Phage Display-Derived Monoclonal Antibodies Against Internalins A and B Allow Specific Detection of Listeria monocytogenes.

Listeria monocytogenes is the causative agent of listeriosis, a highly lethal disease initiated after the ingestion of Listeria-contaminated food. This species comprises different serovars, from which 4b, 1/2a, and 1/2b cause most of the infections. Among the different proteins involved in pathogenesis, the internalins A (InlA) and B (InlB) are the best characterized, since they play a major role in the enterocyte entry of Listeria cells during early infection. Due to their covalent attachment to the cell wall and location on the bacterial surface, along with their exclusive presence in the pathogenic L. monocytogenes, these proteins are also used as detection targets for this species. Even though huge advancements were achieved in the enrichment steps for subsequent Listeria detection, few studies have focused on the improvement of the antibodies for immunodetection. In the present study, recombinant InlA and InlB produced in Escherichia coli were used as targets to generate antibodies via phage display using the human naïve antibody libraries HAL9 and HAL10. A set of five recombinant antibodies (four against InlA, and one against InlB) were produced in scFv-Fc format and tested in indirect ELISA against a panel of 19 Listeria strains (17 species; including the three main serovars of L. monocytogenes) and 16 non-Listeria species. All five antibodies were able to recognize L. monocytogenes with 100% sensitivity (CI 29.24-100.0) and specificity (CI 88.78-100.0) in all three analyzed antibody concentrations. These findings show that phage display-derived antibodies can improve the biological tools to develop better immunodiagnostics for L. monocytogenes.

Pyruvate dehydrogenase complex-enzyme 2, a new target for Listeria spp. detection identified using combined phage display technologies.

The genus Listeria comprises ubiquitous bacteria, commonly present in foods and food production facilities. In this study, three different phage display technologies were employed to discover targets, and to generate and characterize novel antibodies against Listeria: antibody display for biomarker discovery and antibody generation; ORFeome display for target identification; and single-gene display for epitope characterization. With this approach, pyruvate dehydrogenase complex-enzyme 2 (PDC-E2) was defined as a new detection target for Listeria, as confirmed by immunomagnetic separation-mass spectrometry (IMS-MS). Immunoblot and fluorescence microscopy showed that this protein is accessible on the bacterial cell surface of living cells. Recombinant PDC-E2 was produced in E. coli and used to generate 16 additional antibodies. The resulting set of 20 monoclonal scFv-Fc was tested in indirect ELISA against 17 Listeria and 16 non-Listeria species. Two of them provided 100% sensitivity (CI 82.35-100.0%) and specificity (CI 78.20-100.0%), confirming PDC-E2 as a suitable target for the detection of Listeria. The binding region of 18 of these antibodies was analyzed, revealing that ≈ 90% (16/18) bind to the lipoyl domains (LD) of the target. The novel target PDC-E2 and highly specific antibodies against it offer new opportunities to improve the detection of Listeria.

Analysis of 1,000+ Type-Strain Genomes Substantially Improves Taxonomic Classification of Alphaproteobacteria.

The class Alphaproteobacteria is comprised of a diverse assemblage of Gram-negative bacteria that includes organisms of varying morphologies, physiologies and habitat preferences many of which are of clinical and ecological importance. Alphaproteobacteria classification has proved to be difficult, not least when taxonomic decisions rested heavily on a limited number of phenotypic features and interpretation of poorly resolved 16S rRNA gene trees. Despite progress in recent years regarding the classification of bacteria assigned to the class, there remains a need to further clarify taxonomic relationships. Here, draft genome sequences of a collection of genomes of more than 1000 Alphaproteobacteria and outgroup type strains were used to infer phylogenetic trees from genome-scale data using the principles drawn from phylogenetic systematics. The majority of taxa were found to be monophyletic but several orders, families and genera, including taxa recognized as problematic long ago but also quite recent taxa, as well as a few species were shown to be in need of revision. According proposals are made for the recognition of new orders, families and genera, as well as the transfer of a variety of species to other genera and of a variety of genera to other families. In addition, emended descriptions are given for many species mainly involving information on DNA G+C content and (approximate) genome size, both of which are confirmed as valuable taxonomic markers. Similarly, analysis of the gene content was shown to provide valuable taxonomic insights in the class. Significant incongruities between 16S rRNA gene and whole genome trees were not found in the class. The incongruities that became obvious when comparing the results of the present study with existing classifications appeared to be caused mainly by insufficiently resolved 16S rRNA gene trees or incomplete taxon sampling. Another probable cause of misclassifications in the past is the partially low overall fit of phenotypic characters to the sequence-based tree. Even though a significant degree of phylogenetic conservation was detected in all characters investigated, the overall fit to the tree varied considerably.

Influence of disulfide bonds in human beta defensin-3 on its strain specific activity against Gram-negative bacteria.

Antimicrobial peptides (AMPs) play an important role in the host defense against various microbes. One of the most efficient human AMPs is the human beta defensin-3 (hBD-3) which is produced by, e.g. keratinocytes and lung epithelial cells. However, the structure-function relationship for AMPs and in particular for defensins with their typical three disulfide bonds is still poorly understood. In this study the importance of the three disulfide bonds for the activity of the AMPs is investigated with biological assays and with biophysical experiments utilizing different membrane reconstitution systems. The activities of natural hBD-3, hBD-3-c (cyclic variant with one disulfide bond), and hBD-3-l (linear variant without disulfide bonds) and fragments thereof were tested against specific Gram-negative bacteria. Furthermore, hemolytic and cytotoxic activities were analyzed as well as the potency to neutralize immune cell stimulation of lipopolysaccharide (LPS). Experiments using reconstituted lipid matrices composed of phospholipids or LPS purified from the respective Gram-negative bacteria, showed that the membrane activity of all three hBD-3 peptides is decisive for their capability to kill bacteria and to neutralize LPS. In most of the test systems the linear hBD-3-l showed the highest activity. It was also the only peptide significantly active against polymyxin B-resistant Proteus mirabilis R45. However, the stability of hBD-3 against protease activity decreases with decreasing number of disulfide bonds. This study demonstrates that the refining of AMP structures can generate more active compounds against certain strains.

Corrigendum: A Metataxonomic Tool to Investigate the Diversity of Treponema.

[This corrects the article DOI: 10.3389/fmicb.2019.02094.].

Analysis of 1,000 Type-Strain Genomes Improves Taxonomic Classification of Bacteroidetes.

Although considerable progress has been made in recent years regarding the classification of bacteria assigned to the phylum Bacteroidetes, there remains a need to further clarify taxonomic relationships within a diverse assemblage that includes organisms of clinical, piscicultural, and ecological importance. Bacteroidetes classification has proved to be difficult, not least when taxonomic decisions rested heavily on interpretation of poorly resolved 16S rRNA gene trees and a limited number of phenotypic features. Here, draft genome sequences of a greatly enlarged collection of genomes of more than 1,000 Bacteroidetes and outgroup type strains were used to infer phylogenetic trees from genome-scale data using the principles drawn from phylogenetic systematics. The majority of taxa were found to be monophyletic but several orders, families and genera, including taxa proposed long ago such as Bacteroides, Cytophaga, and Flavobacterium but also quite recent taxa, as well as a few species were shown to be in need of revision. According proposals are made for the recognition of new orders, families and genera, as well as the transfer of a variety of species to other genera. In addition, emended descriptions are given for many species mainly involving information on DNA G+C content and (approximate) genome size, both of which can be considered valuable taxonomic markers. We detected many incongruities when comparing the results of the present study with existing classifications, which appear to be caused by insufficiently resolved 16S rRNA gene trees or incomplete taxon sampling. The few significant incongruities found between 16S rRNA gene and whole genome trees underline the pitfalls inherent in phylogenies based upon single gene sequences and the impediment in using ordinary bootstrapping in phylogenomic studies, particularly when combined with too narrow gene selections. While a significant degree of phylogenetic conservation was detected in all phenotypic characters investigated, the overall fit to the tree varied considerably, which is one of the probable causes of misclassifications in the past, much like the use of plesiomorphic character states as diagnostic features.

A Metataxonomic Tool to Investigate the Diversity of Treponema.

The genus Treponema contains a number of human and animal pathogenic as well as symbiotic bacteria that are found in vastly different anatomical and environmental habitats. Our understanding of the species range, evolution, and biology of these important bacteria is still limited. To explore the diversity of treponemes, we established, validated, and tested a novel metataxonomic approach. As the informative nature of the hypervariable regions of the 16S rRNA gene differ, we first analyzed each variable region independently. Considering the in silico results obtained, we established and validated the sequencing of the V4-region of the 16S rRNA gene using known mixtures of Treponema species as well as a selected number of clinical samples. The metataxonomic approach was able to identify Treponema to a near-species level. We demonstrate that using a spirochete-specific enrichment, our method is applicable to complex microbial communities and large variety of biological samples. The metataxonomic approach described provides a useful method to unravel the full diversity and range of Treponema in various ecosystems.

Sneathia vaginalis sp. nov. (Fusobacteriales, Leptotrichiaceae) as a replacement of the species 'Sneathia amnii' Harwich et al. 2012 and 'Leptotrichia amnionii' Shukla et al. 2002, and emended description of Sneathia Collins et al. 2001.

Members of the genus Sneathia are fastidious bacteria that predominantly colonise the female genital tract and are significantly associated with reproductive disorders and genital and neonatal disease. From a taxonomical perspective, the genus only comprises the species Sneathia sanguinegens. Numerous reports on a second species, 'Sneathia amnii', have been published, but the name has never been validated. The same is the case for 'Leptotrichia amnionii', which was previously shown to belong to the same species as 'Sneathia amnii'. We studied strains DSM 16631T and DSM 16630, which have been identified and deposited as 'Leptotrichia amnionii' previously. At the time of isolation, these strains were found to be most closely related to, but clearly different from, Sneathia sanguinegens based on 16S rRNA gene sequence similarities. Both strains proved to be almost indistinguishable from 'Sneathia amnii' based on molecular, morphological and physiological traits. The 16S rRNA gene sequence analysis revealed that strain DSM 16631T was assigned to the genus Sneathia with a sequence similarity of 95.47 % to Sneathia sanguinegens CCUG 41628T, followed by type strains of Caviibacter abscessus (93.03 %), Oceanivirga salmonicida (92.68 %) and Oceanivirga miroungae (91.97 %) as the next closely related members of the Leptotrichiaceae. The novel species was also clearly differentiated from other related taxa by core genome phylogeny, average nucleotide and amino acid identities, in silico DNA-DNA hybridization and MALDI-TOF MS. With respect to chemotaxonomic and physiological patterns, strains DSM 16631T and DSM 16630 were again highly similar to Sneathia sanguinegens. On the basis of these data, we propose the novel species Sneathia vaginalis sp. nov. with the type strain DSM 16631T (=CCUG 52977T=CCUG 52889AT) and a second strain DSM 16630 (=CCUG 52976=CCUG 52888) that were both isolated from bloodstream infections in women with puerperal fever in France. The G+C content of the DNA of the type strain is 28.4 mol% and the genome size is 1.28 Mbp. Based on the observed extremely high similarities of genotypic and phenotypic traits of the novel proposed species to those reported for 'Sneathia amnii', we recommend using this new name in all further publications on this taxon.

Rhizobium tumorigenes sp. nov., a novel plant tumorigenic bacterium isolated from cane gall tumors on thornless blackberry.

Four plant tumorigenic strains 932, 1019, 1078T and 1081 isolated from cane gall tumors on thornless blackberry (Rubus sp.) were characterized. They shared low sequence identity with related Rhizobium spp. based on comparisons of 16S rRNA gene (≤98%) and housekeeping genes atpD, recA and rpoB (<90%). Phylogenetic analysis indicated that the strains studied represent a novel species within the genus Rhizobium, with Rhizobium tubonense CCBAU 85046T as their closest relative. Furthermore, obtained average nucleotide identity (ANI) and in silico DNA-DNA hybridization (DDH) values calculated for whole-genome sequences of strain 1078T and related Rhizobium spp. confirmed the authenticity of the novel species. The ANI-Blast (ANIb), ANI-MUMmer (ANIm) and in silico DDH values between strain 1078T and most closely related R. tubonense CCBAU 85046T were 76.17%, 84.11% and 21.3%, respectively. The novel species can be distinguished from R. tubonense based on phenotypic and chemotaxonomic properties. Here, we demonstrated that four strains studied represent a novel species of the genus Rhizobium, for which the name Rhizobium tumorigenes sp. nov. is proposed (type strain 1078T = DSM 104880T = CFBP 8567T). R. tumorigenes is a new plant tumorigenic species carrying the tumor-inducing (Ti) plasmid.

Manual curation and reannotation of the genomes of Clostridium difficile 630Δerm and C. difficile 630.

PURPOSE: We resequenced the genome of Clostridium difficile 630Δerm (DSM 28645), a model strain commonly used for the generation of insertion mutants. METHODOLOGY: The genome sequence was obtained by a combination of single-molecule real-timeand Illumina sequencing technology. RESULTS: Detailed manual curation and comparison to the previously published genomic sequence revealed sequence differences including inverted regions and the presence of plasmid pCD630. Manual curation of our previously deposited genome sequence of the parental strain 630 (DSM 27543) led to an improved genome sequence. In addition, the sequence of the transposon Tn5397 was completely identified. We manually revised the current manual annotation of the initial sequence of strain 630 and modified either gene names, gene product names or assigned EC numbers of 57 % of genes. The number of hypothetical and conserved hypothetical proteins was reduced by 152. This annotation was used as a template to annotate the most recent genome sequences of the strains 630Δerm and 630. CONCLUSION: Based on the genomic analysis, several new metabolic features of C. difficile are proposed and could be supported by literature and subsequent experiments.

A Clostridioides difficile bacteriophage genome encodes functional binary toxin-associated genes.

Pathogenic clostridia typically produce toxins as virulence factors which cause severe diseases in both humans and animals. Whereas many clostridia like e.g., Clostridium perfringens, Clostridium botulinum or Clostridium tetani were shown to contain toxin-encoding plasmids, only toxin genes located on the chromosome were detected in Clostridioides difficile so far. In this study, we determined, annotated, and analyzed the complete genome of the bacteriophage phiSemix9P1 using single-molecule real-time sequencing technology (SMRT). To our knowledge, this represents the first C. difficile-associated bacteriophage genome that carries a complete functional binary toxin locus in its genome.

Harnessing the landscape of microbial culture media to predict new organism-media pairings.

Culturing microorganisms is a critical step in understanding and utilizing microbial life. Here we map the landscape of existing culture media by extracting natural-language media recipes into a Known Media Database (KOMODO), which includes >18,000 strain-media combinations, >3300 media variants and compound concentrations (the entire collection of the Leibniz Institute DSMZ repository). Using KOMODO, we show that although media are usually tuned for individual strains using biologically common salts, trace metals and vitamins/cofactors are the most differentiating components between defined media of strains within a genus. We leverage KOMODO to predict new organism-media pairings using a transitivity property (74% growth in new in vitro experiments) and a phylogeny-based collaborative filtering tool (83% growth in new in vitro experiments and stronger growth on predicted well-scored versus poorly scored media). These resources are integrated into a web-based platform that predicts media given an organism's 16S rDNA sequence, facilitating future cultivation efforts.

Complete Genome Sequence of the Clostridium difficile Type Strain DSM 1296T.

In this study, we sequenced the complete genome of the Clostridium difficile type strain DSM 1296(T). A combination of single-molecule real-time (SMRT) and Illumina sequencing technology revealed the presence of one chromosome and two extrachromosomal elements, the bacteriophage phiCDIF1296T and a putative plasmid-like structure harboring genes of another bacteriophage.

Spirochaeta sinaica sp. nov., a halophilic spirochaete isolated from a cyanobacterial mat.

A strain of free-living obligately anaerobic, halophilic spirochaete, SLT, was isolated from a sample of a cyanobacterial mat of the hypersaline Solar Lake, Sinai shore. The strain had motile helical cells, 0.35-0.40 × 6-10 µm. Strain SLT exhibited high resistance to NaCl among known halophilic spirochaetes growing at NaCl concentrations from 2 to 12% (optimum growth at 7%). The strain grew at temperatures from 10 to 32 °C (optimum at 28 °C) and pH from 6 to 8.5 (optimum at pH 7.0-7.5). Carbohydrates, but not alcohols, organic acids or nitrogenous compounds (peptone, yeast extract and amino acids), were used as energy substrates for growth. Ethanol, acetate, lactate, H2 and CO2 were the products of glucose fermentation. Sulfide was produced in the presence of S0 or thiosulfate in the medium. The DNA G+C content was 44.7 mol%. Based on 16S rRNA gene sequence analysis, strain SLT clustered within the genus Spirochaeta, exhibiting 94.2 and 93.7% similarity with its closest relatives, Spirochaeta bajacaliforniensis DSM 160554T and Spirochaeta smaragdinae DSM 11293T, respectively; similarity with other species did not exceed 86%. The phenotypic and chemotaxonomic characteristics of the strain, as well as the results of phylogenetic analysis support the classification of strain SLT as representing a novel species of the genus Spirochaeta, for which the name Spirochaeta sinaica sp. nov. is proposed. The type strain is SLT ( = DSM 14994 = NIQEM U 783).

Complete Genome Sequence of the Novel Temperate Clostridium difficile Phage phiCDIF1296T.

Clostridium difficile contains many integrated and extrachromosomal genetic elements. In this study, we determined, annotated, and analyzed the complete genome of the C. difficile bacteriophage phiCDIF1296T using single-molecule real-time sequencing technology. To our knowledge, this represents the largest genome (131 kb) of a temperate C. difficile phage recognized so far.

Genome Resequencing of the Virulent and Multidrug-Resistant Reference Strain Clostridium difficile 630.

We resequenced the complete genome of the virulent and multidrug-resistant pathogen Clostridium difficile strain 630. A combination of single-molecule real-time and Illumina sequencing technology revealed the presence of an additional rRNA gene cluster, additional tRNAs, and the absence of a transposon in comparison to the published and reannotated genome sequence.

Genome sequence of the phylogenetically isolated spirochete Leptonema illini type strain (3055(T)).

Leptonema illini Hovind-Hougen 1979 is the type species of the genus Leptonema, family Leptospiraceae, phylum Spirochaetes. Organisms of this family have a Gram-negative-like cell envelope consisting of a cytoplasmic membrane and an outer membrane. The peptidoglycan layer is associated with the cytoplasmic rather than the outer membrane. The two flagella of members of Leptospiraceae extend from the cytoplasmic membrane at the ends of the bacteria into the periplasmic space and are necessary for their motility. Here we describe the features of the L. illini type strain, together with the complete genome sequence, and annotation. This is the first genome sequence (finished at the level of Improved High Quality Draft) to be reported from of a member of the genus Leptonema and a representative of the third genus of the family Leptospiraceae for which complete or draft genome sequences are now available. The three scaffolds of the 4,522,760 bp draft genome sequence reported here, and its 4,230 protein-coding and 47 RNA genes are part of the G enomic E ncyclopedia of Bacteria and Archaea project.

Genome sequence of the free-living aerobic spirochete Turneriella parva type strain (H(T)), and emendation of the species Turneriella parva.

Turneriella parva Levett et al. 2005 is the only species of the genus Turneriella which was established as a result of the reclassification of Leptospira parva Hovind-Hougen et al. 1982. Together with Leptonema and Leptospira, Turneriella constitutes the family Leptospiraceae, within the order Spirochaetales. Here we describe the features of this free-living aerobic spirochete together with the complete genome sequence and annotation. This is the first complete genome sequence of a member of the genus Turneriella and the 13(th) member of the family Leptospiraceae for which a complete or draft genome sequence is now available. The 4,409,302 bp long genome with its 4,169 protein-coding and 45 RNA genes is part of the G enomic E ncyclopedia of Bacteria and Archaea project.

Complete genome sequence of the bile-resistant pigment-producing anaerobe Alistipes finegoldii type strain (AHN2437(T)).

Alistipes finegoldii Rautio et al. 2003 is one of five species of Alistipes with a validly published name: family Rikenellaceae, order Bacteroidetes, class Bacteroidia, phylum Bacteroidetes. This rod-shaped and strictly anaerobic organism has been isolated mostly from human tissues. Here we describe the features of the type strain of this species, together with the complete genome sequence, and annotation. A. finegoldii is the first member of the genus Alistipes for which the complete genome sequence of its type strain is now available. The 3,734,239 bp long single replicon genome with its 3,302 protein-coding and 68 RNA genes is part of the G enomic E ncyclopedia of Bacteria and Archaea project.

Genome sequence of the thermophilic fresh-water bacterium Spirochaeta caldaria type strain (H1(T)), reclassification of Spirochaeta caldaria, Spirochaeta stenostrepta, and Spirochaeta zuelzerae in the genus Treponema as Treponema caldaria comb. nov., Treponema stenostrepta comb. nov., and Treponema zuelzerae comb. nov., and emendation of the genus Treponema.

Spirochaeta caldaria Pohlschroeder et al. 1995 is an obligately anaerobic, spiral-shaped bacterium that is motile via periplasmic flagella. The type strain, H1(T), was isolated in 1990 from cyanobacterial mat samples collected at a freshwater hot spring in Oregon, USA, and is of interest because it enhances the degradation of cellulose when grown in co-culture with Clostridium thermocellum. Here we provide a taxonomic re-evaluation for S. caldaria based on phylogenetic analyses of 16S rRNA sequences and whole genomes, and propose the reclassification of S. caldaria and two other Spirochaeta species as members of the emended genus Treponema. Whereas genera such as Borrelia and Sphaerochaeta possess well-distinguished genomic features related to their divergent lifestyles, the physiological and functional genomic characteristics of Spirochaeta and Treponema appear to be intermixed and are of little taxonomic value. The 3,239,340 bp long genome of strain H1(T) with its 2,869 protein-coding and 59 RNA genes is a part of the G enomic E ncyclopedia of Bacteria and Archaea project.