scholarly journals Identification of isolated or mixed strains from long reads: a challenge met on Streptococcus thermophilus using a MinION sequencer

2021 ◽  
Vol 7 (11) ◽  
Author(s):  
Grégoire Siekaniec ◽  
Emeline Roux ◽  
Téo Lemane ◽  
Eric Guédon ◽  
Jacques Nicolas
Keyword(s):  
Memory Management ◽  
Bacterial Species ◽  
Streptococcus Thermophilus ◽  
Bacterial Strains ◽  
Content Type ◽  
Long Reads ◽  
Detailed Classification ◽  
Long Read ◽  
Identification Of Bacteria

This study aimed to provide efficient recognition of bacterial strains on personal computers from MinION (Nanopore) long read data. Thanks to the fall in sequencing costs, the identification of bacteria can now proceed by whole genome sequencing. MinION is a fast, but highly error-prone sequencing device and it is a challenge to successfully identify the strain content of unknown simple or complex microbial samples. It is heavily constrained by memory management and fast access to the read and genome fragments. Our strategy involves three steps: indexing of known genomic sequences for a given or several bacterial species; a request process to assign a read to a strain by matching it to the closest reference genomes; and a final step looking for a minimum set of strains that best explains the observed reads. We have applied our method, called ORI, on 77 strains of Streptococcus thermophilus . We worked on several genomic distances and obtained a detailed classification of the strains, together with a criterion that allows merging of what we termed ‘sibling’ strains, only separated by a few mutations. Overall, isolated strains can be safely recognized from MinION data. For mixtures of several non-sibling strains, results depend on strain abundance.

scholarly journals Novel Variants of Streptococcus thermophilus Bacteriophages Are Indicative of Genetic Recombination among Phages from Different Bacterial Species

2016 ◽  
Vol 83 (5) ◽  
Author(s):  
Paula Szymczak ◽  
Thomas Janzen ◽  
Ana Rute Neves ◽  
Witold Kot ◽  
Lars H. Hansen ◽  
...  
Keyword(s):  
Genetic Recombination ◽  
Genetic Relationships ◽  
Bacterial Species ◽  
Streptococcus Thermophilus ◽  
Starter Cultures ◽  
Sequencing Analysis ◽  
Content Type ◽  
Novel Variants ◽  
Positive Results

ABSTRACT Bacteriophages are the main cause of fermentation failures in dairy plants. The majority of Streptococcus thermophilus phages can be divided into either cos- or pac-type phages and are additionally characterized by examining the V2 region of their antireceptors. We screened a large number of S. thermophilus phages from the Chr. Hansen A/S collection, using PCR specific for the cos- or pac-type phages, as well as for the V2 antireceptor region. Three phages did not produce positive results with the assays. Analysis of phage morphologies indicated that two of these phages, CHPC577 and CHPC926, had shorter tails than the traditional S. thermophilus phages. The third phage, CHPC1151, had a tail size similar to those of the cos- or pac-type phages, but it displayed a different baseplate structure. Sequencing analysis revealed the genetic similarity of CHPC577 and CHPC926 with a subgroup of Lactococcus lactis P335 phages. Phage CHPC1151 was closely related to the atypical S. thermophilus phage 5093, homologous with a nondairy streptococcal prophage. By testing adsorption of the related streptococcal and lactococcal phages to the surface of S. thermophilus and L. lactis strains, we revealed the possibility of cross-interactions. Our data indicated that the use of S. thermophilus together with L. lactis, extensively applied for dairy fermentations, triggered the recombination between phages infecting different bacterial species. A notable diversity among S. thermophilus phage populations requires that a new classification of the group be proposed. IMPORTANCE Streptococcus thermophilus is a component of thermophilic starter cultures commonly used for cheese and yogurt production. Characterizing streptococcal phages, understanding their genetic relationships, and studying their interactions with various hosts are the necessary steps for preventing and controlling phage attacks that occur during dairy fermentations.

scholarly journals Complete Genome Sequences of 12 Quinolone-Resistant Escherichia coli Strains Containing qnrS1 Based on Hybrid Assemblies

2021 ◽  
Vol 10 (4) ◽  
Author(s):  
Håkon Kaspersen ◽  
Thomas H. A. Haverkamp ◽  
Hanna Karin Ilag ◽  
Øivind Øines ◽  
Camilla Sekse ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Complete Genome ◽  
Flow Cell ◽  
Hybrid Assembly ◽  
Genome Sequences ◽  
Content Type ◽  
Short Reads ◽  
Long Reads ◽  
Long Read ◽  
Hybrid Assemblies

ABSTRACT In total, 12 quinolone-resistant Escherichia coli (QREC) strains containing qnrS1 were submitted to long-read sequencing using a FLO-MIN106 flow cell on a MinION device. The long reads were assembled with short reads (Illumina) and analyzed using the MOB-suite pipeline. Six of these QREC genome sequences were closed after hybrid assembly.

scholarly journals Rapid Mycobacterium tuberculosis spoligotyping from uncorrected long reads using Galru

2020 ◽  
Author(s):  
Andrew J. Page ◽  
Nabil-Fareed Alikhan ◽  
Michael Strinden ◽  
Thanh Le Viet ◽  
Timofey Skvortsov
Keyword(s):  
Mycobacterium Tuberculosis ◽  
State Of The Art ◽  
Sequence Data ◽  
Human Pathogen ◽  
Sequencing Data ◽  
Short Read ◽  
Short Read Sequencing ◽  
Long Reads ◽  
Long Read

AbstractSpoligotyping of Mycobacterium tuberculosis provides a subspecies classification of this major human pathogen. Spoligotypes can be predicted from short read genome sequencing data; however, no methods exist for long read sequence data such as from Nanopore or PacBio. We present a novel software package Galru, which can rapidly detect the spoligotype of a Mycobacterium tuberculosis sample from as little as a single uncorrected long read. It allows for near real-time spoligotyping from long read data as it is being sequenced, giving rapid sample typing. We compare it to the existing state of the art software and find it performs identically to the results obtained from short read sequencing data. Galru is freely available from https://github.com/quadram-institute-bioscience/galru under the GPLv3 open source licence.

scholarly journals Spatiotemporal Distribution of Pseudomonas aeruginosa Alkyl Quinolones under Metabolic and Competitive Stress

mSphere ◽  
2020 ◽  
Vol 5 (4) ◽  
Author(s):  
Tianyuan Cao ◽  
Jonathan V. Sweedler ◽  
Paul W. Bohn ◽  
Joshua D. Shrout
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Bacterial Species ◽  
Metabolic Stress ◽  
Opportunistic Pathogen ◽  
Cell Interaction ◽  
Chemical Imaging ◽  
Carbon Limitation ◽  
Bacterial Strains ◽  
Content Type ◽  
High Level

ABSTRACT Pseudomonas aeruginosa is an opportunistic human pathogen important to diseases such as cystic fibrosis. P. aeruginosa has multiple quorum-sensing (QS) systems, one of which utilizes the signaling molecule 2-heptyl-3-hydroxy-4-quinolone (Pseudomonas quinolone signal [PQS]). Here, we use hyperspectral Raman imaging to elucidate the spatiotemporal PQS distributions that determine how P. aeruginosa regulates surface colonization and its response to both metabolic stress and competition from other bacterial strains. These chemical imaging experiments illustrate the strong link between environmental challenges, such as metabolic stress caused by nutritional limitations or the presence of another bacterial species, and PQS signaling. Metabolic stress elicits a complex response in which limited nutrients induce the bacteria to produce PQS earlier, but the bacteria may also pause PQS production entirely if the nutrient concentration is too low. Separately, coculturing P. aeruginosa in the proximity of another bacterial species, or its culture supernatant, results in earlier production of PQS. However, these differences in PQS appearance are not observed for all alkyl quinolones (AQs) measured; the spatiotemporal response of 2-heptyl-4-hydroxyquinoline N-oxide (HQNO) is highly uniform for most conditions. These insights on the spatiotemporal distributions of quinolones provide additional perspective on the behavior of P. aeruginosa in response to different environmental cues. IMPORTANCE Alkyl quinolones (AQs), including Pseudomonas quinolone signal (PQS), made by the opportunistic pathogen Pseudomonas aeruginosa have been associated with both population density and stress. The regulation of AQ production is known to be complex, and the stimuli that modulate AQ responses are not fully clear. Here, we have used hyperspectral Raman chemical imaging to examine the temporal and spatial profiles of AQs exhibited by P. aeruginosa under several potentially stressful conditions. We found that metabolic stress, effected by carbon limitation, or competition stress, effected by proximity to other species, resulted in accelerated PQS production. This competition effect did not require cell-to-cell interaction, as evidenced by the fact that the addition of supernatants from either Escherichia coli or Staphylococcus aureus led to early appearance of PQS. Lastly, the fact that these modulations were observed for PQS but not for all AQs suggests a high level of complexity in AQ regulation that remains to be discerned.

scholarly journals Unleashing Natural Competence in Lactococcus lactis by Induction of the Competence Regulator ComX

2017 ◽  
Vol 83 (20) ◽  
Author(s):  
Joyce Mulder ◽  
Michiel Wels ◽  
Oscar P. Kuipers ◽  
Michiel Kleerebezem ◽  
Peter A. Bron
Keyword(s):  
Lactococcus Lactis ◽  
Genetic Manipulation ◽  
Bacterial Species ◽  
Streptococcus Thermophilus ◽  
Starter Cultures ◽  
Natural Competence ◽  
Content Type ◽  
Competence Gene ◽  
Gene Sets ◽  
Novel Traits

ABSTRACT In biotechnological workhorses like Streptococcus thermophilus and Bacillus subtilis, natural competence can be induced, which facilitates genetic manipulation of these microbes. However, in strains of the important dairy starter Lactococcus lactis, natural competence has not been established to date. However, in silico analysis of the complete genome sequences of 43 L. lactis strains revealed complete late competence gene sets in 2 L. lactis subsp. cremoris strains (KW2 and KW10) and at least 10 L. lactis subsp. lactis strains, including the model strain IL1403 and the plant-derived strain KF147. The remainder of the strains, including all dairy isolates, displayed genomic decay in one or more of the late competence genes. Nisin-controlled expression of the competence regulator comX in L. lactis subsp. lactis KF147 resulted in the induction of expression of the canonical competence regulon and elicited a state of natural competence in this strain. In contrast, comX expression in L. lactis NZ9000, which was predicted to encode an incomplete competence gene set, failed to induce natural competence. Moreover, mutagenesis of the comEA-EC operon in strain KF147 abolished the comX-driven natural competence, underlining the involvement of the competence machinery. Finally, introduction of nisin-inducible comX expression into nisRK-harboring derivatives of strains IL1403 and KW2 allowed the induction of natural competence in these strains also, expanding this phenotype to other L. lactis strains of both subspecies. IMPORTANCE Specific bacterial species are able to enter a state of natural competence in which DNA is taken up from the environment, allowing the introduction of novel traits. Strains of the species Lactococcus lactis are very important starter cultures for the fermentation of milk in the cheese production process, where these bacteria contribute to the flavor and texture of the end product. The activation of natural competence in this industrially relevant organism can accelerate research aiming to understand industrially relevant traits of these bacteria and can facilitate engineering strategies to harness the natural biodiversity of the species in optimized starter strains.

scholarly journals Neurotoxin Gene Profiling of Clostridium botulinum Types C and D Native to Different Countries within Europe

2012 ◽  
Vol 78 (9) ◽  
pp. 3120-3127 ◽  
Author(s):  
Cedric Woudstra ◽  
Hanna Skarin ◽  
Fabrizio Anniballi ◽  
Lucia Fenicia ◽  
Luca Bano ◽  
...  
Keyword(s):  
Clostridium Botulinum ◽  
Limit Of Detection ◽  
Bacterial Species ◽  
Simultaneous Detection ◽  
Geographical Location ◽  
Gene Profiling ◽  
Animal Origin ◽  
Bacterial Strains ◽  
Content Type ◽  
Pcr Assays

ABSTRACTClostridium botulinumtypes C and D, as well as their mosaic variants C-D and D-C, are associated with avian and mammalian botulism. This study reports on the development of low-density macroarrays based on the GeneDisc cycler platform (Pall-GeneDisc Technologies) applied to the simultaneous detection of theC. botulinumsubtypes C, C-D, D, and D-C. The limit of detection of the PCR assays was 38 fg of total DNA, corresponding to 15 genome copies. Artificially contaminated samples of cecum showed a limit of detection below 50 spores/g. The tests were performed with a large variety of bacterial strains, includingC. botulinumtypes C (n= 12), C-D (n= 29), D (n= 5), and D-C (n= 10), other botulinum neurotoxin (BoNT)-producingClostridiumstrains (n= 20), non-BoNT-producing clostridia (n= 20), and other bacterial species (n= 23), and showed a high specificity. These PCR assays were compared to previously published real-time PCRs for the detection ofC. botulinumin 292 samples collected from cases of botulism events in four European regions. The majority of the samples originated from wild birds (n= 108), poultry (n= 60), and bovines (n= 56). Among the 292 samples, 144 were positive for either thebont/C-D or thebont/D-C gene by using the GeneDisc arrays. The reliability of the results tallied to 97.94%. Interestingly, only BoNT mosaics, types C-D and D-C, were found in naturally contaminated samples whatever their animal origin and their geographical location. Further investigations should now be performed in order to check that mosaic types dominate in Europe and that acquisition of mosaic types helps in survival or adaptation to particular niche.

scholarly journals Metagenomic Exploration of the Marine Sponge Mycale hentscheli Uncovers Multiple Polyketide-Producing Bacterial Symbionts

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Mathew A. Storey ◽  
Sarah K. Andreassend ◽  
Joe Bracegirdle ◽  
Alistair Brown ◽  
Robert A. Keyzers ◽  
...  
Keyword(s):  
Marine Sponge ◽  
Bacterial Species ◽  
Gene Clusters ◽  
Taxonomic Diversity ◽  
Marine Sponges ◽  
Metagenomic Sequencing ◽  
Content Type ◽  
Defensive Symbiosis ◽  
Long Read ◽  
Mycale Hentscheli

ABSTRACT Marine sponges have been a prolific source of unique bioactive compounds that are presumed to act as a deterrent to predation. Many of these compounds have potential therapeutic applications; however, the lack of efficient and sustainable synthetic routes frequently limits clinical development. Here, we describe a metagenomic investigation of Mycale hentscheli, a chemically gifted marine sponge that possesses multiple distinct chemotypes. We applied shotgun metagenomic sequencing, hybrid assembly of short- and long-read data, and metagenomic binning to obtain a comprehensive picture of the microbiome of five specimens, spanning three chemotypes. Our data revealed multiple producing species, each having relatively modest secondary metabolomes, that contribute collectively to the chemical arsenal of the holobiont. We assembled complete genomes for multiple new genera, including two species that produce the cytotoxic polyketides pateamine and mycalamide, as well as a third high-abundance symbiont harboring a proteusin-type biosynthetic pathway that appears to encode a new polytheonamide-like compound. We also identified an additional 188 biosynthetic gene clusters, including a pathway for biosynthesis of peloruside. These results suggest that multiple species cooperatively contribute to defensive symbiosis in M. hentscheli and reveal that the taxonomic diversity of secondary-metabolite-producing sponge symbionts is larger and richer than previously recognized. IMPORTANCE Mycale hentscheli is a marine sponge that is rich in bioactive small molecules. Here, we use direct metagenomic sequencing to elucidate highly complete and contiguous genomes for the major symbiotic bacteria of this sponge. We identify complete biosynthetic pathways for the three potent cytotoxic polyketides which have previously been isolated from M. hentscheli. Remarkably, and in contrast to previous studies of marine sponges, we attribute each of these metabolites to a different producing microbe. We also find that the microbiome of M. hentscheli is stably maintained among individuals, even over long periods of time. Collectively, our data suggest a cooperative mode of defensive symbiosis in which multiple symbiotic bacterial species cooperatively contribute to the defensive chemical arsenal of the holobiont.

scholarly journals Implications of Genome-Based Discrimination between Clostridium botulinum Group I and Clostridium sporogenes Strains for Bacterial Taxonomy

2015 ◽  
Vol 81 (16) ◽  
pp. 5420-5429 ◽  
Author(s):  
Michael R. Weigand ◽  
Angela Pena-Gonzalez ◽  
Timothy B. Shirey ◽  
Robin G. Broeker ◽  
Maliha K. Ishaq ◽  
...  
Keyword(s):  
Clostridium Botulinum ◽  
Bacterial Species ◽  
Phylogenetic Reconstruction ◽  
Bacterial Taxonomy ◽  
Clostridium Sporogenes ◽  
Content Type ◽  
Group I ◽  
Taxonomic Organization ◽  
Different Strains

ABSTRACTTaxonomic classification ofClostridium botulinumis based on the production of botulinum neurotoxin (BoNT), while closely related, nontoxic organisms are classified asClostridium sporogenes. However, this taxonomic organization does not accurately mirror phylogenetic relationships between these species. A phylogenetic reconstruction using 2,016 orthologous genes shared among strains ofC. botulinumgroup I andC. sporogenesclearly separated these two species into discrete clades which showed ∼93% average nucleotide identity (ANI) between them. Clustering of strains based on the presence of variable orthologs revealed 143C. sporogenesclade-specific genetic signatures, a subset of which were further evaluated for their ability to correctly classify a panel of presumptiveC. sporogenesstrains by PCR. Genome sequencing of severalC. sporogenesstrains lacking these signatures confirmed that they clustered withC. botulinumstrains in a core genome phylogenetic tree. Our analysis also identifiedC. botulinumstrains that containedC. sporogenesclade-specific signatures and phylogenetically clustered withC. sporogenesstrains. The genome sequences of twobont/B2-containing strains belonging to theC. sporogenesclade contained regions with similarity to abont-bearing plasmid (pCLD), while two different strains belonging to theC. botulinumclade carriedbont/B2on the chromosome. These results indicate thatbont/B2was likely acquired byC. sporogenesstrains through horizontal gene transfer. The genome-based classification of these species used to identify candidate genes for the development of rapid assays for molecular identification may be applicable to additional bacterial species that are challenging with respect to their classification.

scholarly journals Iridescence of a Marine Bacterium and Classification of Prokaryotic Structural Colors

2012 ◽  
Vol 78 (7) ◽  
pp. 2092-2099 ◽  
Author(s):  
Betty Kientz ◽  
Peter Vukusic ◽  
Stephen Luke ◽  
Eric Rosenfeld
Keyword(s):  
Marine Bacterium ◽  
Structural Color ◽  
Growth Media ◽  
Bacterial Strains ◽  
Content Type ◽  
Color Changes ◽  
Structural Colors ◽  
Direct Illumination ◽  
Higher Eukaryotes

ABSTRACTIridescence is a property of structural color that is occasionally encountered in higher eukaryotes but that has been poorly documented in the prokaryotic kingdom. In the present work, we describe a marine bacterium, identified asCellulophaga lytica, isolated from the surface of an anemone, that exhibits bright green iridescent colonies under direct epi-illumination. This phenomenon has not previously been investigated in detail. In this study, color changes ofC. lyticacolonies were observed at various angles of direct illumination or observation. Its iridescent green appearance was dominant on various growth media. Red and violet colors were also discerned on colony edges. RemarkableC. lyticabacterial iridescence was revealed and characterized using high-resolution optical spectrometry. In addition to this, by culturing other bacterial strains to which various forms of faintly iridescent traits have previously been attributed, we identify four principal appearance characteristics of structural color in prokaryotes. A new general classification of bacterial iridescence is therefore proposed in this study. Furthermore, a specific separate class is described for iridescentC. lyticastrains because they exhibit what is so far a unique intense glitter-like iridescence in reflection.C. lyticais the first prokaryote discovered to produce the same sort of intense iridescence under direct illumination as that associated with higher eukaryotes, like some insects and birds. Due to the nature of bacterial biology, cultivation, and ubiquity, this discovery may be of significant interest for both ecological and nanoscience endeavors.

scholarly journals Tracking the Taxonomy of the GenusBifidobacteriumBased on a Phylogenomic Approach

2017 ◽  
Vol 84 (4) ◽  
Author(s):  
Gabriele Andrea Lugli ◽  
Christian Milani ◽  
Sabrina Duranti ◽  
Leonardo Mancabelli ◽  
Marta Mangifesta ◽  
...  
Keyword(s):  
New Technologies ◽  
Bacterial Species ◽  
Bacterial Genome ◽  
Bacterial Taxonomy ◽  
Genome Sequences ◽  
Content Type ◽  
Complete Bacterial Genome ◽  
Novel Taxa

ABSTRACTFor decades, bacterial taxonomy has been based onin vitromolecular biology techniques and comparison of molecular marker sequences to measure the degree of genetic similarity and deduce phylogenetic relatedness of novel bacterial species to reference microbial taxa. Due to the advent of the genomic era, access to complete bacterial genome contents has become easier, thereby presenting the opportunity to precisely investigate the overall genetic diversity of microorganisms. Here, we describe a high-accuracy phylogenomic approach to assess the taxonomy of members of the genusBifidobacteriumand identify apparent misclassifications in current bifidobacterial taxonomy. The developed method was validated by the classification of seven novel taxa belonging to the genusBifidobacteriumby employing their overall genetic content. The results of this study demonstrate the potential of this whole-genome approach to become the gold standard for phylogenomics-based taxonomic classification of bacteria.IMPORTANCENowadays, next-generation sequencing has given access to genome sequences of the currently known bacterial taxa. The public databases constructed by means of these new technologies allowed comparison of genome sequences between microorganisms, providing information to perform genomic, phylogenomic, and evolutionary analyses. In order to avoid misclassifications in the taxonomy of novel bacterial isolates, new (bifido)bacterial taxons should be validated with a phylogenomic assessment like the approach presented here.

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