Genome-Based Taxonomy of Species in the Pseudomonas syringae and Pseudomonas lutea Phylogenetic Groups and Proposal of Pseudomonas maioricensis sp. nov., Isolated from Agricultural Soil.

Species in the phylogenetic group Pseudomonas syringae are considered the most relevant plant pathogenic bacteria, but their taxonomy is still controversial. Twenty named species are validated in the current taxonomy of this group and in recent years many strains have been genome-sequenced, putative new species have been proposed and an update in the taxonomy is needed. A taxonomic study based on the core-genome phylogeny, genomic indices (ANI and GGDC) and gene content (phyletic pattern and Jaccard index) have been applied to clarify the taxonomy of the group. A phylogenomic analysis demonstrates that at least 50 phylogenomic species can be delineated within the group and that many strains whose genomes have been deposited in the databases are not correctly classified at the species level. Other species names, like "Pseudomonas coronafaciens", have been proposed but are not validated yet. One of the putative new species is taxonomically described, and the name Pseudomonas maioricensis sp. nov. is proposed. The taxonomies of Pseudomonas avellanae and Pseudomonas viridiflava are discussed in detail as case studies. Correct strain identification is a prerequisite for many studies, and therefore, criteria are given to facilitate identification.

Stutzerimonas decontaminans sp. nov. isolated from marine polluted sediments.

Strains 19SMN4T and ST27MN3 were isolated from marine sediments after enrichment with 2-methylnaphthalene and were classified as Pseudomonas stutzeri genomovar 4. Four other strains, BG 2, HT20, HT24, and A7, were isolated from sulphide-oxidizing bioreactors or activated sludge affiliated with the same clade in the 16S rRNA phylogenetic tree. P. stutzeri has been recently reclassified as a new genus, Stutzerimonas, and a preliminary analysis indicated that the strains in this study were distinct from any classified Stutzerimonas and are considered representatives of phylogenomic species 4 (pgs4). Strains 19SMN4T and ST27MN3 were extensively characterized with phenotypic, chemotaxonomic, genomic and phylogenomic data. Strain 19SMN4T had a well-characterized naphthalene degradative plasmid that has been compared with other plasmids, while in strain ST27MN3, the naphthalene degradative genes were detected in the chromosome sequence. Phylogenomic analysis of the core gene sequences showed that strains 19SMN4T and ST27MN3 shared 3,995 genes and were closely related to members of the species "Stutzerimonas songnenensis" and Stutzerimonas perfectomarina, as well as to the Stutzerimonas phylogenomic species, pgs9, pgs16 and pgs24. The aggregate average nucleotide identity (ANI) indicated that strains 19SMN4T and ST27MN3 belonged to the same genomic species, whereas the genomic indices with their closest-related type strains were below the accepted species threshold (95 %). We therefore conclude that strains 19SMN4T and ST27MN3 represent a novel species of Stutzerimonas, for which the name Stutzerimonas decontaminans is proposed; the type strain is 19SMN4T (=CCUG44593T = DSM6084T = LMG18521T).

Genome-Based Taxonomy of the Genus Stutzerimonas and Proposal of S. frequens sp. nov. and S. degradans sp. nov. and Emended Descriptions of S. perfectomarina and S. chloritidismutans.

Stutzerimonas is a recently proposed genus within the Pseudomonadaceae comprising strains in the formerly phylogenetic group of Pseudomonas stutzeri. At least sixteen named species have to be included in the genus, together with 22 genomovars of Stutzerimonas stutzeri. To clarify the taxonomy of Stutzerimonas, a core-genome phylogeny of 200 strains in the genus was inferred and monophyletic strains with average nucleotide identities (ANIb) with values equal to or higher than 95 were grouped in the same phylogenomic species. A total of 45 phylogenomic species within the genus Stutzerimonas were detected in the present study. Sixteen phylogenomic species correspond to already named species, although three of them are not yet validated and two are proposed in the present study. A synonymy was detected between P. kunmingensis and S. chloritidismutans, both members of phylogenomic species 3, with a prevalence of the S. chloritidismutans name. The correspondence of the phylogenomic species to the genome taxonomy database classification (GTDB taxonomy) is discussed. Combining phylogenomic and phenotypic data, two novel species are described (Stutzerimonas frequens and Stutzerimonas degradans) and two species descriptions are emended (Stutzerimonas perfectomarina and Stutzerimonas chloritidismutans).

Common and distinctive genomic features of Klebsiella pneumoniae thriving in the natural environment or in clinical settings.

The Klebsiella pneumoniae complex is comprised of ubiquitous bacteria that can be found in soils, plants or water, and as humans' opportunistic pathogens. This study aimed at inferring common and distinctive features in clinical and environmental K. pneumoniae. Whole genome sequences of members of the K. pneumoniae complex (including K. variicola, n = 6; and K. quasipneumoniae, n = 7), of clinical (n = 78) and environmental (n = 61) origin from 21 countries were accessed from the GenBank. These genomes were compared based on phylogeny, pangenome and selected clinically relevant traits. Phylogenetic analysis based on 2704 genes of the core genome showed close relatedness between clinical and environmental strains, in agreement with the multi-locus sequence typing. Eight out of the 62 sequence types (STs) identified, included both clinical and environmental genomes (ST11, ST14, ST15, ST37, ST45, ST147, ST348, ST437). Pangenome-wide association studies did not evidence significant differences between clinical and environmental genomes. However, the genomes of clinical isolates presented significantly more exclusive genes related to antibiotic resistance/plasmids, while the environmental isolates yielded significantly higher allelic diversity of genes related with functions such as efflux or oxidative stress. The study suggests that K. pneumoniae can circulate among the natural environment and clinical settings, probably under distinct adaptation pressures.

Third generation cephalosporin-resistant Klebsiella pneumoniae thriving in patients and in wastewater: what do they have in common?

BACKGROUND: Klebsiella pneumoniae are ubiquitous bacteria and recognized multidrug-resistant opportunistic pathogens that can be released into the environment, mainly through sewage, where they can survive even after wastewater treatment. A major question is if once released into wastewater, the selection of lineages missing clinically-relevant traits may occur. Wastewater (n = 25) and clinical (n = 34) 3rd generation cephalosporin-resistant K. pneumoniae isolates were compared based on phenotypic, genotypic and genomic analyses. RESULTS: Clinical and wastewater isolates were indistinguishable based on phenotypic and genotypic characterization. The analysis of whole genome sequences of 22 isolates showed that antibiotic and metal resistance or virulence genes, were associated with mobile genetic elements, mostly transposons, insertion sequences or integrative and conjugative elements. These features were variable among isolates, according to the respective genetic lineage rather than the origin. CONCLUSIONS: It is suggested that once acquired, clinically relevant features of K. pneumoniae may be preserved in wastewater, even after treatment. This evidence highlights the high capacity of K. pneumoniae for spreading through wastewater, enhancing the risks of transmission back to humans.

Past, present and future of the boundaries of the Pseudomonas genus: Proposal of Stutzerimonas gen. Nov.

Pseudomonas is one the best studied bacterial genera, and it is the genus with the highest number of species among the gram-negative bacteria. Pseudomonas spp. are widely distributed and play relevant ecological roles; several species are commensal or pathogenic to humans, animals and plants. The main aim of the present minireview is the discussion of how the Pseudomonas taxonomy has evolved with the development of bacterial taxonomy since the first description of the genus in 1894. We discuss how the successive implementation of novel methodologies has influenced the taxonomy of the genus and, vice versa, how the taxonomic studies developed in Pseudomonas have introduced novel tools and concepts to bacterial taxonomy. Current phylogenomic analyses of the family Pseudomonadaceae demonstrate that a considerable number of named Pseudomonas spp. are not monophyletic with P. aeruginosa, the type species of the genus, and that a reorganization of several genera can be foreseen. Phylogenomics of Pseudomonas, Azomonas and Azotobacter within the Pseudomonadaceae is presented as a case study. Five new genus names are delineated to accommodate five well-defined phylogenetic branches that are supported by the shared genes in each group, and two of them can be differentiated by physiological and ecological properties: the recently described genus Halopseudomonas and the genus Stutzerimonas proposed in the present study. Five former Pseudomonas species are transferred to Halopseudomonas and 10 species to Stutzerimonas.

Pseudomonas arcuscaelestis sp. nov., isolated from rainbow trout and water.

Cells of strains P66T, V1 and W15Feb18 are Gram-stain-negative short rods and motile by one polar flagellum. Strain P66T was isolated from rainbow trout (Oncorhynchus mykiss) cultivated at a fish farm in Turkey. Strain V1 was isolated from sand of an intertidal shore on the Galicia coast in Spain and strain W15Feb18 was isolated from water collected at the Woluwe River in Belgium. Based on 16S rRNA sequence similarity values, the strains were grouped under the genus Pseudomonas and the Pseudomonas putida phylogenetic group of species. The DNA G+C content ranged from 58.5 to 58.9 mol%. The strains were characterized phenotypically by the API 20NE and Biolog GEN III tests, and chemotaxonomically by their whole-cell MALDI-TOF MS protein profiles and fatty acid contents. The absence of the hydrolysis of gelatin and the assimilation of arabinose, mannose and mannitol differentiated these strains from the closest species, Pseudomonas alkylphenolica. The major fatty acid components were C16:0 (29.91-31.68 %) and summed feature 3 (36.44-37.55 %). Multilocus sequence analysis with four and 83 housekeeping gene sequences and a core proteome analysis showed that these strains formed a phylogenetic cluster in the P. putida group of species. Genome comparisons by the average nucleotide identity based on blast and the Genome-to-Genome Distance Calculator demonstrated that the three strains belonged to the same genomic species and were distant from any known species, with similarity values lower than the thresholds established for species in the genus Pseudomonas. These data permitted us to conclude that strains P66T, V1 and W15Feb18 belong to a novel species in the genus Pseudomonas, for which the name Pseudomonas arcuscaelestis sp. nov. is proposed. The type strain is P66T (=CECT 30176T=CCUG 74872T). The other strains have been deposited in the CECT with the corresponding collection numbers: V1 (=CECT 30356) and W15Feb18 (=CECT 30355).

Pseudomonas lalucatii sp. nov. isolated from Vallgornera, a karstic cave in Mallorca, Western Mediterranean.

Caves are extreme underground environments colonized by oligotrophic bacterial communities that influence mineral transformations. The identification at the species level is important and this study aims to the taxonomic characterisation of four bacterial strains previously isolated from rock surfaces and water samples from a karstic cave located on Mallorca (Spain) that were assigned to the genus Pseudomonas according to 16S rRNA nucleotide sequence analysis. Sequence analysis of the RNA polymerase sigma factor gene (rpoD) allocated these strains to the P. fluorescens lineage within the P. anguilliseptica phylogenetic group, close to the P. benzenivorans type strain. A polyphasic taxonomic approach included phenotypic characterization, fatty acid composition analysis, and whole-cell protein profiling, together with phylogenomic data. The results supported the proposal of a new species in the Pseudomonas genus. Characteristic fatty acid methyl esters of members of the Pseudomonas genus were present (C16:0, C10:0 3-OH, C12:0 2-OH and C12:0 3-OH) and the C12:1 3OH content differentiated these strains from P. benzenivorans. The genomic G + C mol% content of the four sequenced genomes was 66.9%. The average nucleotide indices based on BLAST analysis and the calculation of genome-to-genome distance with respect to their closest relative were lower than 88% and 30%, respectively. These data confirm that the four isolates, R1b-4, R1b-52A, A2bC-1 and R1b-54T, represent a new species, for which the name Pseudomonas lalucatii is proposed, with strain R1b-54T as the type strain (=CCUG 74754T = CECT 30179T). This is the first species in the P. anguilliseptica group isolated from this extreme habitat.

Pseudomonas anatoliensis sp. nov and Pseudomonas iridis sp. nov. isolated from fish.

In a study carried out between 2013 and 2018 in fish farms in Turkey, several putative novel species were isolated. The 16S rRNA nucleotide sequences of fourteen strains of Gram-negative rods, which were isolated from asymptomatic and symptomatic rainbow trouts (Onchorhynchus mykiss), placed them under the genus Pseudomonas. The similarity values of the concatenated nucleotide sequences of the rpoD, rpoB, gyrB and 16S rRNA genes clustered these isolates into the P. fluorescens phylogenetic group of species and into the Pseudomonas koreensis subgroup, close to Pseudomonas helmanticensis and Pseudomonas baetica. An isolate of a totally different origin, strain CCUG 67011, clustered with these isolates. Phenotypic characterization, together with the chemotaxonomic data, whole-cell MALDI-TOF MS and fatty acids methyl esters analyses were performed. The DNA G + C content was 58.7 mol% for isolate P9T and 58.8 mol% for isolate P42T. The phylogenomic analysis and whole genome nucleotide sequences of four of these isolates confirmed that the isolates P9T, P25 and P141, represent a novel species for which the name Pseudomonas anatoliensis sp. nov. is proposed, with P9T as the type strain (=CCUG 74755T = CECT 3172T). The isolates P1, P2, P10, P27, P30, P24a, P42T, P117, P139, P152 and CCUG 67011 represent another novel sècies, for which the name Pseudomonas iridis sp. nov. is proposed, with P42T as the type strain (=CCUG 74870T = CECT 3174T).

Phylogenetic inference enables reconstruction of a long-overlooked outbreak of almond leaf scorch disease (Xylella fastidiosa) in Europe.

The recent introductions of the bacterium Xylella fastidiosa (Xf) into Europe are linked to the international plant trade. However, both how and when these entries occurred remains poorly understood. Here, we show how almond scorch leaf disease, which affects ~79% of almond trees in Majorca (Spain) and was previously attributed to fungal pathogens, was in fact triggered by the introduction of Xf around 1993 and subsequently spread to grapevines (Pierce's disease). We reconstructed the progression of almond leaf scorch disease by using broad phylogenetic evidence supported by epidemiological data. Bayesian phylogenetic inference predicted that both Xf subspecies found in Majorca, fastidiosa ST1 (95% highest posterior density, HPD: 1990-1997) and multiplex ST81 (95% HPD: 1991-1998), shared their most recent common ancestors with Californian Xf populations associated with almonds and grapevines. Consistent with this chronology, Xf-DNA infections were identified in tree rings dating to 1998. Our findings uncover a previously unknown scenario in Europe and reveal how Pierce's disease reached the continent.

Pseudomonas Species Diversity Along the Danube River Assessed by rpoD Gene Sequence and MALDI-TOF MS Analyses of Cultivated Strains.

A collection of 611 Pseudomonas isolated from 14 sampling sites along the Danube River were identified previously by MALDI-TOF MS with the VITEK MS system and were grouped in 53 clusters by their main protein profiles. The strains were identified in the present study at the phylospecies level by rpoD gene sequencing. Partial sequences of the rpoD gene of 190 isolates representatives of all clusters were analyzed. Strains in the same MALDI-TOF cluster were grouped in the same phylospecies when they shared a minimum 95% similarity in their rpoD sequences. The sequenced strains were assigned to 34 known species (108 strains) and to 32 possible new species (82 strains). The 611 strains were identified at the phylospecies level combining both methods. Most strains were assigned to phylospecies in the Pseudomonas putida phylogenetic group of species. Special attention was given to 14 multidrug resistant strains that could not be assigned to any known Pseudomonas species and were considered environmental reservoir of antibiotic resistance genes. Coverage indices and rarefaction curves demonstrated that at least 50% of the Pseudomonas species in the Danube River able to grow in the isolation conditions have been identified at the species level. Main objectives were the confirmation of the correlation between the protein profile clusters detected by MALDI-TOF MS and the phylogeny of Pseudomonas strains based on the rpoD gene sequence, the assessment of the higher species discriminative power of the rpoD gene sequence, as well as the estimation of the high diversity of Pseudomonas ssp. along the Danube river. This study highlights the Pseudomonas species diversity in freshwater ecosystems and the usefulness of the combination of MALDI-TOF mass spectrometry for the dereplication of large sets of strains and the rpoD gene sequences for rapid and accurate identifications at the species level.

Pseudomonas sivasensis sp. nov. isolated from farm fisheries in Turkey.

A study of 91 isolates from fish farms in Turkey showed that isolates P7T, P11, P24b, P29, P72, P73 and P158 belonged to the genus Pseudomonas according to 16S rRNA nucleotide sequence analysis. The analysis of the sequences of the RNA polymerase sigma factor gene (rpoD) located these strains in the Pseudomonas fluorescens lineage of species within the P. fluorescens subgroup, close to the cluster composed of the species Pseudomonas grimontii, Pseudomonas marginalis and Pseudomonas panacis. Based on similarities in the 16S rRNA and rpoD gene sequences of three previously isolated strains from other origins (CCUG 57209, CCUG 62357 and W5.2-93) linked them to the same cluster. A polyphasic taxonomic approach including phenotypic characterization, fatty acid composition, and multilocus sequence analysis, together with whole-cell MALDI-TOF data, corroborated this assumption. The genome G+C mol% contents were 59.48 and 59.71, respectively. The average nucleotide indices based on BLAST analysis and the genome-to-genome distance calculation for the P7T and CCUG 57209 strains with their closest relative, P. grimontii, were 88.16-88.29% and 38.10-38.20%, respectively. These data confirm that isolates P7T, P11, P24b, P29, P72, P73, P158, CCUG 57209, CCUG 62357 and W5.2-93 represent a new species for which the name Pseudomonas sivasensis is proposed, with P7T as a type strain (=CCUG 74260T= and CECT30107T).

Complete genome sequences of Streptococcus pyogenes type strain reveal 100%-match between PacBio-solo and Illumina-Oxford Nanopore hybrid assemblies.

We present the first complete, closed genome sequences of Streptococcus pyogenes strains NCTC 8198T and CCUG 4207T, the type strain of the type species of the genus Streptococcus and an important human pathogen that causes a wide range of infectious diseases. S. pyogenes NCTC 8198T and CCUG 4207T are derived from deposit of the same strain at two different culture collections. NCTC 8198T was sequenced, using a PacBio platform; the genome sequence was assembled de novo, using HGAP. CCUG 4207T was sequenced and a de novo hybrid assembly was generated, using SPAdes, combining Illumina and Oxford Nanopore sequence reads. Both strategies yielded closed genome sequences of 1,914,862 bp, identical in length and sequence identity. Combining short-read Illumina and long-read Oxford Nanopore sequence data circumvented the expected error rate of the nanopore sequencing technology, producing a genome sequence indistinguishable to the one determined with PacBio. Sequence analyses revealed five prophage regions, a CRISPR-Cas system, numerous virulence factors and no relevant antibiotic resistance genes. These two complete genome sequences of the type strain of S. pyogenes will effectively serve as valuable taxonomic and genomic references for infectious disease diagnostics, as well as references for future studies and applications within the genus Streptococcus.

A Pangenome Approach for Discerning Species-Unique Gene Markers for Identifications of Streptococcus pneumoniae and Streptococcus pseudopneumoniae.

Correct identifications of isolates and strains of the Mitis-Group of the genus Streptococcus are particularly difficult, due to high genetic similarity, resulting from horizontal gene transfer and homologous recombination, and unreliable phenotypic and genotypic biomarkers for differentiating the species. Streptococcus pneumoniae and Streptococcus pseudopneumoniae are the most closely related species of the clade. In this study, publicly-available genome sequences for Streptococcus pneumoniae and S. pseudopneumoniae were analyzed, using a pangenomic approach, to find candidates for species-unique gene markers; ten species-unique genes for S. pneumoniae and nine for S. pseudopneumoniae were identified. These species-unique gene marker candidates were verified by PCR assays for identifying S. pneumoniae and S. pseudopneumoniae strains isolated from clinical samples. All determined species-level unique gene markers for S. pneumoniae were detected in all S. pneumoniae clinical isolates, whereas fewer of the unique S. pseudopneumoniae gene markers were present in more than 95% of the clinical isolates. In parallel, taxonomic identifications of the clinical isolates were confirmed, using conventional optochin sensitivity testing, targeted PCR-detection for the "Xisco" gene, as well as genomic ANIb similarity analyses for the genome sequences of selected strains. Using mass spectrometry-proteomics, species-specific peptide matches were observed for four of the S. pneumoniae gene markers and for three of the S. pseudopneumoniae gene markers. Application of multiple species-level unique biomarkers of S. pneumoniae and S. pseudopneumoniae, is proposed as a protocol for the routine clinical laboratory for improved, reliable differentiation, and identification of these pathogenic and commensal species.

Genomics in Bacterial Taxonomy: Impact on the Genus Pseudomonas.

The introduction of genomics is profoundly changing current bacterial taxonomy. Phylogenomics provides accurate methods for delineating species and allows us to infer the phylogeny of higher taxonomic ranks as well as those at the subspecies level. We present as a model the currently accepted taxonomy of the genus Pseudomonas and how it can be modified when new taxonomic methodologies are applied. A phylogeny of the species in the genus deduced from analyses of gene sequences or by whole genome comparison with different algorithms allows three main conclusions: (i) several named species are synonymous and have to be reorganized in a single genomic species; (ii) many strains assigned to known species have to be proposed as new genomic species within the genus; and (iii) the main phylogenetic groups defined by 4-, 100- and 120-gene multilocus sequence analyses are concordant with the groupings in the whole genome analyses. Moreover, the boundaries of the genus Pseudomonas are also discussed based on phylogenomic analyses in relation to other genera in the family Pseudomonadaceae. The new technologies will result in a substantial increase in the number of species and probably split the current genus into several genera or subgenera, although these classifications have to be supported by a polyphasic taxonomic approach.

Discovery of Species-unique Peptide Biomarkers of Bacterial Pathogens by Tandem Mass Spectrometry-based Proteotyping.

Mass spectrometry (MS) and proteomics offer comprehensive characterization and identification of microorganisms and discovery of protein biomarkers that are applicable for diagnostics of infectious diseases. The use of biomarkers for diagnostics is widely applied in the clinic and the use of peptide biomarkers is increasingly being investigated for applications in the clinical laboratory. Respiratory-tract infections are a predominant cause for medical treatment, although, clinical assessments and standard clinical laboratory protocols are time-consuming and often inadequate for reliable diagnoses. Novel methods, preferably applied directly to clinical samples, excluding cultivation steps, are needed to improve diagnostics of infectious diseases, provide adequate treatment and reduce the use of antibiotics and associated development of antibiotic resistance. This study applied nano-liquid chromatography (LC) coupled with tandem MS, with a bioinformatics pipeline and an in-house database of curated high-quality reference genome sequences to identify species-unique peptides as potential biomarkers for four bacterial pathogens commonly found in respiratory tract infections (RTIs): Staphylococcus aureus; Moraxella catarrhalis; Haemophilus influenzae and Streptococcus pneumoniae The species-unique peptides were initially identified in pure cultures of bacterial reference strains, reflecting the genomic variation in the four species and, furthermore, in clinical respiratory tract samples, without prior cultivation, elucidating proteins expressed in clinical conditions of infection. For each of the four bacterial pathogens, the peptide biomarker candidates most predominantly found in clinical samples, are presented. Data are available via ProteomeXchange with identifier PXD014522. As proof-of-principle, the most promising species-unique peptides were applied in targeted tandem MS-analyses of clinical samples and their relevance for identifications of the pathogens, i.e. proteotyping, was validated, thus demonstrating their potential as peptide biomarker candidates for diagnostics of infectious diseases.

Pseudomonas nosocomialis sp. nov., isolated from clinical specimens.

Strains A31/70T, CCUG 58779 and SD129 were Gram-stain-negative, short rods, motile by one polar flagellum and isolated from clinical specimens in Botswana, Sweden and Spain, respectively. The 16S rRNA sequence similarity values grouped them in the Pseudomonas stutzeri phylogenetic group of species. The DNA G+C content ranged from 65.5 to 65.7 mol%. The strains were characterized phenotypically by the API 20NE and Biolog GEN III tests, and chemotaxonomically by their whole-cell MALDI-TOF MS protein profiles and by their fatty acid contents. The absence of the arginine dihydrolase and the hydrolysis of gelatin differentiated these strains from the closest species, Pseudomonas azotifigens. The major fatty acid contents were summed feature 8 (38.6 %), C16 : 0 (22.6 %), summed feature 3 (20.5 %) and C12 : 0 (8.4 %). Multilocus sequence analysis with three housekeeping gene sequences (rpoD, gyrB and 16S rRNA) together with whole-genome comparisons indicated that these strains cluster together in the phylogenetic analysis and their similarity values were lower than the thresholds established for species in the genus Pseudomonas. These results permit us to conclude that strains A31/70T, CCUG 58779 and SD129 belong to a novel species in the genus Pseudomonas for which the name Pseudomonas nosocomialis sp. nov. is proposed. The type strain is A31/70T (=CECT 9981T=CCUG 73638T).