Development and validation of a quick, automated, and reproducible ATR FT-IR spectroscopy machine-learning model for Klebsiella pneumoniae typing.

The reliability of Fourier-transform infrared (FT-IR) spectroscopy for Klebsiella pneumoniae typing and outbreak control has been previously assessed, but issues remain in standardization and reproducibility. We developed and validated a reproducible FT-IR with attenuated total reflectance (ATR) workflow for the identification of K. pneumoniae lineages. We used 293 isolates representing multidrug-resistant K. pneumoniae lineages causing outbreaks worldwide (2002-2021) to train a random forest classification (RF) model based on capsular (KL)-type discrimination. This model was validated with 280 contemporaneous isolates (2021-2022), using wzi sequencing and whole-genome sequencing as references. Repeatability and reproducibility were tested in different culture media and instruments throughout time. Our RF model allowed the classification of 33 capsular (KL)-types and up to 36 clinically relevant K. pneumoniae lineages based on the discrimination of specific KL- and O-type combinations. We obtained high rates of accuracy (89%), sensitivity (88%), and specificity (92%), including from cultures obtained directly from the clinical sample, allowing to obtain typing information the same day bacteria are identified. The workflow was reproducible in different instruments throughout time (>98% correct predictions). Direct colony application, spectral acquisition, and automated KL prediction through Clover MS Data analysis software allow a short time-to-result (5 min/isolate). We demonstrated that FT-IR ATR spectroscopy provides meaningful, reproducible, and accurate information at a very early stage (as soon as bacterial identification) to support infection control and public health surveillance. The high robustness together with automated and flexible workflows for data analysis provide opportunities to consolidate real-time applications at a global level. IMPORTANCE We created and validated an automated and simple workflow for the identification of clinically relevant Klebsiella pneumoniae lineages by FT-IR spectroscopy and machine-learning, a method that can be extremely useful to provide quick and reliable typing information to support real-time decisions of outbreak management and infection control. This method and workflow is of interest to support clinical microbiology diagnostics and to aid public health surveillance.

Citrobacter meridianamericanus sp. nov., isolated from a soil sample.

A Gram-stain-negative strain, designated BR102T, isolated from a soil sample in Brazil was characterized by a polyphasic approach. Comparative 16S rRNA gene sequences indicated that strain BR102T belonged to the genus Citrobacter. The recN- and whole-genome-based phylogeny, and multilocus sequence analysis based on concatenated partial fusA, leuS, pyrG and rpoB sequences strongly supported a clade encompassing strain BR102T and a strain from public database that was distinct from currently recognized species of the genus Citrobacter. Average nucleotide identity and digital DNA-DNA hybridization values between strain BR102T and the closest relative Citrobacter freundii ATCC 8090T were 91.8 and 48.8 %, respectively. The ability to metabolize different compounds further discriminated strain BR102T from other closely related species of the genus Citrobacter. The novel variants bla CMY-179 and qnrB97, which encoded a CMY-2-like ß-lactamase and a QnrB-type protein, respectively, were identified in strain BR102T. BR102T was resistant to ampicillin, amoxicillin/clavulanate and cefoxitin. The DNA G+C content of strain BR102T is 51.3 mol%. Based on these results, strain BR102T represents a novel species of the genus Citrobacter, for which the name Citrobacter meridianamericanus sp. nov. is proposed. The type strain is BR102T (=MUM 22.55T=IMI 507229T).

Gardnerella pickettii sp. nov. (formerly Gardnerella genomic species 3) and Gardnerella greenwoodii sp. nov. (formerly Gardnerella genomic species 8) isolated from female urinary microbiome.

During an ongoing female urinary microbiome research study, strains c17Ua_112T and c31Ua_26T isolated from urine samples of a patient diagnosed with overactive bladder and a healthy postmenopausal woman, respectively, could not be allocated to any Gardnerella species with valid names. In this work, we aimed to characterize these strains. The 16S rRNA gene sequences confirmed that these strains are members of the genus Gardnerella. Phylogenetic analysis based on cpn60 strongly supported two clades, one encompassing c17Ua_112T and nine other strains from the public database, and the other including c31Ua_26T and three other strains, which were distinct from currently recognized species of the genus Gardnerella. Likewise, the phylogenomic tree also showed that strains c17Ua_112T and c31Ua_26T formed independent and robust clusters. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between c17Ua_112T and c31Ua_26T were 79.27 and 27.4 %, respectively. Strain c17Ua_112T showed the highest ANI (94.8 %) and dDDH values (59.8 %) with Gardnerella piotii UGent 18.01T, and strain c31Ua_26T revealed highest ANI (84.2 %) and dDDH (29.1 %) values with Gardnerella swidsinskii GS 9838-1T. Based on the data presented here, the two strains c17Ua_112T and c31Ua_26T represent two novel species of the genus Gardnerella, for which the names Gardnerella pickettii (c17Ua_112T=DSM 113414T=CCP 71T) and Gardnerella greenwoodii (c31Ua_26T=DSM 113415T=CCP 72T) are proposed.

Citrobacter telavivum sp. nov. with chromosomal mcr-9 from hospitalized patients.

Strains 6105T and 6106, recovered from colonized patients in a hospital in Tel-Aviv, Israel, were compared with currently known species of the genus Citrobacter by a polyphasic taxonomic approach. Strains were characterized by whole-genome sequencing, 16S rRNA and recN gene sequencing, multilocus sequence analysis (MLSA), average nucleotide identity (ANI), Genome-to-Genome Distance Calculator (GGDC), and biochemical tests. The location and genetic surrounding of antibiotic resistance genes were investigated, and antibiotic susceptibility profiles were determined by broth microdilution or agar dilution methods. Phylogenetic analysis based on recN and MLSA revealed that both strains formed a distinct cluster from all currently recognized species. The ANI and GGDC were 90.7% and 54.3% with Citrobacter farmeri, respectively. The ability to metabolize various compounds also differentiated both strains from closely related Citrobacter species. Chromosomes of the isolates contained locus encoding a novel class A ß-lactamase (TEL-1; 90.5% amino acid identity with CdiA of Citrobacter koseri) plus a LysR-like transcriptional regulator (TEL-R) and an ~ 25.5-kb mcr-9 mosaic region. The direct mcr-9 context matched with those previously identified in several plasmids and chromosomes of diverse Enterobacteriaceae, yet similarity with the plasmidic loci extended further. Untypeable plasmids, pCTEL-2 (~ 235 kb) and pCTEL-1 (~ 114 kb), devoid of resistance genes, were identified in the strains. The isolates were non-susceptible to ß-lactams. The name Citrobacter telavivum sp. nov. is proposed, with 6105T (CECT 9989T or DSM 110286T) as the type strain. C. telavivum may represent a bacterial species adapting to hospital settings, able to disseminate and acquire antimicrobial resistance genes.

The status of the species Lactobacillus fornicalis Dicks et al. 2000. Request for an opinion.

During a recent study on members of the genus Lactobacillus we realized that cultures of Lactobacillus fornicalis TV 1018T (=DSM 13171T=ATCC 700934T) are no longer available from the online catalogue of the German Collection of Microorganisms and Cell Cultures GmbH, being displayed as Lactobacillus plantarum at the American Type Culture Collection. Based on data currently available, the organism deposited as ATCC 700934T is a member of the species Lactobacillus plantarum subs. plantarum. Therefore, the type strain of Lactobacillus fornicalis cannot be included in any further scientific comparative study. This matter is referred to the Judicial Commission, asking for an opinion on the status of the species.

Lactobacillus mulieris sp. nov., a new species of Lactobacillus delbrueckii group.

One Gram-stain-positive, non-motile, non-spore-forming, catalase-negative, and coccobacilli-shaped strain, designated c10Ua161MT, was isolated from a urine sample from a reproductive-age healthy woman. Comparative 16S rRNA gene sequence analysis indicated that strain c10Ua161MT belonged to the genus Lactobacillus. Phylogenetic analysis based on pheS and rpoA gene sequences strongly supported a clade encompassing strains c10Ua161MT and eight other strains from public databases, distinct from currently recognized species of the genus Lactobacillus. In silico Average Nucleotide Identity (ANI) and Genome-to-Genome Distance Calculator (GGDC), showed 87.9 and 34.3 % identity to the closest relative Lactobacillus jensenii, respectively. The major fatty acids of strain c10Ua161MT were C18 : 1ω9c (65.0%), C16 : 0 (17.8%), and summed feature 8 (10.2 %; comprising C18 : 1ω7c, and/or C18 : 1ω6c). The DNA G+C content of the strains is 34.2 mol%. On the basis of data presented here, strain c10Ua161MT represents a novel species of the genus Lactobacillus, for which the name Lactobacillus mulieris sp. nov. is proposed. The type strain is c10Ua161MT (=CECT 9755T=DSM 108704T).

Limosilactobacillus urinaemulieris sp. nov. and Limosilactobacillus portuensis sp. nov. isolated from urine of healthy women.

Two Gram-stain-positive strains, c9Ua_26_MT and c11Ua_112_MT, were isolated from voided urine samples from two healthy women. Comparative 16S rRNA gene sequences demonstrated that these novel strains were members of the genus Limosilactobacillus. Phylogenetic analysis based on pheS gene sequences and core genomes showed that each strain formed a separated branch and are closest to Limosilactobacillus vaginalis DSM 5837T. The average nucleotide identity (ANI) and Genome-to-Genome Distance Calculator (GGDC) values between c9Ua_26_MT and the closest relative DSM 5837T were 90.7 and 42.9 %, respectively. The ANI and GGDC values between c11Ua_112_MT and the closest relative DSM 5837T were 91.2 and 45.0 %, and those among the strains were 92.9% and 51,0 %, respectively. The major fatty acids were C12 : 0 (40.2 %), C16 : 0 (26.7 %) and C18 : 1 ω9c (17.7 %) for strain c9Ua_26_MT, and C18 : 1 ω9c (38.0 %), C16 : 0 (33.3 %) and C12 : 0 (17.6 %) for strain c11Ua_112_MT. The genomic DNA G+C content of strains c9Ua_26_MT and c11Ua_112_MT was 39.9 and 39.7 mol%, respectively. On the basis of the data presented here, strains c9Ua_26_MT and c11Ua_112_MT represent two novel species of the genus Limosilactobacillus, for which the names Limosilactobacillus urinaemulieris sp. nov. (c9Ua_26_MT=CECT 30144T=LMG 31899T) and Limosilactobacillus portuensis sp. nov. (c11Ua_112_MT=CECT 30145T=LMG 31898T) are proposed.

Citrobacter europaeus sp. nov., isolated from water and human faecal samples.

Strains 97/79T and A121, recovered respectively from human faeces and well water, were compared to currently known species of the genus Citrobacter using genotypic and phenotypic approaches. Multilocus sequence analysis based on housekeeping genes fusA, leuS, pyrG, rpoB and recN, showed that the two strains formed a distinct phylogenetic lineage within the genus Citrobacter. Average nucleotide identity (ANI) between strains 97/79T and A121 was 99.2 %, whereas ANI values of strain 97/79T with the type strains of closely related species of the genus Citrobacter, C. werkmanii, C. braakii, C. freundii, C. youngae and C. pasteurii, were all below 93.0 %. The ability to metabolize different compounds also discriminated strains 97/79T and A121 from other species of the genus Citrobacter. Based on these results, strains 97/79T and A121 represent a novel species of the genus Citrobacter, for which the name Citrobacter europaeus sp. nov. is proposed, with strain 97/79T (=CIP 106467T=DSM 103031T) as the type strain. The DNA G+C content of strain 97/79T is 52.0 %.

Citrobacter portucalensis sp. nov., isolated from an aquatic sample.

A Gram-stain-negative strain, A60T, isolated from a water well sample in Portugal, was characterized phenotypically, genotypically and phylogenetically. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain A60T belonged to the genus Citrobacter, and recN gene phylogeny revealed one strongly supported clade encompassing strain A60T and 13 other strains from public databases, distinct from currently recognized species of the genus Citrobacter. Furthermore, multilocus sequence analysis (MLSA) based on concatenated partial fusA, leuS, pyrG and rpoB sequences confirmed the classification obtained with the recN sequence. In silico genomic comparisons, including average nucleotide identity (ANI) and the genome-to-genome distance calculator (GGDC), showed 94.6 % and 58.4 % identity to the closest relative Citrobacter freundii ATCC 8090T, respectively. The ability to metabolize different compounds further discriminated strain A60T from other species of the genus Citrobacter. The G+C content of strain A60T is 52.0 %. The results obtained support the description of a novel species within the genus Citrobacter, for which the name Citrobacter portucalensis sp. nov. is proposed, with the type strain A60T (=DSM 104542T=CECT 9236T).

Characterization of GlobalFiler loci in Angolan and Guinean populations inhabiting Southern Portugal.

We analyzed the GlobalFiler short tandem repeat (STR) loci for 152 and 70 unrelated individuals from Angolan and Guinean immigrant populations inhabiting Southern Portugal, respectively. After Bonferroni correction, no significant deviations from the Hardy-Weinberg equilibrium and linkage disequilibrium were detected for either population. For the Angolan population, SE33 was the most informative marker. In contrast, D5S818 and D13S317 were the least informative loci. The combined power of discrimination was 99.9999999999999999999999961907%. For the Guinean population, SE33 and D21S1 were the most informative loci, while D13S317 was the least. The combined power of discrimination was 99.99999999999999999999997915%. No significant differences were observed between Angolan, Guinean, and Afro-American populations for any of the analyzed STRs. The South African population presented significant differences at D22S1045 and D10S1248 when compared to Angola, and at D22S1045 when compared to Guinea-Bissau. The MDS plot and neighbor-joining tree analysis revealed that Angolan and Guinean populations are genetically close to African-American and South African populations, and genetically different from Korean, Mexican, European (including American-Caucasian), and Middle Eastern populations.

Study of genetic markers of CODIS and ESS systems in a population of individuals from Cabo Verde living in Lisboa.

Twenty-two autosomal short tandem repeats included in the PowerPlex® Fusion System Amplification kit (Promega Corporation) were genotyped in a population sample of 500 unrelated individuals from Cabo Verde living in Lisboa. Allelic frequency data and forensic and statistical parameters were calculated and evaluated in this work. The genetic relationship among immigrant population from Cabo Verde living in Lisboa and other populations, such as Brazilian and Angola immigrants living in Lisboa; Afro-Americans, Caucasians, Hispanics and Asians living in the USA and the population from Lisboa was assessed, and a multidimensional scaling plot was drown to show these results.

Study of InDel genetic markers with forensic and ancestry informative interest in PALOP's immigrant populations in Lisboa.

The migratory phenomenon in Portugal has become one of the main factors for the genetic variability. In the last few years, a new class of autosomal insertion/deletion markers-InDel-has attracted interest in forensic genetics. Since there is no data for InDel markers of Portuguese-speaking African countries (PALOP) immigrants living in Lisboa, our aim is the characterization of those groups of individuals by typing them with at least 30 InDel markers and to compare different groups of individuals/populations. We studied 454 bloodstain samples belonging to immigrant individuals from Angola, Guinea-Bissau, and Mozambique. DNA extraction was performed with the Chelex® 100 method. After extraction, all samples were typed with the Investigator® DIPplex method. Through the obtained results, allelic frequencies show that all markers are at Hardy-Weinberg equilibrium, and we can confirm that those populations show significant genetic distances between themselves, between them, and the host Lisboa population. Because of this, they introduce genetic variability in Lisboa population.

Phylogeny and Comparative Genomics Unveil Independent Diversification Trajectories of qnrB and Genetic Platforms within Particular Citrobacter Species.

To gain insights into the diversification trajectories of qnrB genes, a phylogenetic and comparative genomics analysis of these genes and their surrounding genetic sequences was performed. For this purpose, Citrobacter sp. isolates (n = 21) and genome or plasmid sequences (n = 56) available in public databases harboring complete or truncated qnrB genes were analyzed. Citrobacter species identification was performed by phylogenetic analysis of different genotypic markers. The clonal relatedness among isolates, the location of qnrB genes, and the genetic surroundings of qnrB genes were investigated by pulsed-field gel electrophoresis (PFGE), S1-/I-CeuI-PFGE and hybridization, and PCR mapping and sequencing, respectively. Identification of Citrobacter isolates was achieved using leuS and recN gene sequences, and isolates characterized in this study were diverse and harbored chromosomal qnrB genes. Phylogenetic analysis of all known qnrB genes revealed seven main clusters and two branches, with most of them included in two clusters. Specific platforms (comprising pspF and sapA and varying in synteny and/or identity of other genes and intergenic regions) were associated with each one of these qnrB clusters, and the reliable identification of all Citrobacter isolates revealed that each platform evolved in different recognizable (Citrobacter freundii, C. braakii, C. werkmanii, and C. pasteurii) and putatively new species. A high identity was observed between some of the platforms identified in the chromosome of Citrobacter spp. and in different plasmids of Enterobacteriaceae. Our data corroborate Citrobacter as the origin of qnrB and further suggest divergent evolution of closely related qnrB genes/platforms in particular Citrobacter spp., which were delineated using particular genotypic markers.

The family 6 Carbohydrate Binding Module (CtCBM6) of glucuronoxylanase (CtXynGH30) of Clostridium thermocellum binds decorated and undecorated xylans through cleft A.

CtCBM6 of glucuronoxylan-xylanohydrolase (CtXynGH30) from Clostridium thermocellum was cloned, expressed and purified as a soluble ~14 kDa protein. Quantitative binding analysis with soluble polysaccharides by affinity electrophoresis and ITC revealed that CtCBM6 displays similar affinity towards decorated and undecorated xylans by binding wheat- and rye-arabinoxylans, beechwood-, birchwood- and oatspelt-xylan. Protein melting studies confirmed thermostable nature of CtCBM6 and that Ca(2+) ions did not affect its structure stability and binding affinity significantly. The CtCBM6 structure was modeled and refined and CD spectrum displayed 44% ß-strands supporting the predicted structure. CtCBM6 displays a jelly roll ß-sandwich fold presenting two potential carbohydrate binding clefts, A and B. The cleft A, is located between two loops connecting ß4-ß5 and ß8-ß9 strands. Tyr28 and Phe84 present on these loops make a planar hydrophobic binding surface to accommodate sugar ring of ligand. The cleft B, is located on concave surface of ß-sandwich fold. Tyr34 and Tyr104 make a planar hydrophobic platform, which may be inaccessible to ligand due to hindrance by Pro68. Site-directed mutagenesis revealed Tyr28 and Phe84 in cleft A, playing a major role in ligand binding. The results suggest that CtCBM6 interacts with carbohydrates through cleft A, which recognizes equally well both decorated and un-decorated xylans.

Prevalence of Mycobacterium avium subsp. paratuberculosis and Escherichia coli in blood samples from patients with inflammatory bowel disease.

Mycobacterium avium subsp. paratuberculosis (MAP) and adherent-invasive Escherichia coli (AIEC) have been implicated as primary triggers in Crohn's disease (CD). In this study, we evaluated the prevalence of MAP and E. coli (EC) DNA in peripheral blood from 202 inflammatory bowel disease (IBD) patients at various disease periods and compared against 24 cirrhotic patients with ascites (CIR) (non-IBD controls) and 29 healthy controls (HC). MAP DNA was detected by IS900-specific nested PCR, EC DNA by malB-specific nested PCR and AIEC identity, in selected samples, by sequencing of fimH gene. CD patients with active disease showed the highest MAP DNA prevalence among IBD patients (68 %). Infliximab treatment resulted in decreased MAP detection. CIR patients had high individual and coinfection rates (75 % MAP, 88 % EC and 67 % MAP and EC), whilst HC controls had lower MAP prevalence (38 %) and EC was undetectable in this control group. EC DNA prevalence in IBD patients was highly associated with CD, and 80 % of EC from the selected samples of CD patients analyzed carried the fimH30 allele, with a mutation strongly associated with AIEC. Our results show that coinfection with MAP and AIEC is common and persistent in CD, although the high MAP and EC detection in CIR patients suggested that colonization is, at least, partially dependent on increased gut permeability. Nevertheless, facilitative mechanisms between a susceptible host and these two potential human pathogens may allow their implication in CD pathogenesis.

Assessment of IrisPlex-based multiplex for eye and skin color prediction with application to a Portuguese population.

DNA phenotyping research is one of the most emergent areas of forensic genetics. Predictions of externally visible characteristics are possible through analysis of single nucleotide polymorphisms. These tools can provide police with "intelligence" in cases where there are no obvious suspects and unknown biological samples found at the crime scene do not result in any criminal DNA database hits. IrisPlex, an eye color prediction assay, revealed high prediction rates for blue and brown eye color in European populations. However, this is less predictive in some non-European populations, probably due to admixing. When compared to other European countries, Portugal has a relatively admixed population, resulting from a genetic influx derived from its proximity to and historical relations with numerous African territories. The aim of this work was to evaluate the utility of IrisPlex in the Portuguese population. Furthermore, the possibility of supplementing this multiplex with additional markers to also achieve skin color prediction within this population was evaluated. For that, IrisPlex was augmented with additional SNP loci. Eye and skin color prediction was estimated using the multinomial logistic regression and binomial logistic regression models, respectively. The results demonstrated eye color prediction accuracies of the IrisPlex system of 90 and 60% for brown and blue eye color, respectively, and 77% for intermediate eye color, after allele frequency adjustment. With regard to skin color, it was possible to achieve a prediction accuracy of 93%. In the future, phenotypic determination multiplexes must include additional loci to permit skin color prediction as presented in this study as this can be an advantageous tool for forensic investigation.

A comprehensive assessment of the transcriptome of cork oak (Quercus suber) through EST sequencing.

BACKGROUND: Cork oak (Quercus suber) is one of the rare trees with the ability to produce cork, a material widely used to make wine bottle stoppers, flooring and insulation materials, among many other uses. The molecular mechanisms of cork formation are still poorly understood, in great part due to the difficulty in studying a species with a long life-cycle and for which there is scarce molecular/genomic information. Cork oak forests are of great ecological importance and represent a major economic and social resource in Southern Europe and Northern Africa. However, global warming is threatening the cork oak forests by imposing thermal, hydric and many types of novel biotic stresses. Despite the economic and social value of the Q. suber species, few genomic resources have been developed, useful for biotechnological applications and improved forest management. RESULTS: We generated in excess of 7 million sequence reads, by pyrosequencing 21 normalized cDNA libraries derived from multiple Q. suber tissues and organs, developmental stages and physiological conditions. We deployed a stringent sequence processing and assembly pipeline that resulted in the identification of ~159,000 unigenes. These were annotated according to their similarity to known plant genes, to known Interpro domains, GO classes and E.C. numbers. The phylogenetic extent of this ESTs set was investigated, and we found that cork oak revealed a significant new gene space that is not covered by other model species or EST sequencing projects. The raw data, as well as the full annotated assembly, are now available to the community in a dedicated web portal at http://www.corkoakdb.org. CONCLUSIONS: This genomic resource represents the first trancriptome study in a cork producing species. It can be explored to develop new tools and approaches to understand stress responses and developmental processes in forest trees, as well as the molecular cascades underlying cork differentiation and disease response.

Blastic Plasmacytoid Dendritic Cell Neoplasm: A Rare Entity in Clinical Practice.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an exceedingly rare and aggressive hematologic malignancy. In the current World Health Organization classification, it is classified among histiocytic/dendritic cell neoplasms. This report describes the case of an 85-year-old female with a complex medical history, including rheumatoid arthritis, who presented with a one-month history of low-grade fever, anorexia, and unexplained weight loss. The diagnosis of BPDCN was confirmed following an immunophenotyping analysis of a bone marrow aspirate. With this report, the authors intend to shed some light on BPDCN's clinical presentation, diagnostic journey, therapeutic approaches, and patient outcomes, and denote the significance of early detection and interdisciplinary collaboration in enhancing patient care.

Expanding the Bacterial Diversity of the Female Urinary Microbiome: Description of Eight New Corynebacterium Species.

The genus Corynebacterium is frequently found in the female urinary microbiome (FUM). In-depth characterization of Corynebacterium at the species level has been barely exploited. During ongoing FUM research studies, eight strains (c8Ua_144T, c8Ua_172T, c8Ua_174T, c8Ua_181T, c9Ua_112T, c19Ua_109T, c19Ua_121T, and c21Ua_68T) isolated from urine samples of healthy women or diagnosed with overactive bladder could not be allocated to any valid Corynebacterium species. In this work, we aimed to characterize these strains based on a polyphasic approach. The strains were Gram stain positive, rod to coccoid shaped, nonmotile, catalase positive, and oxidase negative. Phylogenetic analysis based on 16S rRNA and rpoB gene sequences indicated that all strains belonged to the genus Corynebacterium. The average nucleotide identity and digital DNA-DNA hybridization values among the genomes of the above eight strains and closely related type strains of the Corynebacterium genus were <95 (74.1%-93.9%) and <70% (22.2%-56.5%), respectively. Mycolic acids were identified in all strains. MK-8(H2) and/or MK-9(H2) were identified as the major menaquinones. The polar lipids' pattern mostly consisted of diphosphatidylglycerol, phosphatidylglycerol, and glycophospholipids. The major fatty acid was C18:1ω9c. Corynebacterium lehmanniae (c8Ua_144T = DSM 113405T = CCP 74T), Corynebacterium meitnerae (c8Ua_172T = DSM 113406T = CCP 75T), Corynebacterium evansiae (c8Ua_174T = DSM 113407T = CCP 76T), Corynebacterium curieae (c8Ua_181T = DSM 113408T = CCP 77T), Corynebacterium macclintockiae (c9Ua_112T = DSM 113409T = CCP 78T), Corynebacterium hesseae (c19Ua_109T = DSM 113410T= CCP 79T), Corynebacterium marquesiae (c19Ua_121T = DSM 113411T = CCP 80T), and Corynebacterium yonathiae (c21Ua_68T = DSM 113412T = CCP 81T) are proposed. This study evidenced that commonly used methodologies on FUM research presented limited resolution for discriminating Corynebacterium at the species level. Future research studying the biological mechanisms of the new Corynebacterium species here described may shed light on their possible beneficial role for healthy FUM.

Diploid Nuclei Occur throughout the Life Cycles of Pucciniales Fungi.

Within Eukaryotes, fungi are the typical representatives of haplontic life cycles. Basidiomycota fungi are dikaryotic in extensive parts of their life cycle, but diploid nuclei are known to form only in basidia. Among Basidiomycota, the Pucciniales are notorious for presenting the most complex life cycles, with high host specialization, and for their expanded genomes. Using cytogenomic (flow cytometry and cell sorting on propidium iodide-stained nuclei) and cytogenetic (FISH with rDNA probe) approaches, we report the widespread occurrence of replicating haploid and diploid nuclei (i.e., 1C, 2C and a small proportion of 4C nuclei) in diverse life cycle stages (pycnial, aecial, uredinial, and telial) of all 35 Pucciniales species analyzed, but not in sister taxa. These results suggest that the Pucciniales life cycle is distinct from any cycle known, i.e., neither haplontic, diplontic nor haplodiplontic, corroborating patchy and disregarded previous evidence. However, the biological basis and significance of this phenomenon remain undisclosed. IMPORTANCE Within Eukaryotes, fungi are the typical representatives of haplontic life cycles, contrasting with plants and animals. As such, fungi thus contain haploid nuclei throughout their life cycles, with sexual reproduction generating a single diploid cell upon karyogamy that immediately undergoes meiosis, thus resuming the haploid cycle. In this work, using cytogenetic and cytogenomic tools, we demonstrate that a vast group of fungi presents diploid nuclei throughout their life cycles, along with haploid nuclei, and that both types of nuclei replicate. Moreover, haploid nuclei are absent from urediniospores. The phenomenon appears to be transversal to the organisms in the order Pucciniales (rust fungi) and it does not occur in neighboring taxa, but a biological explanation or function for it remains elusive.