even broader resistance than tested. Results of this study confirmed and thoroughly characterized two novel psychrotrophic Flavobacterium species, for which the names Flavobacterium flabelliforme sp. nov. and Flavobacterium geliluteum sp. nov. are proposed.202134745033
509740.9907Comparing Graph Sample and Aggregation (SAGE) and Graph Attention Networks in the Prediction of Drug-Gene Associations of Extended-Spectrum Beta-Lactamases in Periodontal Infections and Resistance. INTRODUCTION: Gram-negative bacteria exhibit more antibiotic resistance than gram-positive bacteria due to their cell wall structure and composition differences. Porins, or protein channels in these bacteria, can allow small, hydrophilic antibiotics to diffuse, affecting their susceptibility. Mutations in porin protein genes can also impair antibiotic entry. Predicting drug-gene associations of extended-spectrum beta-lactamases (ESBLs) is crucial as they confer resistance to beta-lactam antibiotics, challenging the treatment of infections. This aids clinicians in selecting suitable treatments, optimizing drug usage, enhancing patient outcomes, and controlling antibiotic resistance in healthcare settings. Graph-based neural networks can predict drug-gene associations in periodontal infections and resistance. The aim of the study was to predict drug-gene associations of ESBLs in periodontal infections and resistance. METHODS: The study focuses on analyzing drug-gene associations using probes and drugs. The data was converted into graph language, assigning nodes and edges for drugs and genes. Graph neural networks (GNNs) and similar algorithms were implemented using Google Colab and Python. Cytoscape and CytoHubba are open-source software platforms used for network analysis and visualization. GNNs were used for tasks like node classification, link prediction, and graph-level prediction. Three graph-based models were used: graph convolutional network (GCN), Graph SAGE, and graph attention network (GAT). Each model was trained for 200 epochs using the Adam optimizer with a learning rate of 0.01 and a weight decay of 5e-4. RESULTS: The drug-gene association network has 57 nodes, 79 edges, and a 2.730 characteristic path length. Its structure, organization, and connectivity are analyzed using the GCN and Graph SAGE, which show high accuracy, precision, recall, and an F1-score of 0.94. GAT's performance metrics are lower, with an accuracy of 0.68, precision of 0.47, recall of 0.68, and F1-score of 0.56, suggesting that it may not be as effective in capturing drug-gene relationships. CONCLUSION: Compared to ESBLs, both GCN and Graph SAGE demonstrate excellent performance with accuracy, precision, recall, and an F1-score of 0.94. These results indicate that GCN and Graph SAGE are highly effective in predicting drug-gene associations related to ESBLs. GCN and Graph SAGE outperform GAT in predicting drug-gene associations for ESBLs. Improvements include data augmentation, regularization, and cross-validation. Ethical considerations, fairness, and open-source implementations are crucial for future research in precision periodontal treatment.202439347119
616050.9907Comparative transcriptional profiling of tildipirosin-resistant and sensitive Haemophilus parasuis. Numerous studies have been conducted to examine the molecular mechanism of Haemophilus parasuis resistance to antibiotic, but rarely to tildipirosin. In the current study, transcriptional profiling was applied to analyse the variation in gene expression of JS0135 and tildipirosin-resistant JS32. The growth curves showed that JS32 had a higher growth rate but fewer bacteria than JS0135. The cell membranes of JS32 and a resistant clinical isolate (HB32) were observed to be smoother than those of JS0135. From the comparative gene expression profile 349 up- and 113 downregulated genes were observed, covering 37 GO and 63 KEGG pathways which are involved in biological processes (11), cellular components (17), molecular function (9), cellular processes (1), environmental information processing (4), genetic information processing (9) and metabolism (49) affected in JS32. In addition, the relative overexpression of genes of the metabolism pathway (HAPS_RS09315, HAPS_RS09320), ribosomes (HAPS_RS07815) and ABC transporters (HAPS_RS10945) was detected, particularly the metabolism pathway, and verified with RT-qPCR. Collectively, the gene expression profile in connection with tildipirosin resistance factors revealed unique and highly resistant determinants of H. parasuis to macrolides that warrant further attention due to the significant threat of bacterial resistance.201728790420
844160.9906Genomic and phenotypic attributes of novel salinivibrios from stromatolites, sediment and water from a high altitude lake. BACKGROUND: Salinivibrios are moderately halophilic bacteria found in salted meats, brines and hypersaline environments. We obtained three novel conspecific Salinivibrio strains closely related to S. costicola, from Socompa Lake, a high altitude hypersaline Andean lake (approx. 3,570 meters above the sea level). RESULTS: The three novel Salinivibrio spp. were extremely resistant to arsenic (up to 200 mM HAsO42-), NaCl (up to 15%), and UV-B radiation (19 KJ/m2, corresponding to 240 minutes of exposure) by means of phenotypic tests. Our subsequent draft genome ionsequencing and RAST-based genome annotation revealed the presence of genes related to arsenic, NaCl, and UV radiation resistance. The three novel Salinivibrio genomes also had the xanthorhodopsin gene cluster phylogenetically related to Marinobacter and Spiribacter. The genomic taxonomy analysis, including multilocus sequence analysis, average amino acid identity, and genome-to-genome distance revealed that the three novel strains belong to a new Salinivibrio species. CONCLUSIONS: Arsenic resistance genes, genes involved in DNA repair, resistance to extreme environmental conditions and the possible light-based energy production, may represent important attributes of the novel salinivibrios, allowing these microbes to thrive in the Socompa Lake.201424927949
773770.9905Distinctive signatures of pathogenic and antibiotic resistant potentials in the hadal microbiome. BACKGROUND: Hadal zone of the deep-sea trenches accommodates microbial life under extreme energy limitations and environmental conditions, such as low temperature, high pressure, and low organic matter down to 11,000 m below sea level. However, microbial pathogenicity, resistance, and adaptation therein remain unknown. Here we used culture-independent metagenomic approaches to explore the virulence and antibiotic resistance in the hadal microbiota of the Mariana Trench. RESULTS: The results indicate that the 10,898 m Challenger Deep bottom sediment harbored prosperous microbiota with contrasting signatures of virulence factors and antibiotic resistance, compared with the neighboring but shallower 6038 m steep wall site and the more nearshore 5856 m Pacific basin site. Virulence genes including several famous large translocating virulence genes (e.g., botulinum neurotoxins, tetanus neurotoxin, and Clostridium difficile toxins) were uniquely detected in the trench bottom. However, the shallower and more nearshore site sediment had a higher abundance and richer diversity of known antibiotic resistance genes (ARGs), especially for those clinically relevant ones (e.g., fosX, sul1, and TEM-family extended-spectrum beta-lactamases), revealing resistance selection under anthropogenic stresses. Further analysis of mobilome (i.e., the collection of mobile genetic elements, MGEs) suggests horizontal gene transfer mediated by phage and integrase as the major mechanism for the evolution of Mariana Trench sediment bacteria. Notably, contig-level co-occurring and taxonomic analysis shows emerging evidence for substantial co-selection of virulence genes and ARGs in taxonomically diverse bacteria in the hadal sediment, especially for the Challenger Deep bottom where mobilized ARGs and virulence genes are favorably enriched in largely unexplored bacteria. CONCLUSIONS: This study reports the landscape of virulence factors, antibiotic resistome, and mobilome in the sediment and seawater microbiota residing hadal environment of the deepest ocean bottom on earth. Our work unravels the contrasting and unique features of virulence genes, ARGs, and MGEs in the Mariana Trench bottom, providing new insights into the eco-environmental and biological processes underlying microbial pathogenicity, resistance, and adaptative evolution in the hadal environment.202235468809
837480.9905Importance of RpoD- and Non-RpoD-Dependent Expression of Horizontally Acquired Genes in Cupriavidus metallidurans. The genome of the metal-resistant, hydrogen-oxidizing bacterium Cupriavidus metallidurans contains a large number of horizontally acquired plasmids and genomic islands that were integrated into its chromosome or chromid. For the C. metallidurans CH34 wild-type strain growing under nonchallenging conditions, 5,763 transcriptional starting sequences (TSSs) were determined. Using a custom-built motif discovery software based on hidden Markov models, patterns upstream of the TSSs were identified. The pattern TTGACA, -35.6 ± 1.6 bp upstream of the TSSs, in combination with a TATAAT sequence 15.8 ± 1.4 bp upstream occurred frequently, especially upstream of the TSSs for 48 housekeeping genes, and these were assigned to promoters used by RNA polymerase containing the main housekeeping sigma factor RpoD. From patterns upstream of the housekeeping genes, a score for RpoD-dependent promoters in C. metallidurans was derived and applied to all 5,763 TSSs. Among these, 2,572 TSSs could be associated with RpoD with high probability, 373 with low probability, and 2,818 with no probability. In a detailed analysis of horizontally acquired genes involved in metal resistance and not involved in this process, the TSSs responsible for the expression of these genes under nonchallenging conditions were assigned to RpoD- or non-RpoD-dependent promoters. RpoD-dependent promoters occurred frequently in horizontally acquired metal resistance and other determinants, which should allow their initial expression in a new host. However, other sigma factors and sense/antisense effects also contribute-maybe to mold in subsequent adaptation steps the assimilated gene into the regulatory network of the cell. IMPORTANCE In their natural environment, bacteria are constantly acquiring genes by horizontal gene transfer. To be of any benefit, these genes should be expressed. We show here that the main housekeeping sigma factor RpoD plays an important role in the expression of horizontally acquired genes in the metal-resistant hydrogen-oxidizing bacterium C. metallidurans. By conservation of the RpoD recognition consensus sequence, a newly arriving gene has a high probability to be expressed in the new host cell. In addition to integrons and genes travelling together with that for their sigma factor, conservation of the RpoD consensus sequence may be an important contributor to the overall evolutionary success of horizontal gene transfer in bacteria. Using C. metallidurans as an example, this publication sheds some light on the fate and function of horizontally acquired genes in bacteria.202235311568
908190.9905Identification and reconstruction of novel antibiotic resistance genes from metagenomes. BACKGROUND: Environmental and commensal bacteria maintain a diverse and largely unknown collection of antibiotic resistance genes (ARGs) that, over time, may be mobilized and transferred to pathogens. Metagenomics enables cultivation-independent characterization of bacterial communities but the resulting data is noisy and highly fragmented, severely hampering the identification of previously undescribed ARGs. We have therefore developed fARGene, a method for identification and reconstruction of ARGs directly from shotgun metagenomic data. RESULTS: fARGene uses optimized gene models and can therefore with high accuracy identify previously uncharacterized resistance genes, even if their sequence similarity to known ARGs is low. By performing the analysis directly on the metagenomic fragments, fARGene also circumvents the need for a high-quality assembly. To demonstrate the applicability of fARGene, we reconstructed β-lactamases from five billion metagenomic reads, resulting in 221 ARGs, of which 58 were previously not reported. Based on 38 ARGs reconstructed by fARGene, experimental verification showed that 81% provided a resistance phenotype in Escherichia coli. Compared to other methods for detecting ARGs in metagenomic data, fARGene has superior sensitivity and the ability to reconstruct previously unknown genes directly from the sequence reads. CONCLUSIONS: We conclude that fARGene provides an efficient and reliable way to explore the unknown resistome in bacterial communities. The method is applicable to any type of ARGs and is freely available via GitHub under the MIT license.201930935407
8678100.9904Metagenomics-Guided Discovery of Potential Bacterial Metallothionein Genes from the Soil Microbiome That Confer Cu and/or Cd Resistance. Metallothionein (MT) genes are valuable genetic materials for developing metal bioremediation tools. Currently, a limited number of prokaryotic MTs have been experimentally identified, which necessitates the expansion of bacterial MT diversity. In this study, we conducted a metagenomics-guided analysis for the discovery of potential bacterial MT genes from the soil microbiome. More specifically, we combined resistance gene enrichment through diversity loss, metagenomic mining with a dedicated MT database, evolutionary trace analysis, DNA chemical synthesis, and functional genomic validation to identify novel MTs. Results showed that Cu stress induced a compositional change in the soil microbiome, with an enrichment of metal-resistant bacteria in soils with higher Cu concentrations. Shotgun metagenomic sequencing was performed to obtain the gene pool of environmental DNA (eDNA), which was subjected to a local BLAST search against an MT database for detecting putative MT genes. Evolutional trace analysis led to the identification of 27 potential MTs with conserved cysteine/histidine motifs different from those of known prokaryotic MTs. Following chemical synthesis of these 27 potential MT genes and heterologous expression in Escherichia coli, six of them were found to improve the hosts' growth substantially and enhanced the hosts' sorption of Cu, Cd, and Zn, among which MT5 led to a 13.7-fold increase in Cd accumulation. Furthermore, four of them restored Cu and/or Cd resistance in two metal-sensitive E. coli strains.IMPORTANCE The metagenomics-guided procedure developed here bypasses the difficulties encountered in classic PCR-based approaches and led to the discovery of novel MT genes, which may be useful in developing bioremediation tools. The procedure used here expands our knowledge on the diversity of bacterial MTs in the environment and may also be applicable to identify other functional genes from eDNA.202032111593
8723110.9904Unraveling the Basis of Neonicotinoid Resistance in Whitefly Species Complex: Role of Endosymbiotic Bacteria and Insecticide Resistance Genes. Bemisia tabaci (whitefly) is one of the most detrimental agricultural insect pests and vectors of many plant viruses distributed worldwide. Knowledge of the distribution patterns and insecticide resistance of this cryptic species is crucial for its management. In this study, genetic variation of mitochondrial cytochrome oxidase subunit 1 (MtCoI) gene of B. tabaci was analyzed followed by a study of the infection profile of various endosymbionts in 26 whitefly populations collected from West Bengal, India. Phylogenetic analysis revealed Asia I as the major cryptic species (65.38%), followed by Asia II 5, China 3, and Asia II 7, which were diversified into 20 different haplotypes. In addition to the primary endosymbiont (C. poriera), each of the four whitefly species showed a variable population of three secondary endosymbionts, majorly Arsenophonus with the highest infection rate (73.07%), followed by Wolbachia and Rickettsia. Further phylogenetic analyses revealed the presence of two subgroups of Arsenophonus, viz., A1 and A2, and one each in Wolbachia (W1) and Rickettsia (R3). Resistance to thiamethoxam, imidacloprid, and acetamiprid insecticides was analyzed for a clear picture of pesticide resistance status. The highest susceptibility was noted toward thiamethoxam (LC(50) = 5.36 mg/L), followed by imidacloprid and acetamiprid. The whitefly population from Purulia and Hooghly districts bearing Asia II 7 and Asia II 5 cryptic species, respectively, shows maximum resistance. The differences in mean relative titer of four symbiotic bacteria among field populations varied considerably; however, a significant positive linear correlation was observed between the resistance level and relative titer of Arsenophonus and Wolbachia in the case of imidacloprid and thiamethoxam, while only Wolbachia was found in case of acetamiprid. Expression analysis demonstrated differential upregulation of insecticide resistance genes with Purulia and Hooghly populations showing maximally upregulated P450 genes. Moreover, thiamethoxam and imidacloprid resistance ratio (RR) showed a significant correlation with CYP6CM1, CYP6DZ7, and CYP4C64 genes, while acetamiprid RR correlated with CYP6CX1, CYP6DW2, CYP6DZ7, and CYP4C64 genes. Taken together, these findings suggested that P450 mono-oxygenase and symbiotic bacteria together affected whitefly resistance to neonicotinoids. Hence, a symbiont-oriented management programme could be a better alternative to control or delay resistance development in whitefly and can be used for pesticide clean-up in an agricultural field.202235814684
8728120.9904Identification of the defense-related gene VdWRKY53 from the wild grapevine Vitis davidii using RNA sequencing and ectopic expression analysis in Arabidopsis. BACKGROUND: Grapevine is an important fruit crop grown worldwide, and its cultivars are mostly derived from the European species Vitis vinifera, which has genes for high fruit quality and adaptation to a wide variety of climatic conditions. Disease resistance varies substantially across grapevine species; however, the molecular mechanisms underlying such variation remain uncharacterized. RESULTS: The anatomical structure and disease symptoms of grapevine leaves were analyzed for two grapevine species, and the critical period of resistance of grapevine to pathogenic bacteria was determined to be 12 h post inoculation (hpi). Differentially expressed genes (DEGs) were identified from transcriptome analysis of leaf samples obtained at 12 and 36 hpi, and the transcripts in four pathways (cell wall genes, LRR receptor-like genes, WRKY genes, and pathogenesis-related (PR) genes) were classified into four co-expression groups by using weighted correlation network analysis (WGCNA). The gene VdWRKY53, showing the highest transcript level, was introduced into Arabidopsis plants by using a vector containing the CaMV35S promoter. These procedures allowed identifying the key genes contributing to differences in disease resistance between a strongly resistant accession of a wild grapevine species Vitis davidii (VID) and a susceptible cultivar of V. vinifera, 'Manicure Finger' (VIV). Vitis davidii, but not VIV, showed a typical hypersensitive response after infection with a fungal pathogen (Coniella diplodiella) causing white rot disease. Further, 20 defense-related genes were identified, and their differential expression between the two grapevine species was confirmed using quantitative real-time PCR analysis. VdWRKY53, showing the highest transcript level, was selected for functional analysis and therefore over-expressed in Arabidopsis under the control of the CaMV35S promoter. The transgenic plants showed enhanced resistance to C. diplodiella and to two other pathogens, Pseudomonas syringae pv. tomato DC3000 and Golovinomyces cichoracearum. CONCLUSION: The consistency of the results in VID and transgenic Arabidopsis indicated that VdWRKY53 might be involved in the activation of defense-related genes that enhance the resistance of these plants to pathogens. Thus, the over-expression of VdWRKY53 in transgenic grapevines might improve their resistance to pathogens.201931057347
9080130.9904Comparison of de-novo assembly tools for plasmid metagenome analysis. BACKGROUND: With the advent of next-generation sequencing techniques, culture-independent metagenome approaches have now made it possible to predict possible presence of genes in the environmental bacteria most of which may be non-cultivable. Short reads obtained from the deep sequencing can be assembled into long contigs some of which include plasmids. Plasmids are the circular double stranded DNA in bacteria and known as one of the major carriers of antibiotic resistance genes. OBJECTIVE: Metagenomic analyses, especially focused on plasmids, could help us predict dissemination mechanisms of antibiotic resistance genes in the environment. However, with the availability of a myriad of metagenomic assemblers, the selection of the most appropriate metagenome assembler for the plasmid metagenome study might be challenging. Therefore, in this study, we compared five open source assemblers to suggest most effective way of plasmid metagenome analysis. METHODS: IDBA-UD, MEGAHIT, SPAdes, SOAPdenovo2, and Velvet are compared for conducting plasmid metagenome analyses using two water samples. RESULTS: Our results clearly showed that abundance and types of antibiotic resistance genes on plasmids varied depending on the selection of assembly tools. IDBA-UD and MEGAHIT demonstrated the overall best assembly statistics with high N50 values with higher portion of longer contigs. CONCLUSION: These two assemblers also detected more diverse plasmids. Among the two, MEGAHIT showed more memory efficient assembly, therefore we suggest that the use of MEGAHIT for plasmid metagenome analysis may offer more diverse plasmids with less computer resource required. Here, we also summarized a fundamental plasmid metagenome work flow, especially for antibiotic resistance gene investigation.201931187446
6581140.9904Do wastewater treatment plants increase antibiotic resistant bacteria or genes in the environment? Protocol for a systematic review. BACKGROUND: Antibiotic resistance is a global public health threat. Water from human activities is collected at wastewater treatment plants where processes often do not sufficiently neutralize antibiotic resistant bacteria and genes, which are further shed into the local environment. This protocol outlines the steps to conduct a systematic review based on the Population, Exposure, Comparator and Outcome (PECO) framework, aiming at answering the question "Are antimicrobial-resistant enterobacteriaceae and antimicrobial resistance genes present (O) in air and water samples (P) taken either near or downstream or downwind or down-gradient from wastewater treatment plants (E), as compared to air and water samples taken either further away or upstream or upwind or up-gradient from such wastewater treatment plant (C)?" Presence of antimicrobial-resistant bacteria and genes will be quantitatively measured by extracting their prevalence or concentration, depending on the reviewed study. METHODS: We will search PubMed, EMBASE, the Cochrane database and Web of Science for original articles published from 1 Jan 2000 to 3 Sep 2018 with language restriction. Articles will undergo a relevance and a design screening process. Data from eligible articles will be extracted by two independent reviewers. Further, we will perform a risk of bias assessment using a decision matrix. We will synthesize and present results in narrative and tabular form and will perform a meta-analysis if heterogeneity of results allows it. DISCUSSION: Antibiotic resistance in environmental samples around wastewater treatment plants may pose a risk of exposure to workers and nearby residents. Results from the systematic review outlined in this protocol will allow to estimate the extend of exposure, to inform policy making and help to design future studies.201931806019
9079150.9904Review, Evaluation, and Directions for Gene-Targeted Assembly for Ecological Analyses of Metagenomes. Shotgun metagenomics has greatly advanced our understanding of microbial communities over the last decade. Metagenomic analyses often include assembly and genome binning, computationally daunting tasks especially for big data from complex environments such as soil and sediments. In many studies, however, only a subset of genes and pathways involved in specific functions are of interest; thus, it is not necessary to attempt global assembly. In addition, methods that target genes can be computationally more efficient and produce more accurate assembly by leveraging rich databases, especially for those genes that are of broad interest such as those involved in biogeochemical cycles, biodegradation, and antibiotic resistance or used as phylogenetic markers. Here, we review six gene-targeted assemblers with unique algorithms for extracting and/or assembling targeted genes: Xander, MegaGTA, SAT-Assembler, HMM-GRASPx, GenSeed-HMM, and MEGAN. We tested these tools using two datasets with known genomes, a synthetic community of artificial reads derived from the genomes of 17 bacteria, shotgun sequence data from a mock community with 48 bacteria and 16 archaea genomes, and a large soil shotgun metagenomic dataset. We compared assemblies of a universal single copy gene (rplB) and two N cycle genes (nifH and nirK). We measured their computational efficiency, sensitivity, specificity, and chimera rate and found Xander and MegaGTA, which both use a probabilistic graph structure to model the genes, have the best overall performance with all three datasets, although MEGAN, a reference matching assembler, had better sensitivity with synthetic and mock community members chosen from its reference collection. Also, Xander and MegaGTA are the only tools that include post-assembly scripts tuned for common molecular ecology and diversity analyses. Additionally, we provide a mathematical model for estimating the probability of assembling targeted genes in a metagenome for estimating required sequencing depth.201931749830
6345160.9903Transfer RNA gene numbers may not be completely responsible for the codon usage bias in asparagine, isoleucine, phenylalanine, and tyrosine in the high expression genes in bacteria. It is generally believed that the effect of translational selection on codon usage bias is related to the number of transfer RNA genes in bacteria, which is more with respect to the high expression genes than the whole genome. Keeping this in the background, we analyzed codon usage bias with respect to asparagine, isoleucine, phenylalanine, and tyrosine amino acids. Analysis was done in seventeen bacteria with the available gene expression data and information about the tRNA gene number. In most of the bacteria, it was observed that codon usage bias and tRNA gene number were not in agreement, which was unexpected. We extended the study further to 199 bacteria, limiting to the codon usage bias in the two highly expressed genes rpoB and rpoC which encode the RNA polymerase subunits β and β', respectively. In concordance with the result in the high expression genes, codon usage bias in rpoB and rpoC genes was also found to not be in agreement with tRNA gene number in many of these bacteria. Our study indicates that tRNA gene numbers may not be the sole determining factor for translational selection of codon usage bias in bacterial genomes.201223053196
5803170.9903Face mask sampling reveals antimicrobial resistance genes in exhaled aerosols from patients with chronic obstructive pulmonary disease and healthy volunteers. INTRODUCTION: The degree to which bacteria in the human respiratory tract are aerosolised by individuals is not established. Building on our experience sampling bacteria exhaled by individuals with pulmonary tuberculosis using face masks, we hypothesised that patients with conditions frequently treated with antimicrobials, such as chronic obstructive pulmonary disease (COPD), might exhale significant numbers of bacteria carrying antimicrobial resistance (AMR) genes and that this may constitute a previously undefined risk for the transmission of AMR. METHODS: Fifteen-minute mask samples were taken from 13 patients with COPD (five paired with contemporaneous sputum samples) and 10 healthy controls. DNA was extracted from cell pellets derived from gelatine filters mounted within the mask. Quantitative PCR analyses directed to the AMR encoding genes: blaTEM (β-lactamase), ErmB (target methylation), mefA (macrolide efflux pump) and tetM (tetracycline ribosomal protection protein) and six additional targets were investigated. Positive signals above control samples were obtained for all the listed genes; however, background signals from the gelatine precluded analysis of the additional targets. RESULTS: 9 patients with COPD (69%), aerosolised cells containing, in order of prevalence, mefA, tetM, ErmB and blaTEM, while three healthy controls (30%) gave weak positive signals including all targets except blaTEM. Maximum estimated copy numbers of AMR genes aerosolised per minute were mefA: 3010, tetM: 486, ErmB: 92 and blaTEM: 24. The profile of positive signals found in sputum was not concordant with that in aerosol in multiple instances. DISCUSSION: We identified aerosolised AMR genes in patients repeatedly exposed to antimicrobials and in healthy volunteers at lower frequencies and levels. The discrepancies between paired samples add weight to the view that sputum content does not define aerosol content. Mask sampling is a simple approach yielding samples from all subjects and information distinct from sputum analysis. Our results raise the possibility that patient-generated aerosols may be a significant means of AMR dissemination that should be assessed further and that consideration be given to related control measures.201830271606
4761180.9903Antimicrobial resistance and biofilm formation of penile prosthesis isolates: insights from in-vitro analysis. BACKGROUND: Inflatable penile prostheses (IPPs) have been shown to harbor biofilms in the presence and absence of infection despite exposure to various antimicrobials. Microbes persisting on IPPs following antibiotic exposure have not been adequately studied to assess biofilm formation capacity and antibiotic resistance. AIM: In this study, we aimed to assess these properties of microbes obtained from explanted infected and non-infected IPPS using an in vitro model. METHODS: 35 bacterial isolates were grown and tested against various single-agent or multiple agent antibiotic regimens including: bacitracin, cefaclor, cefazolin, gentamicin, levofloxacin, trimethoprim-sulfamethoxazole, tobramycin, vancomycin, piperacillin/tazobactam, gentamicin + piperacillin/tazobactam, gentamicin + cefazolin, and gentamicin + vancomycin. Zones of inhibition were averaged for each sample site and species. Statistics were analyzed with Holm's corrected, one-sample t-tests against a null hypothesis of 0. Isolates were also allowed to form biofilms in a 96-well polyvinyl plate and absorbance was tested at 570 nm using a microplate reader. OUTCOMES: Resistance was determined via clinical guidelines or previously established literature, and the mean and standard deviation of biofilm absorbance values were calculated and normalized to the optical density600 of the bacterial inoculum. RESULTS: Every species tested was able to form robust biofilms with the exception of Staphylococcus warneri. As expected, most bacteria were resistant to common perioperative antimicrobial prophylaxis. Gentamicin dual therapy demonstrated somewhat greater efficacy. STRENGTHS AND LIMITATIONS: This study examines a broad range of antimicrobials against clinically obtained bacterial isolates. However, not all species and antibiotics tested had standardized breakpoints, requiring the use of surrogate values from the literature. The microbes included in this study and their resistance genes are expectedly biased towards those that survived antibiotic exposure, and thus reflect the types of microbes which might "survive" in vivo exposure following revisional surgery. CLINICAL TRANSLATION: Despite exposure to antimicrobials, bacteria isolated during penile prosthesis revision for both infected and non-infected cases exhibit biofilm forming capacity and extensive antibiotic resistance patterns in vitro. These microbes merit further investigation to understand when simple colonization vs re-infection might occur. CONCLUSIONS: Although increasing evidence supports the concept that all IPPs harbor biofilms, even in the absence of infection, a deeper understanding of the characteristics of bacteria that survive revisional surgery is warranted. This study demonstrated extensive biofilm forming capabilities, and resistance patterns among bacteria isolated from both non-infected and infected IPP revision surgeries. Further investigation is warranted to determine why some devices become infected while others remain colonized but non-infected.202540062463
172190.9903Molecular characterization influencing metal resistance in the Cupriavidus/Ralstonia genomes. Our environment is stressed with a load of heavy and toxic metals. Microbes, abundant in our environment, are found to adapt well to this metal-stressed condition. A comparative study among five Cupriavidus/Ralstonia genomes can offer a better perception of their evolutionary mechanisms to adapt to these conditions. We have studied codon usage among 1051 genes common to all these organisms and identified 15 optimal codons frequently used in highly expressed genes present within 1051 genes. We found the core genes of Cupriavidus metallidurans CH34 have a different optimal codon choice for arginine, glycine and alanine in comparison with the other four bacteria. We also found that the synonymous codon usage bias within these 1051 core genes is highly correlated with their gene expression. This supports that translational selection drives synonymous codon usage in the core genes of these genomes. Synonymous codon usage is highly conserved in the core genes of these five genomes. The only exception among them is C. metallidurans CH34. This genomewide shift in synonymous codon choice in C. metallidurans CH34 may have taken place due to the insertion of new genes in its genomes facilitating them to survive in heavy metal containing environment and the co-evolution of the other genes in its genome to achieve a balance in gene expression. Structural studies indicated the presence of a longer N-terminal region containing a copper-binding domain in the cupC proteins of C. metallidurans CH3 that helps it to attain higher binding efficacy with copper in comparison with its orthologs.201526156561