Multiple Bacterial Genomes Research Articles

Detection And Quantification Of Treponema Denticola In Subgingival Plaque Of Humans By Polymerase Chain Reaction

Introduction: Inflammation of the supporting tissues which is also known as periodontitis contributes to the major etiological factor for tooth loss. Consortium of pathogenic bacteria with elevated levels lead to onset and progression of periodontal disease. Gram negative bacteria, primarily severe anaerobes, are responsible for most of it. Aims and Objectives: The present study aimed to assess the presence of Treponema denticola in periodontal disease in different subgingival plaque samples collected from the oral cavity of 30 patients diagnosed with chronic periodontitis. The plaque samples were compared with similar plaque samples of 15 healthy patients. Method: Subgingival dental plaque samples from shallow and deep crevices were examined for the presence of bacterial DNA using the PCR method, which enables the simultaneous identification of multiple bacterial genomes. Results: PCR results show amplification and quantification of T. denticola in 10 out of 15 patients in group-1, 9 out of 15 patients in group-2, and 4 out of 15 patients in group-3. The statistical analyses of the results show that they are statistically significant. Conclusion: According to the aforementioned study, PCR microorganism identification is sensitive and specific. For early infection diagnosis and subsequently for the prevention and treatment of periodontal disease, a rapid and accurate assessment of periodonto-pathogens is crucial. Bangladesh Journal of Medical Science Vol.22 (Special Issue) 2023 p.93-99

Rearrangement analysis of multiple bacterial genomes

BackgroundGenomes are subjected to rearrangements that change the orientation and ordering of genes during evolution. The most common rearrangements that occur in uni-chromosomal genomes are inversions (or reversals) to adapt to the changing environment. Since genome rearrangements are rarer than point mutations, gene order with sequence data can facilitate more robust phylogenetic reconstruction. Helicobacter pylori is a good model because of its unique evolution in niche environment.ResultsWe have developed a method to identify genome rearrangements by comparing almost-conserved genes among closely related strains. Orthologous gene clusters, rather than the gene sequences, are used to align the gene order so that comparison of large number of genomes becomes easier. Comparison of 72 Helicobacter pylori strains revealed shared as well as strain-specific reversals, some of which were found in different geographical locations.ConclusionDegree of genome rearrangements increases with time. Therefore, gene orders can be used to study the evolutionary relationship among species and strains. Multiple genome comparison helps to identify the strain-specific as well as shared reversals. Identification of the time course of rearrangements can provide insights into evolutionary events.

Determination of Repeat Number and Expression States of Phase-Variable Loci Through Next Generation Sequencing and Bioinformatic Analysis.

Phase variation (PV) enables high frequency, reversible switches in expression of genetic loci across numerous species of bacteria. A major mechanism of PV in bacteria is the use of slipped strand mispairing across simple sequence repeats (SSRs). The generation and online availability of genomic datasets enables a comprehensive analysis of the distribution and composition of SSRs across multiple bacterial genomes of a species. PhasomeIt is a program that was developed to rapidly identify SSRs, to determine whether these SSRs mediate PV and to find homologous PV loci across multiple genomes. We describe use of this program for analysis of neisserial genomes. We further describe a method to reassemble specific PV loci to allow analysis of large repeat tracts which are often poorly assembled due to inherent drawbacks of the Illumina next generation sequencing (NGS) platform. These methodologies allow for rapid analysis of a major mechanism of PV across numerous species of Neisseria and other bacterial species.

The use of Oxford Nanopore native barcoding for complete genome assembly.

BackgroundThe Oxford Nanopore Technologies MinION(TM) is a mobile DNA sequencer that can produce long read sequences with a short turn-around time. Here we report the first demonstration of single contig genome assembly using Oxford Nanopore native barcoding when applied to a multiplexed library of 12 samples and combined with existing Illumina short read data. This paves the way for the closure of multiple bacterial genomes from a single MinION(TM) sequencing run, given the availability of existing short read data. The strain we used, MHO_001, represents the important community-acquired methicillin-resistant Staphylococcus aureus lineage USA300.FindingsUsing a hybrid assembly of existing short read and barcoded long read sequences from multiplexed data, we completed a genome of the S. aureus USA300 strain MHO_001. The long read data represented only ∼5% to 10% of an average MinION(TM) run (∼7x genomic coverage), but, using standard tools, this was sufficient to complete the circular chromosome of S. aureus strain MHO_001 (2.86 Mb) and two complete plasmids (27 Kb and 3 Kb). Minor differences were noted when compared to USA300 reference genome, USA300_FPR3757, including the translocation, loss, and gain of mobile genetic elements.ConclusionHere we demonstrate that MinION(TM) reads, multiplexed using native barcoding, can be used in combination with short read data to fully complete a bacterial genome. The ability to complete multiple genomes, for which short read data is already available, from a single MinION(TM) run is set to impact our understanding of accessory genome content, plasmid diversity, and genome rearrangements.

Identifying contamination with advanced visualization and analysis practices: metagenomic approaches for eukaryotic genome assemblies.

High-throughput sequencing provides a fast and cost-effective mean to recover genomes of organisms from all domains of life. However, adequate curation of the assembly results against potential contamination of non-target organisms requires advanced bioinformatics approaches and practices. Here, we re-analyzed the sequencing data generated for the tardigrade Hypsibius dujardini, and created a holistic display of the eukaryotic genome assembly using DNA data originating from two groups and eleven sequencing libraries. By using bacterial single-copy genes, k-mer frequencies, and coverage values of scaffolds we could identify and characterize multiple near-complete bacterial genomes from the raw assembly, and curate a 182 Mbp draft genome for H. dujardini supported by RNA-Seq data. Our results indicate that most contaminant scaffolds were assembled from Moleculo long-read libraries, and most of these contaminants have differed between library preparations. Our re-analysis shows that visualization and curation of eukaryotic genome assemblies can benefit from tools designed to address the needs of today’s microbiologists, who are constantly challenged by the difficulties associated with the identification of distinct microbial genomes in complex environmental metagenomes.

Discovering novel enzymes by functional screening of plurigenomic libraries from alga-associated Flavobacteriia and Gammaproteobacteria

Alga-associated microorganisms, in the context of their numerous interactions with the host and the complexity of the marine environment, are known to produce diverse hydrolytic enzymes with original biochemistry. We recently isolated several macroalgal-polysaccharide-degrading bacteria from the surface of the brown alga Ascophyllum nodosum. These active isolates belong to two classes: the Flavobacteriia and the Gammaproteobacteria. In the present study, we constructed two “plurigenomic” (with multiple bacterial genomes) libraries with the 5 most interesting isolates (regarding their phylogeny and their enzymatic activities) of each class (Fv and Gm libraries). Both libraries were screened for diverse hydrolytic activities. Five activities, out of the 48 previously identified in the natural polysaccharolytic isolates, were recovered by functional screening: a xylanase (GmXyl7), a beta-glucosidase (GmBg1), an esterase (GmEst7) and two iota-carrageenases (Fvi2.5 and Gmi1.3). We discuss here the potential role of the used host-cell, the average DNA insert-sizes and the used restriction enzymes on the divergent screening yields obtained for both libraries and get deeper inside the “great screen anomaly”. Interestingly, the discovered esterase probably stands for a novel family of homoserine o-acetyltransferase-like-esterases, while the two iota-carrageenases represent new members of the poorly known GH82 family (containing only 19 proteins since its description in 2000). These original results demonstrate the efficiency of our uncommon “plurigenomic” library approach and the underexplored potential of alga-associated cultivable microbiota for the identification of novel and algal-specific enzymes.

Reconstructing rare soil microbial genomes using in situ enrichments and metagenomics

Despite extensive direct sequencing efforts and advanced analytical tools, reconstructing microbial genomes from soil using metagenomics have been challenging due to the tremendous diversity and relatively uniform distribution of genomes found in this system. Here we used enrichment techniques in an attempt to decrease the complexity of a soil microbiome prior to sequencing by submitting it to a range of physical and chemical stresses in 23 separate microcosms for 4 months. The metagenomic analysis of these microcosms at the end of the treatment yielded 540 Mb of assembly using standard de novo assembly techniques (a total of 559,555 genes and 29,176 functions), from which we could recover novel bacterial genomes, plasmids and phages. The recovered genomes belonged to Leifsonia (n = 2), Rhodanobacter (n = 5), Acidobacteria (n = 2), Sporolactobacillus (n = 2, novel nitrogen fixing taxon), Ktedonobacter (n = 1, second representative of the family Ktedonobacteraceae), Streptomyces (n = 3, novel polyketide synthase modules), and Burkholderia (n = 2, includes mega-plasmids conferring mercury resistance). Assembled genomes averaged to 5.9 Mb, with relative abundances ranging from rare (<0.0001%) to relatively abundant (>0.01%) in the original soil microbiome. Furthermore, we detected them in samples collected from geographically distant locations, particularly more in temperate soils compared to samples originating from high-latitude soils and deserts. To the best of our knowledge, this study is the first successful attempt to assemble multiple bacterial genomes directly from a soil sample. Our findings demonstrate that developing pertinent enrichment conditions can stimulate environmental genomic discoveries that would have been impossible to achieve with canonical approaches that focus solely upon post-sequencing data treatment.

Normalizing alternate representations of large sequence variants across multiple bacterial genomes

Background and description Variant-focused comparative genomics enables researchers to study the evolution of distinct genetic characteristics in bacterial populations, while avoiding the difficulties of whole-genome assembly and alignment. A major challenge in using this method is that many variant detecting tools are largely limited to predicting single nucleotide variants (SNVs) and small indels. This is a challenge because bacterial organisms do not only possess SNVs but also harbor much larger sequence variants (LSVs), such as large indels and substitutions (>25 nt), when compared to a reference genome. LSVs have been shown to play a role in shaping important biological aspects such as virulence and drug resistance as well as reporting on population structure [1-3]. Recent variant callers, such as Pilon http://www.broadinstitute. org/software/pilon/, can identify LSVs with single nucleotide accuracy in microbial genomes. However, one remaining challenge is that identical LSVs can be represented non-identically by a single variant detecting tool; this generally results from similarity in the flanking sequence of the variant and variability of the read quality and alignment information in that region across the different strains. As a result, alternate representations of large variants make it difficult to perform downstream analyses such as association studies that depend on consistent representations of variants. We present Emu, an algorithm that resolves alternate representations of LSVs by comparing variant calls across genomes. Results To evaluate Emu’s ability to resolve alternate representations of LSVs, we introduced 179 simulated LSVs into the H37Rv genome–a carefully curated and finished reference genome for Mycobacterium tuberculosis (Mtb). We then used Pilon to identify variants in a set of 146 clinical samples of Mtb that were collected in China using the modified H37Rv genome as a reference [4]. We identified a total of 10,001 unique variant representations. The average number of non-identical representations of each simulated LSV was 56 (in the range of 1 to 145). We then applied Emu to identify the non-identical representations across the genomes of the 146 clinical samples and canonicalize them to a single form. Emu reduced the total number of non-identical representations to 676 LSVs bringing the average number of non-identical representations at each LSV to 4, with 15 LSVs reduced to a single representation and no LSV having more than 25 representations. We then investigated how Emu’s ability to resolve alternate representations might impact association analyses, e.g., associating LSVs with population structure. We ran Pilon again on the set of 161 clinical samples from China, but used the unmodified H37Rv genome. Pilon identified a total of 20,512 distinct LSVs when compared to the unmodified H37Rv genome. By applying Emu, the number of distinct LSVs decreased by almost 50% to 10,936 LSVs. Emu also increased the power of association tests on the LSVs. While we initially identified a total number of 69 LSVs that were significantly associated (p < 0.01) with membership to a specific clade, after processing with Emu that number increased to 94.

Natural products in complex microbiomes – Assembling multiple bacterial genomes directly through shotgun metagenomics

Natural products in complex microbiomes – Assembling multiple bacterial genomes directly through shotgun metagenomics

Sequences of multiple bacterial genomes and a Chlamydia trachomatis genotype from direct sequencing of DNA derived from a vaginal swab diagnostic specimen

Ultra-deep Illumina sequencing was performed on whole genome amplified DNA derived from a Chlamydia trachomatis-positive vaginal swab. Alignment of reads with reference genomes allowed robust SNP identification from the C. trachomatis chromosome and plasmid. This revealed that the C. trachomatis in the specimen was very closely related to the sequenced urogenital, serovar F, clade T1 isolate F-SW4. In addition, high genome-wide coverage was obtained for Prevotella melaninogenica, Gardnerella vaginalis, Clostridiales genomosp. BVAB3 and Mycoplasma hominis. This illustrates the potential of metagenome data to provide high resolution bacterial typing data from multiple taxa in a diagnostic specimen.

New players in the same old game: a system level in silico study to predict type III secretion system and effector proteins in bacterial genomes reveals common themes in T3SS mediated pathogenesis

BackgroundType III secretion system (T3SS) plays an important role in virulence or symbiosis of many pathogenic or symbiotic bacteria [CHM 2:291–294, 2007; Physiology (Bethesda) 20:326–339, 2005]. T3SS acts like a tunnel between a bacterium and its host through which the bacterium injects ‘effector’ proteins into the latter [Nature 444:567–573, 2006; COSB 18:258–266, 2008]. The effectors spatially and temporally modify the host signalling pathways [FEMS Microbiol Rev 35:1100–1125, 2011; Cell Host Microbe5:571–579, 2009]. In spite its crucial role in host-pathogen interaction, the study of T3SS and the associated effectors has been limited to a few bacteria [Cell Microbiol 13:1858–1869, 2011; Nat Rev Microbiol 6:11–16, 2008; Mol Microbiol 80:1420–1438, 2011]. Before one set out to perform systematic experimental studies on an unknown set of bacteria it would be beneficial to identify the potential candidates by developing an in silico screening algorithm. A system level study would also be advantageous over traditional laboratory methods to extract an overriding theme for host-pathogen interaction, if any, from the vast resources of data generated by sequencing multiple bacterial genomes.ResultsWe have developed an in silico protocol in which the most conserved set of T3SS proteins was used as the query against the entire bacterial database with increasingly stringent search parameters. It enabled us to identify several uncharacterized T3SS positive bacteria. We adopted a similar strategy to predict the presence of the already known effectors in the newly identified T3SS positive bacteria. The huge resources of biochemical data [FEMS Microbiol Rev 35:1100–1125, 2011; Cell Host Microbe 5:571–579, 2009; BMC Bioinformatics 7(11):S4, 2010] on the T3SS effectors enabled us to search for the common theme in T3SS mediated pathogenesis. We identified few cellular signalling networks in the host, which are manipulated by most of the T3SS containing pathogens. We went on to look for correlation, if any, between the biological quirks of a particular class of bacteria with the effectors they harbour. We could pin point few effectors, which were enriched in certain classes of bacteria.ConclusionThe current study would open up new avenues to explore many uncharacterized T3SS positive bacteria. The experimental validation of the predictions from this study will unravel a generalized mechanism for T3SS positive bacterial infection into host cell.

Eukaryote to gut bacteria transfer of a glycoside hydrolase gene essential for starch breakdown in plants

Lateral gene transfer (LGT) between bacteria constitutes a strong force in prokaryote evolution, transforming the hierarchical tree of life into a network of relationships between species. In contrast, only a few cases of LGT from eukaryotes to prokaryotes have been reported so far. The distal animal intestine is predominantly a bacterial ecosystem, supplying the host with energy from dietary polysaccharides through carbohydrate-active enzymes absent from its genome. It has been suggested that LGT is particularly important for the human microbiota evolution. Here we show evidence for the first eukaryotic gene identified in multiple gut bacterial genomes. We found in the genome sequence of several gut bacteria, a typically eukaryotic glycoside-hydrolase necessary for starch breakdown in plants. The distribution of this gene is patchy in gut bacteria with presence otherwise detected only in a few environmental bacteria. We speculate that the transfer of this gene to gut bacteria occurred by a sequence of two key LGT events; first, an original eukaryotic gene was transferred probably from Archaeplastida to environmental bacteria specialized in plant polysaccharides degradation and second, the gene was transferred from the environmental bacteria to gut microbes.

Optically Mapping Multiple Bacterial Genomes Simultaneously in a Single Run

Optical mapping of bacterial chromosomes provides an unambiguous low-resolution sequence scaffold of the entire chromosome. In comparison to some techniques, such as pulse field gel electrophoresis, cost and throughput limit the application of this technique outside of genome finishing. We have demonstrated the production of multiple bacterial maps using a single set of consumables; this significantly reduces the time and expense of map production.

CAMBerVis: visualization software to support comparative analysis of multiple bacterial strains

A number of inconsistencies in genome annotations are documented among bacterial strains. Visualization of the differences may help biologists to make correct decisions in spurious cases. We have developed a visualization tool, CAMBerVis, to support comparative analysis of multiple bacterial strains. The software manages simultaneous visualization of multiple bacterial genomes, enabling visual analysis focused on genome structure annotations. The CAMBerVis software is freely available at the project website: http://bioputer.mimuw.edu.pl/camber. Input datasets for Mycobacterium tuberculosis and Staphylocacus aureus are integrated with the software as examples. m.wozniak@mimuw.edu.pl Supplementary data are available at Bioinformatics online.

A high-throughput pipeline for the design of real-time PCR signatures

BackgroundPathogen diagnostic assays based on polymerase chain reaction (PCR) technology provide high sensitivity and specificity. However, the design of these diagnostic assays is computationally intensive, requiring high-throughput methods to identify unique PCR signatures in the presence of an ever increasing availability of sequenced genomes.ResultsWe present the Tool for PCR Signature Identification (TOPSI), a high-performance computing pipeline for the design of PCR-based pathogen diagnostic assays. The TOPSI pipeline efficiently designs PCR signatures common to multiple bacterial genomes by obtaining the shared regions through pairwise alignments between the input genomes. TOPSI successfully designed PCR signatures common to 18 Staphylococcus aureus genomes in less than 14 hours using 98 cores on a high-performance computing system.ConclusionsTOPSI is a computationally efficient, fully integrated tool for high-throughput design of PCR signatures common to multiple bacterial genomes. TOPSI is freely available for download at http://www.bhsai.org/downloads/topsi.tar.gz.

Oral probiotics in preterm infants and bacterial signatures in consecutive stool samples

Introduction: In preterm infants the risk of Necrotizing Enterocolitis (NEC) is reduced by oral probiotics. The protective effect could be associated with changes of the gut flora (direct effects) or by indirect effects modulating bacterial-host-interaction at the mucosal interface or the intestinal or systemic immune system. Material and Methods: In the present prospective randomized cross-over study we analysed the development of the bacterial community in stool samples of preterm infants for the first 6 weeks of life. 21 preterm infants at a gestational age &lt;32 weeks and with birth-weight &lt;1500g were included. Groups supplemented with Lactobacillus acidophilum and Bifidobacterium infantis (Infloran®) in week 1–3 (group 1) and in week 4–6 (group 2) were compared. The signatures of multiple bacterial genomes were regularly monitored in stool samples by Denaturing Gradient Gel Electrophoresis (PCR-DGGE) and the specific bands were identified by subsequent sequence typing. Bacterial signatures are actually available of &gt;300 samples (10 patients). Results: Bacterial DNA could not be amplified in the first stool sample (meconium), except for few patients with congenital infections. In the first weeks of life the development of an individual bacterial pattern was monitored in stool samples. We could show that the development and the composition of the predominant bacterial species in infants stool were not altered by modulation of probiotic supplementation. In both groups, the development of infants stool flora was initiated by colonisation with gram-negative bacteria, followed by gram-positive cocci and finally anaerobic bacteria. However, antibiotic interventions were associated with changes of bacterial signatures. Interestingly, specific bands for L. acidophilum and B. infantis were almost not detected despite probiotic substitution. Conclusions: Despite the limitations due to the few cases it may be concluded that probiotic supplementation was not associated with significant changes of preterm infants stool flora. Therefore we hypothesize that prevention of NEC might be associated with more indirect effects which remains still to be confirmed in future studies.

MGenomeSubtractor: a web-based tool for parallel in silico subtractive hybridization analysis of multiple bacterial genomes

mGenomeSubtractor performs an mpiBLAST-based comparison of reference bacterial genomes against multiple user-selected genomes for investigation of strain variable accessory regions. With parallel computing architecture, mGenomeSubtractor is able to run rapid BLAST searches of the segmented reference genome against multiple subject genomes at the DNA or amino acid level within a minute. In addition to comparison of protein coding sequences, the highly flexible sliding window-based genome fragmentation approach offered can be used to identify short unique sequences within or between genes. mGenomeSubtractor provides powerful schematic outputs for exploration of identified core and accessory regions, including searches against databases of mobile genetic elements, virulence factors or bacterial essential genes, examination of G+C content and binucleotide distribution bias, and integrated primer design tools. mGenomeSubtractor also allows for the ready definition of species-specific gene pools based on available genomes. Pan-genomic arrays can be easily developed using the efficient oligonucleotide design tool. This simple high-throughput in silico ‘subtractive hybridization’ analytical tool will support the rapidly escalating number of comparative bacterial genomics studies aimed at defining genomic biomarkers of evolutionary lineage, phenotype, pathotype, environmental adaptation and/or disease-association of diverse bacterial species. mGenomeSubtractor is freely available to all users without any login requirement at: http://bioinfo-mml.sjtu.edu.cn/mGS/.

In silico microarray probe design for diagnosis of multiple pathogens

BackgroundWith multiple strains of various pathogens being sequenced, it is necessary to develop high-throughput methods that can simultaneously process multiple bacterial or viral genomes to find common fingerprints as well as fingerprints that are unique to each individual genome. We present algorithmic enhancements to an existing single-genome pipeline that allows for efficient design of microarray probes common to groups of target genomes. The enhanced pipeline takes advantage of the similarities in the input genomes to narrow the search to short, nonredundant regions of the target genomes and, thereby, significantly reduces the computation time. The pipeline also computes a three-state hybridization matrix, which gives the expected hybridization of each probe with each target.ResultsDesign of microarray probes for eight pathogenic Burkholderia genomes shows that the multiple-genome pipeline is nearly four-times faster than the single-genome pipeline for this application. The probes designed for these eight genomes were experimentally tested with one non-target and three target genomes. Hybridization experiments show that less than 10% of the designed probes cross hybridize with non-targets. Also, more than 65% of the probes designed to identify all Burkholderia mallei and B. pseudomallei strains successfully hybridize with a B. pseudomallei strain not used for probe design.ConclusionThe savings in runtime suggest that the enhanced pipeline can be used to design fingerprints for tens or even hundreds of related genomes in a single run. Hybridization results with an unsequenced B. pseudomallei strain indicate that the designed probes might be useful in identifying unsequenced strains of B. mallei and B. pseudomallei.

Large-scale analysis of gene clustering in bacteria

An important strategy to study operons and their evolution is to investigate clustering of related genes across multiple bacterial genomes. Although existing algorithms are available that can identify gene clusters across two or more genomes, very few algorithms are efficient enough to study gene clusters across hundreds of genomes. We observe that a querying strategy can be used to analyze gene clusters across a large number of genomes and develop an efficient algorithm to identify all related clusters on a genome from a given query cluster. We use this algorithm to study gene clustering in 400 bacterial genomes by starting from a well-characterized list of operons in Escherichia coli K12 and perform comparative analysis of operon occurrences, gene orientations, and rearrangements both within and across clusters. We show that important biological insights can be obtained by comparing results across these categories. A software program implementing the algorithm (GCQuery) and supplementary data containing detailed results are available at http://faculty.cs.tamu.edu/shsze/gcquery.

Inference of Bacterial Microevolution Using Multilocus Sequence Data

We describe a model-based method for using multilocus sequence data to infer the clonal relationships of bacteria and the chromosomal position of homologous recombination events that disrupt a clonal pattern of inheritance. The key assumption of our model is that recombination events introduce a constant rate of substitutions to a contiguous region of sequence. The method is applicable both to multilocus sequence typing (MLST) data from a few loci and to alignments of multiple bacterial genomes. It can be used to decide whether a subset of isolates share common ancestry, to estimate the age of the common ancestor, and hence to address a variety of epidemiological and ecological questions that hinge on the pattern of bacterial spread. It should also be useful in associating particular genetic events with the changes in phenotype that they cause. We show that the model outperforms existing methods of subdividing recombinogenic bacteria using MLST data and provide examples from Salmonella and Bacillus. The software used in this article, ClonalFrame, is available from http://bacteria.stats.ox.ac.uk/.