Tigecycline-resistant Klebsiella pneumoniae strains from sewage in Norway carry heavy-metal resistance genes encoding conjugative plasmids.

OBJECTIVES: Tigecycline is a last-resort antibiotic used for treatment of infections with carbapenem-resistant Klebsiella pneumoniae. The aim of the study was to understand the genetic mechanism of resistance and the genetic context of resistance genes in two tigecycline-resistant K. pneumoniae strains isolated from sewage in Bergen, Norway. METHODS: Complete genome sequencing of the two strains was accomplished using a combination of short-read Illumina MiSeq-based and long-read Oxford Nanopore MinION-based sequencing. Conjugation experiments were performed using filter mating and a green fluorescent protein (GFP)-tagged Escherichia coli strain. RESULTS: The complete genome sequences of strain K6-320.1 and strain K7-325 were assembled into two contigs for each strain, one contig representing the complete circular chromosomes of 5 223 440 bp (K6-320.1) and 5 263 092 bp (K7-325), respectively, and the other representing plasmids with sizes of 276 509 bp (pK6-320.1) and 246 731 bp (pK7-325). Strain K6-320.1 belonged to sequence type (ST)869, whereas strain K7-325 belonged to the pathogenic ST307. Both plasmids belonged to the IncFIB(K)/IncFII(K) group and carried several antibiotic resistance genes (ARGs), including tet(A) and blaCTX-M. Both plasmids (pK6-320.1 and pK7-325) were transferred to a GFP-tagged E. coli strain, leading to the acquisition of resistance against multiple classes of antibiotics. Several heavy-metal resistance genes (HMRGs) encoding resistance against silver (sil), copper (pco), and arsenic (ars) were also present on both plasmids. CONCLUSIONS: Our study demonstrates the presence of multidrug-resistant K. pneumoniae strains carrying conjugative plasmids encoding both ARGs and HMRGs that have potential for persistence in the environment and human microbiota.

Exploring the biosynthetic gene clusters in Brevibacterium: a comparative genomic analysis of diversity and distribution.

Exploring Brevibacterium strains from various ecosystems may lead to the discovery of new antibiotic-producing strains. Brevibacterium sp. H-BE7, a strain isolated from marine sediments from Northern Patagonia, Chile, had its genome sequenced to study the biosynthetic potential to produce novel natural products within the Brevibacterium genus. The genome sequences of 98 Brevibacterium strains, including strain H-BE7, were selected for a genomic analysis. A phylogenomic cladogram was generated, which divided the Brevibacterium strains into four major clades. A total of 25 strains are potentially unique new species according to Average Nucleotide Identity (ANIb) values. These strains were isolated from various environments, emphasizing the importance of exploring diverse ecosystems to discover the full diversity of Brevibacterium. Pangenome analysis of Brevibacterium strains revealed that only 2.5% of gene clusters are included within the core genome, and most gene clusters occur either as singletons or as cloud genes present in less than ten strains. Brevibacterium strains from various phylogenomic clades exhibit diverse BGCs. Specific groups of BGCs show clade-specific distribution patterns, such as siderophore BGCs and carotenoid-related BGCs. A group of clade IV-A Brevibacterium strains possess a clade-specific Polyketide synthase (PKS) BGCs that connects with phenazine-related BGCs. Within the PKS BGC, five genes, including the biosynthetic PKS gene, participate in the mevalonate pathway and exhibit similarities with the phenazine A BGC. However, additional core biosynthetic phenazine genes were exclusively discovered in nine Brevibacterium strains, primarily isolated from cheese. Evaluating the antibacterial activity of strain H-BE7, it exhibited antimicrobial activity against Salmonella enterica and Listeria monocytogenes. Chemical dereplication identified bioactive compounds, such as 1-methoxyphenazine in the crude extracts of strain H-BE7, which could be responsible of the observed antibacterial activity. While strain H-BE7 lacks the core phenazine biosynthetic genes, it produces 1-methoxyphenazine, indicating the presence of an unknown biosynthetic pathway for this compound. This suggests the existence of alternative biosynthetic pathways or promiscuous enzymes within H-BE7's genome.

Comparative genomics of Stutzerimonas balearica (Pseudomonas balearica): diversity, habitats, and biodegradation of aromatic compounds.

Stutzerimonas balearica (Pseudomonas balearica) has been found principally in oil-polluted environments. The capability of S. balearica to thrive from the degradation of pollutant compounds makes it a species of interest for potential bioremediation applications. However, little has been reported about the diversity of S. balearica. In this study, genome sequences of S. balearica strains from different origins were analyzed, revealing that it is a diverse species with an open pan-genome that will continue revealing new genes and functionalities as the genomes of more strains are sequenced. The nucleotide signatures and intra- and inter-species variation of the 16S rRNA genes of S. balearica were reevaluated. A strategy of screening 16S rRNA gene sequences in public databases enabled the detection of 158 additional strains, of which only 23% were described as S. balearica. The species was detected from a wide range of environments, although mostly from aquatic and polluted environments, predominantly related to petroleum oil. Genomic and phenotypic analyses confirmed that S. balearica possesses varied inherent capabilities for aromatic compounds degradation. This study increases the knowledge of the biology and diversity of S. balearica and will serve as a basis for future work with the species.

Complete sequence of a new conjugative multidrug-resistance encoding IncFII/IncFIA/IncFIB plasmid carrying NDM-6 metallo-ß-lactamase from pathogenic Escherichia coli sequence type 167 isolated from sewage in Norway.

OBJECTIVES: The aim of the current study was to determine the genomic map of resistance genes and to understand the potential for mobility of a new NDM-6-carrying plasmid from a pathogenic Escherichia coli strain. A complete and closed genome sequence of the E. coli strain was obtained by applying a combination of short-read Illumina and long-read Nanopore-based sequencing. METHODS: Isolation of E. coli was performed, using ECC CHROMagar™, and antibiotic sensitivity patterns were determined, using Sensititre™ EUVSEC3 plates. Whole-genome sequencing was performed, using Illumina MiSeq- and Oxford Nanopore MinION-based sequencing. Conjugation experiments were performed, using filter-mating and a green fluorescent protein (GFP)-tagged E. coli strain. RESULTS: Two carbapenem-resistant E. coli strains were isolated from sewage. These strains (2-331 and 2-333) belonged to sequence type (ST) 167 and carried an NDM-6 carbapenemase. The complete genome of strain 2-331 (GenBank accession no.: CP110117-22.1) was assembled into six contigs, representing a complete circular chromosome of 4 947 178 bp and five plasmids, ranging from 143 596 bp to 1549 bp. Plasmid p2-331_1 (∼144 kb), belonging to the IncFII/IncFIA/IncFIB group, carried multiple antibiotic resistance genes, including mph(A), mrx(A), blaTEM-1, rmtB1, blaNDM-6, ble, sul1, qacEΔ1, aadAΔ, dfrA12, and tet(A). Plasmid p2-331_1 was transferred from strain 2-331, via conjugation, conferring resistance against eight different classes of antibiotics to a GFP-tagged E. coli strain. CONCLUSIONS: Our study showed the emergence of a new conjugative plasmid-carrying NDM-6 carbapenemase from pathogenic E. coli ST167 for the first time in Norway. The importance of population-based sewage-surveillance for understanding the antimicrobial resistance situation within the community is highlighted.

Legionella maioricensis sp. nov., a new species isolated from the hot water distribution systems of a hospital and a shopping center during routine sampling.

Two Legionella-like strains isolated from hot water distribution systems in 2012 have been characterized phenotypically, biochemically and genomically in terms of DNA relatedness. Both strains, HCPI-6T and EUR-108, exhibited biochemical phenotypic profiles typical of Legionella species. Cells were Gram-negative motile rods which grew on BCYEα agar but not on blood agar and displayed phenotypic characteristics typical of the family Legionellaceae, including a requirement for l-cysteine and testing catalase positive. Both strains were negative for oxidase, urease, nitrate reduction and hippurate negative, and non-fermentative. The major ubiquinone was Q12 (59.4 % HCPI-6T) and the dominant fatty acids were C16 : 1 ω7c (28.4 % HCPI-6T, ≈16 % EUR-108), C16 : 0 iso (≈22.5 % and ≈13 %) and C15 : 0 anteiso (19.5 % and ≈23.5 %, respectively). The percent G+C content of genomic DNA was determined to be 39.3 mol %. The 16S rRNA gene, mip sequence and comparative genome sequence-based analyses (average nucleotide identity, ANI; digital DNA-DNA hybridization, dDDH; and phylogenomic treeing) demonstrated that the strains represent a new species of the genus Legionella. The analysis based on the 16S rRNA gene sequences showed that the sequence similarities for both strains ranged from 98.8-90.1 % to other members of the genus. The core genome-based phylogenomic tree (protein-concatemer tree based on concatenation of 418 proteins present in single copy) revealed that these two strains clearly form a separate cluster within the genus Legionella. ANI and dDDH values confirmed the distinctiveness of the strains. Based on the genomic, genotypic and phenotypic findings from a polyphasic study, the isolates are considered to represent a single novel species, for which the name Legionella maioricensis sp. nov. is proposed. The type strain is HCPI-6T (=CCUG 75071T=CECT 30569T).

Seawater from Bergen harbor is a reservoir of conjugative multidrug-resistance plasmids carrying genes for virulence.

Aquatic environments play important roles in the dissemination of clinically-relevant antibiotic resistance genes (ARGs) and pathogens. Limited knowledge exists about the prevalence of clinically-relevant acquired resistance genes in the marine environment, especially in Norway. The aim of the current study was to investigate the presence of and characterize self-transmissible resistance plasmids from Bergen harbor seawater, with exogenous-plasmid capture, using a green fluorescent protein (GFP)-tagged Escherichia coli strain as a recipient. We obtained transconjugants resistant against ampicillin and cefotaxime from four of the 13 samples processed. Nine transconjugants, selected on the basis of antibiotic sensitivity patterns, were sequenced, using Illumina MiSeq and Oxford Nanopore MinION platforms. Ten different plasmids (ranging from 35 kb to 136 kb) belonging to incompatibility groups IncFII/IncFIB/Col156, IncFII, IncI1 and IncB/O/K/Z were detected among these transconjugants. Plasmid p1A1 (IncFII/IncFIB/Col156, 135.7 kb) carried resistance genes blaTEM-1, dfrA17, sul1, sul2, tet(A), mph(A), aadA5, aph(3″)-Ib and aph(6)-Id, conferring resistance against six different classes of antibiotics. Plasmid p1A4 carried blaCTX-M-55, lnu(F), aadA17 and aac(3)-IId. Cephalosporinase blaCMY-2 was detected on plasmids captured from an area impacted by wastewater from a local marine aquarium. Along with ARGs, some plasmids also carried virulence factors, such as enterotoxins, adhesion factors and siderophores. Our study demonstrates the presence of clinically-important multidrug-resistance conjugative plasmids in seawater from Bergen harbor, which have the potential to be transferred to human microbiota. The results highlight the need for surveillance of antibiotic resistance in the environment, as suggested by the World Health Organization, especially in low prevalence settings like Norway.

Novel Plasmid Carrying Mobile Colistin Resistance Gene mcr-4.3 and Mercury Resistance Genes in Shewanella baltica: Insights into Mobilization of mcr-4.3 in Shewanella Species.

Shewanella species have been identified as progenitors of several clinically important antibiotic resistance genes. The aim of our study was to analyze Shewanella baltica strains isolated from the gut contents of wild Atlantic mackerel (Scomber scombrus) for the presence of both known and novel variants of antibiotic resistance genes (ARGs), using Illumina-based whole-genome sequencing (WGS). Thirty-three S. baltica strains were isolated from Atlantic mackerel collected in the northern North Sea. WGS revealed the presence of several new variants of class C and class D beta-lactamases. Nearly 42% (14/33) of the strains carried the mobile colistin resistance gene mcr-4.3. To understand the genetic context of mcr-4.3, we determined the complete genome sequence of strain 11FHM2, using a combination of Oxford Nanopore- and Illumina-based sequencing. The complete genome sequence is 5,406,724 bp long, with one contig representing a chromosome of 5,068,880 bp and three contigs representing novel plasmids (pSBP1, 194,145 bp; pSBP2_mcr4, 86,727 bp; and pSBP3, 56,972 bp). Plasmid pSBP2_mcr4 contains the mobile colistin resistance gene mcr-4.3, as well as the mercury resistance operon merRPAT. Plasmid pSBP1 carries genes encoding resistance against copper, zinc, chromium, and arsenic. Plasmid pSBP3 does not carry any antibiotic or heavy metal resistance genes. Analysis of the flanking region of mcr-4.3 suggests that a phage integrase may be involved in the mobilization of mcr-4.3 in Shewanella spp. Our results provide insights into the mobile mcr-4.3 present in Shewanella spp. and highlight the importance of the marine environment in the emergence and dissemination of clinically important resistance genes. IMPORTANCE We identified two new plasmids in Shewanella baltica isolated from wild Atlantic mackerel (Scomber scombrus) collected from the northern North Sea, one plasmid carrying the mcr-4.3 gene for colistin resistance and the operon merRPAT for mercury resistance and the other carrying multiple heavy metal resistance genes. The marine environment has been recognized as a source of new resistance genes that are found in human pathogens. Selection pressure from heavy metals is seen in the marine environment, especially associated with human activities, such as waste discharge, mining, and in aquaculture settings. This would help maintain and disseminate these plasmids in the environment. Our study provides insights into the mobilization of colistin resistance genes in Shewanella spp. and highlights the importance of the marine environment in the emergence and dissemination of clinically important antibiotic resistance genes.

Identification of Antibiotic Resistance Proteins via MiCId's Augmented Workflow. A Mass Spectrometry-Based Proteomics Approach.

Fast and accurate identifications of pathogenic bacteria along with their associated antibiotic resistance proteins are of paramount importance for patient treatments and public health. To meet this goal from the mass spectrometry aspect, we have augmented the previously published Microorganism Classification and Identification (MiCId) workflow for this capability. To evaluate the performance of this augmented workflow, we have used MS/MS datafiles from samples of 10 antibiotic resistance bacterial strains belonging to three different species: Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. The evaluation shows that MiCId's workflow has a sensitivity value around 85% (with a lower bound at about 72%) and a precision greater than 95% in identifying antibiotic resistance proteins. In addition to having high sensitivity and precision, MiCId's workflow is fast and portable, making it a valuable tool for rapid identifications of bacteria as well as detection of their antibiotic resistance proteins. It performs microorganismal identifications, protein identifications, sample biomass estimates, and antibiotic resistance protein identifications in 6-17 min per MS/MS sample using computing resources that are available in most desktop and laptop computers. We have also demonstrated other use of MiCId's workflow. Using MS/MS data sets from samples of two bacterial clonal isolates, one being antibiotic-sensitive while the other being multidrug-resistant, we applied MiCId's workflow to investigate possible mechanisms of antibiotic resistance in these pathogenic bacteria; the results showed that MiCId's conclusions agree with the published study. The new version of MiCId (v.07.01.2021) is freely available for download at https://www.ncbi.nlm.nih.gov/CBBresearch/Yu/downloads.html.

Responses of carbapenemase-producing and non-producing carbapenem-resistant Pseudomonas aeruginosa strains to meropenem revealed by quantitative tandem mass spectrometry proteomics.

Pseudomonas aeruginosa is an opportunistic pathogen with increasing incidence of multidrug-resistant strains, including resistance to last-resort antibiotics, such as carbapenems. Resistances are often due to complex interplays of natural and acquired resistance mechanisms that are enhanced by its large regulatory network. This study describes the proteomic responses of two carbapenem-resistant P. aeruginosa strains of high-risk clones ST235 and ST395 to subminimal inhibitory concentrations (sub-MICs) of meropenem by identifying differentially regulated proteins and pathways. Strain CCUG 51971 carries a VIM-4 metallo-ß-lactamase or 'classical' carbapenemase; strain CCUG 70744 carries no known acquired carbapenem-resistance genes and exhibits 'non-classical' carbapenem-resistance. Strains were cultivated with different sub-MICs of meropenem and analyzed, using quantitative shotgun proteomics based on tandem mass tag (TMT) isobaric labeling, nano-liquid chromatography tandem-mass spectrometry and complete genome sequences. Exposure of strains to sub-MICs of meropenem resulted in hundreds of differentially regulated proteins, including ß-lactamases, proteins associated with transport, peptidoglycan metabolism, cell wall organization, and regulatory proteins. Strain CCUG 51971 showed upregulation of intrinsic ß-lactamases and VIM-4 carbapenemase, while CCUG 70744 exhibited a combination of upregulated intrinsic ß-lactamases, efflux pumps, penicillin-binding proteins and downregulation of porins. All components of the H1 type VI secretion system were upregulated in strain CCUG 51971. Multiple metabolic pathways were affected in both strains. Sub-MICs of meropenem cause marked changes in the proteomes of carbapenem-resistant strains of P. aeruginosa exhibiting different resistance mechanisms, involving a wide range of proteins, many uncharacterized, which might play a role in the susceptibility of P. aeruginosa to meropenem.

Mass Spectrometry Proteotyping-Based Detection and Identification of Staphylococcus aureus, Escherichia coli, and Candida albicans in Blood.

Bloodstream infections (BSIs), the presence of microorganisms in blood, are potentially serious conditions that can quickly develop into sepsis and life-threatening situations. When assessing proper treatment, rapid diagnosis is the key; besides clinical judgement performed by attending physicians, supporting microbiological tests typically are performed, often requiring microbial isolation and culturing steps, which increases the time required for confirming positive cases of BSI. The additional waiting time forces physicians to prescribe broad-spectrum antibiotics and empirically based treatments, before determining the precise cause of the disease. Thus, alternative and more rapid cultivation-independent methods are needed to improve clinical diagnostics, supporting prompt and accurate treatment and reducing the development of antibiotic resistance. In this study, a culture-independent workflow for pathogen detection and identification in blood samples was developed, using peptide biomarkers and applying bottom-up proteomics analyses, i.e., so-called "proteotyping". To demonstrate the feasibility of detection of blood infectious pathogens, using proteotyping, Escherichia coli and Staphylococcus aureus were included in the study, as the most prominent bacterial causes of bacteremia and sepsis, as well as Candida albicans, one of the most prominent causes of fungemia. Model systems including spiked negative blood samples, as well as positive blood cultures, without further culturing steps, were investigated. Furthermore, an experiment designed to determine the incubation time needed for correct identification of the infectious pathogens in blood cultures was performed. The results for the spiked negative blood samples showed that proteotyping was 100- to 1,000-fold more sensitive, in comparison with the MALDI-TOF MS-based approach. Furthermore, in the analyses of ten positive blood cultures each of E. coli and S. aureus, both the MALDI-TOF MS-based and proteotyping approaches were successful in the identification of E. coli, although only proteotyping could identify S. aureus correctly in all samples. Compared with the MALDI-TOF MS-based approaches, shotgun proteotyping demonstrated higher sensitivity and accuracy, and required significantly shorter incubation time before detection and identification of the correct pathogen could be accomplished.

Analyses of Virulence Genes of Clavibacter michiganensis subsp. michiganensis Strains Reveal Heterogeneity and Deletions That Correlate with Pathogenicity.

Clavibacter michiganensis subsp. michiganensis (Cmm) is the causal agent of bacterial canker of tomato. Differences in virulence between Cmm strains have been reported. The aim of this study was the characterization of nine Cmm strains isolated in Chile to reveal the causes of their differences in virulence. The virulence assays in tomato seedlings revealed different levels of severity associated with the strains, with two highly virulent strains and one causing only mild symptoms. The two most virulent showed increased cellulase activity, and no cellulase activity was observed in the strain causing mild symptoms. In three strains, including the two most virulent strains, PCR amplification of the 10 virulence genes analyzed was observed. In the strain causing mild symptoms, no amplification was observed for five genes, including celA. Sequence and cluster analyses of six virulence genes grouped the strains, as has been previously reported, except for gene pelA1. Gene sequence analysis from the genomes of five Chilean strains revealed the presence of deletions in the virulence genes, celB, xysA, pat-1, and phpA. The results of this study allow us to establish correlations between the differences observed in disease severity and the presence/absence of genes and deletions not previously reported.

Complete Multipartite Genome Sequence of the Cupriavidus basilensis Type Strain, a 2,6-Dichlorophenol-Degrading Bacterium.

We report the complete 8.94-Mb genome sequence of the type strain of Cupriavidus basilensis (DSM 11853T = CCUG 49340T = RK1T), formed by two chromosomes and six putative plasmids, which offers insights into its chloroaromatic-biodegrading capabilities.

Comparative Genomics of Pathogenic Clavibacter michiganensis subsp. michiganensis Strains from Chile Reveals Potential Virulence Features for Tomato Plants.

The genus Clavibacter has been associated largely with plant diseases. The aims of this study were to characterize the genomes and the virulence factors of Chilean C. michiganensis subsp. michiganensis strains VL527, MSF322 and OP3, and to define their phylogenomic positions within the species, Clavibacter michiganensis. VL527 and MSF322 genomes possess 3,396,632 and 3,399,199 bp, respectively, with a pCM2-like plasmid in strain VL527, with pCM1- and pCM2-like plasmids in strain MSF322. OP3 genome is composed of a chromosome and three plasmids (including pCM1- and pCM2-like plasmids) of 3,466,104 bp. Genomic analyses confirmed the phylogenetic relationships of the Chilean strains among C.michiganensis subsp. michiganensis and showed their low genomic diversity. Different virulence levels in tomato plants were observable. Phylogenetic analyses of the virulence factors revealed that the pelA1 gene (chp/tomA region)-that grouped Chilean strains in three distinct clusters-and proteases and hydrolases encoding genes, exclusive for each of the Chilean strains, may be involved in these observed virulence levels. Based on genomic similarity (ANIm) analyses, a proposal to combine and reclassify C. michiganensis subsp. phaseoli and subsp. chilensis at the species level, as C. phaseoli sp. nov., as well as to reclassify C. michiganensis subsp. californiensis as the species C. californiensis sp. nov. may be justified.

Staphylococcus borealis sp. nov., isolated from human skin and blood.

When analysing a large cohort of Staphylococcus haemolyticus, using whole-genome sequencing, five human isolates (four from the skin and one from a blood culture) with aberrant phenotypic and genotypic traits were identified. They were phenotypically similar with yellow colonies, nearly identical 16S rRNA gene sequences and initially speciated as S. haemolyticus based on 16S rRNA gene sequence and MALDI-TOF MS. However, compared to S. haemolyticus, these five strains demonstrate: (i) considerable phylogenetic distance with an average nucleotide identity <95 % and inferred DNA-DNA hybridization <70  %; (ii) a pigmented phenotype; (iii) urease production; and (iv) different fatty acid composition. Based on the phenotypic and genotypic results, we conclude that these strains represent a novel species, for which the name Staphylococcus borealis sp. nov. is proposed. The novel species belong to the genus Staphylococcus and is coagulase- and oxidase-negative and catalase-positive. The type strain, 51-48T, is deposited in the Culture Collection University of Gothenburg (CCUG 73747T) and in the Spanish Type Culture Collection (CECT 30011T).

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.

Acinetobacter portensis sp. nov. and Acinetobacter guerrae sp. nov., isolated from raw meat.

The taxonomic status of six strains of Acinetobacter obtained from meat samples, collected from supermarkets in Porto, Portugal, was investigated using polyphasic analysis. Partial rpoB sequence similarities lower than 95 % to other Acinetobacter species with validly published names led to the hypothesis that these strains represented novel species. This was confirmed based on comparative multilocus sequence analysis, which included the gyrB, recA and 16S rRNA genes, revealing that these strains represented two coherent lineages that were distinct from each other and from all known species. The names Acinetobacter portensis sp. nov. (comprising four strains) and Acinetobacter guerrae sp. nov. (comprising two strains) are proposed for these novel species. The species status of these two groups was confirmed by low (below 95 %) whole-genome sequence average nucleotide identity values and low (below 70 %) digital DNA-DNA hybridization similarities between the whole-genome sequences of the proposed type strains of each novel species and the representatives of the known Acinetobacter species. Phylogenomic treeing from core genome analysis supported these results. The coherence of each new species lineage was supported by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry differentiation of the species at the protein level, by cellular fatty acid profiles, and by unique and differential combinations of metabolic and physiological properties shared by each novel species. The type strain of A. portensis sp. nov. is AC 877T (=CCUG 68672T=CCM 8789T) and the type strain of A. guerrae sp. nov. is AC 1271T (=CCUG 68674T=CCM 8791T).

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.

Genomic and Proteomic Characterization of the Extended-Spectrum ß-Lactamase (ESBL)-Producing Escherichia coli Strain CCUG 73778: A Virulent, Nosocomial Outbreak Strain.

Escherichia coli strain CCUG 78773 is a virulent extended-spectrum ß-lactamase (ESBL)-producing ST131-O25b type strain isolated during an outbreak at a regional university hospital. The complete and closed genome sequence, comprising one chromosome (5,076,638 bp) and six plasmids (1718-161,372 bp), is presented. Characterization of the genomic features detected the presence of 59 potential antibiotic resistance factors, including three prevalent ß-lactamases. Several virulence associated elements were determined, mainly related with adherence, invasion, biofilm formation and antiphagocytosis. Twenty-eight putative type II toxin-antitoxin systems were found. The plasmids were characterized, through in silico analyses, confirming the two ß-lactamase-encoding plasmids to be conjugative, while the remaining plasmids were mobilizable. BLAST analysis of the plasmid sequences showed high similarity with plasmids in E. coli from around the world. Expression of many of the described virulence and AMR factors was confirmed by proteomic analyses, using bottom-up, liquid chromatography-tandem mass spectrometry (LC-MS/MS). The detailed characterization of E. coli strain CCUG 78773 provides a reference for the relevance of genetic elements, as well as the characterization of antibiotic resistance and the spread of bacteria harboring ESBL genes in the hospital environment.

Nanopore sequencing reveals genomic map of CTX-M-type extended-spectrum ß-lactamases carried by Escherichia coli strains isolated from blue mussels (Mytilus edulis) in Norway.

BACKGROUND: Environmental surveillance of antibiotic resistance can contribute towards better understanding and management of human and environmental health. This study applied a combination of long-read Oxford Nanopore MinION and short-read Illumina MiSeq-based sequencing to obtain closed complete genome sequences of two CTX-M-producing multidrug-resistant Escherichia coli strains isolated from blue mussels (Mytilus edulis) in Norway, in order to understand the potential for mobility of the detected antibiotic resistance genes (ARGs). RESULTS: The complete genome sequence of strain 631 (E. coli sequence type 38) was assembled into a circular chromosome of 5.19 Mb and five plasmids (between 98 kb and 5 kb). The majority of ARGs cluster in close proximity to each other on the chromosome within two separate multidrug-resistance determining regions (MDRs), each flanked by IS26 transposases. MDR-1 carries blaTEM-1, tmrB, aac(3)-IId, aadA5, mph(A), mrx, sul1, qacEΔ1 and dfrA17; while MDR-2 harbors aph(3″)-Ib, aph(6)-Id, blaTEM-1, catA1, tet(D) and sul2. Four identical chromosomal copies of blaCTX-M-14 are located outside these regions, flanked by ISEc9 transposases. Strain 1500 (E. coli sequence type 191) exhibited a circular chromosome of 4.73 Mb and two plasmids (91 kb and 4 kb). The 91 kb conjugative plasmid belonging to IncI1 group carries blaCTX-M-15 and blaTEM-1 genes. CONCLUSION: This study confirms the efficacy of combining Nanopore long-read and Illumina short-read sequencing for determining complete bacterial genome sequences, enabling detection and characterization of clinically important ARGs in the marine environment in Norway, with potential for further dissemination. It also highlights the need for environmental surveillance of antibiotic resistance in low prevalence settings like Norway.