Genomic Features Research Articles

In vitro activity and genomic characterization of KPC-producing Klebsiella pneumoniae clinical blood culture isolates resistant to ceftazidime/avibactam, meropenem/vaborbactam, imipenem/relebactam: an Italian nationwide multicentre observational study (2022-23).

To evaluate the in vitro activity of ceftazidime/avibactam, meropenem/vaborbactam, imipenem/relebactam and comparators against KPC-producing Klebsiella pneumoniae (KPC-Kp) clinical isolates collected from a multicentre study in Italy (2022-23) and genomic characterization of the molecular mechanisms causing resistance. Consecutive KPC-Kp isolates from blood cultures (n = 264) were collected from 14 hospital centres in the period 2022-23. Antimicrobial susceptibility testing was performed using broth microdilution. WGS was used to investigate KPC-Kp strains resistant to the new approved β-lactam/β-lactam inhibitor combinations (BLICs). Overall, meropenem/vaborbactam (95.1% susceptible by EUCAST and 93.9% susceptible by CLSI; MIC50 = 0.5 mg/L; MIC90 = 4 mg/L) and imipenem/relebactam (97% susceptible by EUCAST and 92.8% susceptible by CLSI; MIC50 = 0.25 mg/L; MIC90 = 0.5 mg/L) showed similar activity, followed by ceftazidime/avibactam (93.9% susceptible by both EUCAST and CLSI; MIC50 = 2 mg/L; MIC90 = 8 mg/L). Ten out of 13 (76.9%) KPC-Kp resistant to ceftazidime/avibactam carried a blaKPC variant including blaKPC-31, blaKPC-205, blaKPC-203 and blaKPC-93. Among KPC-Kp resistant to meropenem/vaborbactam and imipenem/relebactam, 90.9% (10/11) and 80% (4/5) harboured a WT carbapenemase (i.e. blaKPC-2 or blaKPC-3), respectively. All strains resistant to meropenem/vaborbactam and/or imipenem/relebactam carried truncated OmpK35 and/or mutated (ins135GD) OmpK36. New BLICs were shown to be the most widely active therapeutic option against KPC-Kp clinical isolates collected in Italy. Ceftazidime/avibactam resistance is mainly driven by the expression of KPC variants, whereas the loss of function of the OmpK35 and OmpK36 porins appears to play a key but not exclusive role in the development of meropenem/vaborbactam and/or imipenem/relebactam resistance.

Genomic characterization and histologic analysis of uterine leiomyosarcoma arising from leiomyoma with bizarre nuclei.

Leiomyoma with bizarre nuclei (LM-BN) is a rare variant of leiomyoma with a benign clinical course. In contrast, leiomyosarcoma (LMS) is a high-grade, malignant neoplasm characterized by high recurrence rates and poor survival. While LM-BN and LMS show distinct morphologies, they share similar immunoprofiles and molecular alterations, with both considered 'genomically unstable'. Rare cases of LM-BN associated with LMS have been reported; however, the histogenesis and molecular relationship between these two tumors remains unclear. In this study, we assessed 11 cases of LMS arising in conjunction with LM-BN confirmed by histology and immunohistochemistry (IHC), further analyzed by clinical, histologic, and molecular characteristics of these distinct components. LM-BN and LMS had similar p16 and p53 IHC patterns, but LMS had a higher Ki-67 index and lower estrogen and progesterone eceptor expression. Digital image analysis based on cytologic features revealed spatial relationships between LMS and LM-BN. Genomic copy number alterations (CNAs) demonstrated the same clonal origin of LMS arising from existing LM-BN through conserved CNAs. LMS harbored highly complex CNAs and more frequent losses of the TP53, RB1, and PTEN genomic regions than LM-BN (p = 0.0031), with CDKN2A/B deletion identified in LMS only. Mutational profiling revealed many shared oncogenic alterations in both LM-BN and LMS; however, additional mutations were present within LMS, indicative of tumor progression through progressive genomic instability. Analysis of spatial transcriptomes defined uniquely expressed gene signatures that matched the geographic distribution of LM-BN, LMS, and other cell types. Our findings indicate for the first time that a subset of LMS arises from an existing LM-BN, and highly complex genomic alterations could be potential high risks associated with disease progression in LM-BN. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Segatella asaccharophila sp. nov., an anaerobic pectinophile isolated from a two-phase methane fermentation system.

An obligately anaerobic, Gram-stain-negative, non-spore-forming and non-motile rod (strain LPYR103-PreT) was isolated from a two-phase methane fermentation system. Using 16S rRNA gene sequence-based phylogenetic analysis, strain LPYR103-PreT was classified in the genus Segatella. The 16S rRNA gene sequence similarity, average nucleotide identity and digital DNA-DNA hybridization between strain LPYR103-PreT and its phylogenetically nearest species - Segatella cerevisiae JCM 30867T - were 94.4, 77.9 and 23.4%, respectively. The genome size of strain LPYR103-PreT was 3 326 733 bp, and its genomic DNA G+C content was 44.05%. The most abundant cellular fatty acid was anteiso-C15 : 0. The growth of strain LPYR103-PreT was stimulated by the addition of pectin, d-galacturonate and d-glucuronate; in contrast, the strain exhibited poor growth in the presence of common sugars, such as glucose. Therefore, strain LPYR103-PreT was classified as a pectinophile - a bacterium that shows a preference for pectin and a few related compounds as substrates. Glucose is degraded by type strains of 12 species belonging to the genus Segatella; thus, strain LPYR103-PreT is the first described pectinophile belonging to this genus. Strain LPYR103-PreT produced succinate and acetate as its major metabolic end products. Based on the differences in the phylogenetic, genomic, physiological and chemotaxonomic characteristics of strain LPYR103-PreT and related species, the name Segatella asaccharophila sp. nov. is proposed to accommodate strain LPYR103-PreT (= NRIC 0997T = JCM 37351T=DSM 118531T = KCTC 25923T).

H3K4me3 Genome-Wide Distribution and Transcriptional Regulation of Transposable Elements by RNA Pol2 Deposition

Zygotic genome activation (ZGA) is critical for early embryo development and is meticulously regulated by epigenetic modifications. H3K4me3 is a transcription-permissive histone mark preferentially found at promoters, but its distribution across genome features remains incompletely understood. In this study, we investigated the genome-wide enrichment of H3K4me3 during early embryo development and embryonic stem cells (ESCs) in both sheep and mice. We discovered that broad H3K4me3 domains were present in MII stage oocytes and were progressively diminished, while promoter H3K4me3 enrichment was increased and correlated with gene upregulation during ZGA in sheep. Additionally, we reported the dynamic distribution of H3K4me3 at the transposable elements (TEs) during early embryo development in both sheep and mice. Specifically, the H3K4me3 distribution of LINE1 and ERVL, two subsets of TEs, was associated with their expression during early embryo development in sheep. Furthermore, H3K4me3 enrichment in TEs was greatly increased during ZGA following Kdm5b knockdown, and the distribution of RNA polymerase II (Pol2) in TEs was also markedly increased in Kdm5b knockout ESCs in mice. These findings suggest that H3K4me3 plays important roles in regulating TE expression through interaction with RNA Pol2, providing valuable insights into the regulation of ZGA initiation and cell fate determination by H3K4me3.

Palmitoylation-related gene expression and its prognostic value in ovarian cancer: insights into immune infiltration and therapeutic potential

BackgroundPalmitoylation, a key post-translational modification, plays a significant role in ovarian cancer (OV) progression. However, the impact of palmitoylation-related genes on genomic instability, immune infiltration, and therapeutic response in OV remains poorly understood. This study aimed to investigate these factors to facilitate risk stratification and therapeutic intervention, providing insights into personalized treatment strategies.MethodsData from TCGA and GEO were utilized to develop a prognostic model based on palmitoylation-related genes. Differential expression, functional enrichment, and immune infiltration analyses were performed. Immune cell composition and pathway activities in different risk groups were assessed using CIBERSORT and ssGSEA algorithms. Immunotherapy response was predicted using TIDE and SubMap, while drug sensitivity differences were evaluated using the GDSC database.ResultsUnivariate, LASSO, and multivariate Cox regression analyses identified palmitoylation-related genes with significant prognostic value. The prognostic model effectively stratified patients into high- and low-risk groups, demonstrating significant survival differences. Immune infiltration analysis revealed distinct immune cell compositions and functions between risk groups. Low-risk patients exhibited higher immune scores and increased expression of immune checkpoints (PD-1, CD274, CTLA4), suggesting greater response to immunotherapy. Drug sensitivity analysis identified compounds with differential efficacy between risk groups, highlighting potential targeted treatment options.ConclusionPalmitoylation-related genomic features significantly influence OV progression and the immune landscape, offering potential for improved risk stratification and informing immunotherapy strategies to enhance patient outcomes.

Integrated lipidomics and RNA-seq reveal prognostic biomarkers in well-differentiated and dedifferentiated retroperitoneal liposarcoma

BackgroundRetroperitoneal liposarcoma (RLPS) is a mesenchymal malignant tumor characterized by different degrees of adipocytic differentiation. Well-differentiated liposarcoma (WDLPS) and dedifferentiated liposarcoma (DDLPS) are two of the most common subtypes of RLPS, exhibiting clear differences in biological behaviors and clinical prognosis. The metabolic features and genomic characteristics remain unclear.MethodsThis study employed lipidomic and RNA-seq analyses of RLPS tissues from 19 WDLPS and 29 DDLPS patients. Western blot and immunohistochemistry staining were performed to verify the tumor tissue protein levels of TIMP1, FN1, MMP11, GPNMB, and ECM1. Enzyme-linked immunosorbent assay (ELISA) was performed to evaluate different serum protein levels in 128 blood samples from patients with RLPS. Multivariate analysis was performed to identify the most crucial variables associated with overall survival (OS) and recurrence-free survival (RFS) of the RLPS patients.ResultsLipidomic analysis revealed a significant difference in lipid metabolism, particularly in phosphatidylcholines and triacylglycerides metabolism. RNA sequencing analysis revealed that 1,630 differentially expressed genes (DEGs) were significantly enriched in lipid metabolism, developmental process, and extracellular matrix (ECM) pathways. Integrated lipidomic and transcriptomic analysis identified 29 genes as potential biomarkers between WDLPS and DDLPS. Among the 29 DEGs, we found that TIMP1, FN1, MMP11, GPNMB, and ECM1 were increased in DDLPS tumor tissues than in WDLPS tumor tissues. The receiver operating characteristic (ROC) curve showed high specificity and sensitivity in diagnosing patients using a five-gene combination (AUC = 0.904). ELISA revealed a significant increase in the serum levels of ECM1 and GPNMB in patients with DDLPS compared to patients with WDLPS. ECM1 increased progressively across different FNCLCC Grades, correlating negatively with RFS (P = 0.043). GPNMB levels showed a negative correlation with OS (P = 0.019).ConclusionsOur study reveals different lipid metabolism, several transcriptional pathways between WDLPS and DDLPS, and examines several serum markers associated with the prognosis of RLPS. These findings provide a vital basis for future endeavors in diagnosing and predicting the prognosis of retroperitoneal liposarcoma with different differentiations.

Genomic characteristics of PD-L1-Induced resistance to EGFR-TKIs in lung adenocarcinoma.

The co-occurrence of PD-L1 positivity and EGFR mutations in advanced NSCLC often limits EGFR-TKIs effectiveness, with unclear mechanisms. We analyzed 103 treatment-naive EGFR-mutant LUAD patients from three centers, assessing PD-L1 expression and performing NGS analysis. SMO mutations and MET amplification were significantly higher in the PD-L1 ≥ 1% group versus PD-L1 < 1% group (SMO: 8% vs. 0%, p = 0.048; MET: 18% vs. 7%, p = 0.023). The DNA Damage Response and Repair (DDR) pathogenic deficiency mutations, along with biological processes and signaling pathways related to DNA recombination, cell cycle transition and abnormal phosphorylation, were more prevalent in the PD-L1 ≥ 1% group. PIK3CA and RARA clonal alterations were more common in PD-L1 < 1% group, while TP53 clonal mutations predominated in PD-L1 ≥ 1% group. Retrospective analysis showed EGFR-TKIs plus chemotherapy extended median PFS by 9.8 months, potentially overcoming EGFR-TKI monotherapy resistance. This study elucidates the genomic characteristics of PD-L1-induced resistance to EGFR-TKIs. For patients with concurrent mutations in EGFR and PD-L1 expression, a first-line treatment strategy combining EGFR-TKIs with chemotherapy may offer a more effective alternative.

Draft genome sequences of two distinct Clostridioides difficile isolates coinfecting a patient.

Clostridioides difficile is a spore-forming anaerobic Gram-positive bacillus. In this study, two distinct strains of C. difficile causing coinfection were isolated from the same fecal sample. Whole-genome sequencing was performed on these two strains to elucidate their genomic characteristics.

Genomic Characterizations of Klebsiella variicola: Emerging Pathogens Identified from Sepsis Patients in Ethiopian Referral Hospitals

Healthcare in low- and middle-income countries is becoming problematic due to the emergence of multidrug-resistant bacteria causing serious morbidity and mortality. Klebsiella variicola carrying multiple antimicrobial resistance (AMR) genes was found significantly among sepsis patients in a study done between October 2019 and September 2020 at four Ethiopian hospitals located in the central (Tikur Anbessa and Yekatit 12), southern (Hawassa), and northern (Dessie) parts. Among 1416 sepsis patients, 74 K. variicola isolates were identified using MALDI-TOF, most of them at Dessie (n=44) and Hawassa (n=28) hospitals. Whole genome sequencing showed that K. variicola strains identified at Dessie Hospital displayed phylogenetic clonality, carried an IncM1 plasmid and the majority were ST3924. Many K. variicola identified at Hawassa Hospital were clonally clustered and the majority belonged to novel STs and carried IncFIB(K) and IncFII(K) plasmids concurrently. Fifty K. variicola carried ESBL genes while 2 isolates harbored AmpC. Other frequently found genes were aac(3)-lla, bla CTX-M-15, bla TEM-1B, bla LEN2, bla OXA-1, bla SCO-1, catB3, dfrA14, QnrB1, aac(6')-lb-cr and sul2. Virulence genes detected at both sites were mrk operons for biofilm formation and siderophore ABC transporter operons for iron uptake. Capsular alleles varied, with wzi 269 at Dessie and wzi 582 at Hawassa. The isolation of multidrug-resistant K. variicola as an emerging sepsis pathogen calls for strong infection prevention strategies and antimicrobial stewardship supported by advanced bacterial identification techniques.

Genomic analysis, culturing optimization, and characterization of Escherichia bacteriophage OSYSP, previously studied as effective pathogen control on fresh produce

Advances in bacteriophage genome sequencing and regulatory approvals of some bacteriophages in various applications have renewed interest in these antibacterial viruses as a potential solution to persistent food safety challenges. Here, we analyzed in depth the genome of the previously studied Escherichia bacteriophage OSYSP (phage OSYSP), revealed its application-related characteristics, and optimized its enumeration techniques for facilitating industrial implementation. We previously sequenced phage OSYSP genome completely by combining results from Illumina Miseq and Ion Torrent sequencing platforms and completing the remaining sequence gaps using PCR. Based on the genomics analysis completed herein, phage OSYSP was confirmed as an obligate lytic phage of the Caudoviricetes class. The genome encodes 81 proteins of identifiable functions, including two endolysins and 45 proteins that support host-independent DNA replication, transcription, and repair. Despite its similarities to T5-like phages, unique genome arrangements confirm phage OSYSP’s novelty. The genomic analysis also confirmed the absence of DNA sequences encoding virulence or antibiotic resistance factors. For optimizing phage detection and quantification in the conventional plaque assay, it was observed that decreasing the concentration of agar or agarose, when used as a medium gelling agent, increased phage recovery (p &amp;lt; 0.05), but using agarose resulted in smaller plaque diameters (p &amp;lt; 0.05). Phage OSYSP inactivated pathogenic and non-pathogenic strains of E. coli and some Salmonella enterica serovars, with more pronounced effect against E. coli O157:H7. Phage titers remained fairly unchanged throughout a 24-month storage at 4°C. Incubation for 30 min at 4°C−47°C or pH 4–11 had no significant detrimental effect (p &amp;gt; 0.05) on phage infectivity. In vitro application of phage OSYSP against E. coli O157:H7 EDL933 decreased the pathogen’s viable population by &amp;gt;5.7-log CFU/mL within 80 min, at a multiplicity of infection as low as 0.01. The favorable genome characteristics, combined with improved enumeration methodology, and the proven infectivity stability, make phage OSYSP a promising biocontrol agent against pathogenic E. coli for food or therapeutic applications.

Natural infection of common cranes (Grus grus) with highly pathogenic avian influenza H5N1 in Serbia

IntroductionThe late autumn epizootic of the highly pathogenic avian influenza virus (HPAIV) subtype H5N1 in Serbia in 2023 caused massive mortality in the migratory population of common cranes (Grus Grus). This is the first time HPAIV has been identified in the common crane in Serbia, leading to mass mortality of this bird species.MethodsTo understand the pathological impact of HPAIV in cranes, we evaluated the pathological changes in the tissues of common cranes. Additionally, we report genomic characterization of HPAI/H5N1. In total, 14 juvenile common crane carcasses were examined.ResultsInfected birds primarily exhibited neurologic signs, including ataxia and incoordination. Grossly, necrotizing pancreatitis was the most common finding, while microscopic lesions included necrosis, inflammation and hemorrhages in the lungs, spleen, brain, liver and kidneys. Based on RT-PCR, all birds were infected with the HPAI H5N1 virus, as viral RNA was detected in all 14 selected tissues. Genetic analysis revealed that our H5N1 isolate could be grouped with highly pathogenic avian influenza clade 2.3.4.4b, subgroup DA, and is very closely related to the H5N1 strains isolated from the common crane and turkey from Croatia, the common crane from Italy and the Ural owl from Slovakia.DiscussionOur findings showed that common cranes are highly susceptible to natural infection with the HPAI H5N1 virus of clade 2.3.4.4b and may serve as bio-sentinels for the presence of the HPAI virus in wildlife.

High activity and specificity of bacteriophage cocktails against carbapenem-resistant Klebsiella pneumoniae belonging to the high-risk clones CG258 and ST307

IntroductionThe widespread clinical and environmental dissemination of successful clones of carbapenem-resistant Klebsiella pneumoniae (CRKP) represents a serious global public health threat. In this context, lytic bacteriophages have emerged as a promising alternative for controlling these pathogens. This study describes the biological, structural, and genomic characteristics of lytic bacteriophages against the high-risk CRKP clones CG258 and ST307 and describes their performance in combination.MethodsAn experimental study was carried out. Bacteriophages were isolated from hospital wastewater and from wastewater treatment plants (WWTP). Bacteriophages were isolated using the double layer agar technique and their characterization included host range (individual and cocktail), plating efficiency (EOP), infection or bacterial killing curve, one-step curve, bacteriophage stability at pH and temperature conditions, transmission electron microscopy (TEM) and whole genome sequencing.ResultsAfter purification, five active bacteriophages against CRKP were obtained, three bacteriophages (FKP3, FKP4 and FKP14) had targeted activities against CG258 CRKP and two (FKP10 and FKP12) against ST307 isolates. Seven cocktails were prepared, of which Cocktail 2, made up of the bacteriophages FKP3, FKP10, and FKP14, showed the best activity against 85.7% (n = 36/42) of CRKP isolates belonging to both clones, CG258 (80.8%; n = 21/26) and ST307 (93.8%, n = 15/16). The efficiency of the plating (EOP), infection curve, and one-step growth curve showed that the cocktail phages efficiently infected other CRKP isolates (EOP ≥ 0.5), controlled bacterial growth up to 73.5%, and had short latency periods, respectively, (5–10 min). In addition, they were stable at temperatures between 4°C and 50°C and pH between 4 and 10. All bacteriophages belonged to the Caudoviricetes class, and no genes associated with virulence factors or antibiotic resistance were detected.ConclusionThese findings showed bacteriophages and phage cocktails with high specificity against CRKP belonging to the successful clones CG258 and ST307 with promising characteristics, making them an alternative for controlling these clones in different environmental or health settings, biocontrol agents, or disinfectants in industry and in the field of diagnosis.

Revealing the Dynamic History of Parasitic Plant Plastomes via Structural Characterization, Comparative Analysis, and Phylogenomics

Background: The shift to a parasitic lifestyle in plants often leaves distinct marks on their plastid genomes, given the central role plastids play in photosynthesis. Studying these unique adaptations in parasitic plants is essential for understanding the mechanisms and evolutionary patterns driving plastome reduction in angiosperms. By exploring these changes, we can gain deeper insights into how parasitism reshapes the genomic architecture of plants. Method: This study analyzed and compared the plastomes of 113 parasitic plants from different families. Results: The Orobanchaceae family (hemiparasitic plants) displayed the largest plastome size, while Apodanthaceae exhibited the shortest. Additionally, Orobanchaceae showcased little to no gene loss in their plastomes. However, holoparasitic species typically had reduced plastome sizes. Convolvulaceae exhibited significantly reduced plastome sizes due to high gene loss, and Apodanthaceae retained only a few genes. Gene divergence among different families was also investigated, and rps15, rps18, and rpl33 in Orobanchaceae; accD and ycf1 in Convolvulaceae; atpF and ccsA in Loranthaceae; and rpl32 in Santalaceae showed greater divergence. Additionally, Orobanchaceae had the highest numbers of all repeat types, whereas Loranthaceae and Convolvulaceae exhibited the lowest repeat numbers. Similarly, more simple sequence repeats were reported in Loranthaceae and Santalaceae. Our phylogenetic analysis also uncovered a distinct clade comprising Loranthaceae, with a single Schoepfiaceae species clustering nearby. Contrary to expectations, parasitic and hemiparasitic plants formed mixed groupings instead of segregating into separate clades. Conclusions: These findings offer insights into parasitic plants’ evolutionary relationships, revealing shared and divergent genomic features across diverse lineages.

Characterization of the Mitochondrial Genome of Cambaroides schrenckii (Astacidea: Cambaridae) and Its Phylogenetic Implications

Background: Cambaroides schrenckii is an endangered freshwater crayfish in China, belonging to the genus Cambaroides, that can act as a complementary host for paragonimus. The objective of this study was to examine the complete mitochondrial genome characteristics and their evolutionary relationships within the Astacidea. Methods: The analysis of gene rearrangements and evolutionary relationships was conducted through the sequencing of the mitochondrial genome of C. schrenckii. Results: C. schrenckii mitochondrial genome length was 15,572, comprising thirteen PCGs, two rRNAs, 22 tRNAs, and one d-loop region of C. schrenckii. The mitochondrial genome of C. schrenckii exhibits an A + T content of 69.61% and a G + C content of 30.39%. Among the thirteen PCGs, cytb, nad3, and nad6 have a start codon of ATT, while the other ten PCGs have ATC, ATA, and ATG start codons. All 22 tRNA genes displayed a typical cloverleaf secondary structure. Gene rearrangement analysis showed that seven gene arrangements were identified based on PCGs in the infraorder Astacidea, with type I being the most common. Conclusions: The relationship between the American Cambaridae is closer to Astacidae than the Asian Cambaridae. The present study provides a theoretical basis for further discussions of developmental relationships in the infraorder Astacidea.

Characterization and identification of Pseudomonas sp. AW4, an arsenic-resistant and plant growth-promoting bacteria isolated from the soybean (Glycine max L.) rhizosphere.

Pseudomonas sp. AW4 is a highly arsenic (As) resistant bacterium with plant growth promoting properties, originally isolated from the soybean (Glycine max L.) rhizosphere. In order to safely use this isolate in diverse bioformulations, its characterization needs to be completed and a reliable identification must be provided. In the present work, we analyzed the morpho-physiological, biochemical and genomic characteristics of Pseudomonas sp. AW4. Identification of the isolate varied according to the parameters analyzed, mainly biochemical and physiological tests or individual genes and phylogenetic analyses. In this regard, we performed massive sequencing of its genome, in order to consistently complete its characterization and identification. Pseudomonas sp. AW4 formed a monophyletic clade with P. urmiensis SWRI10, presenting 3.08% of unique genes against this reference isolate. More than 70% of AW4 genes were also shared with P. oryziphila strain 1257 NZ and with P. reidholzensis strain CCOS 865. The search for genes related to As resistance evidenced the presence of the operon arsHRBC. Taken together, results of the present work allow identification of this bacterium as Pseudomonas urmiensis AW4 and open up a number of opportunities to study this strain and understand the mechanisms of arsenic resistance and plant growth promotion.

Multispecies emergence of dual blaKPC/NDM carbapenemase-producing Enterobacterales recovered from invasive infections in Chile.

Carbapenemase-producing carbapenem-resistant Enterobacterales (CP-CRE) represent a significant global threat. The emergence of dual CP-CRE is particularly alarming, as they can potentially compromise the efficacy of newer antibiotics, further decreasing therapeutic alternatives. Herein, we report the emergence of multiple species of CP-CRE recovered from invasive infections in Chile that simultaneously harbor blaKPC and blaNDM and provide an in-depth genomic characterization of these worrisome pathogens. We collected carbapenem-resistant Enterobacterales (CRE) isolates from invasive infections over a 4-year period, across 11 healthcare centers in Chile. Bacterial species and the presence of carbapenemase genes were confirmed using MALDI-TOF and PCR assays, respectively. Antimicrobial susceptibility testing was conducted through disk diffusion and broth microdilution methods. Dual CP-CRE isolates were subjected to short- and long-read whole genome sequencing to perform a detailed genomic characterization of the isolates and of the mobile genetic elements harboring the enzymes. From a total of 1,335 CRE isolates, we observed an increase in the prevalence of CP-CRE, from 11% in 2019 to 38% in 2022. A total of 11 dual CP-CRE isolates were recovered, all of them harboring blaKPC and blaNDM. Species corresponded to Escherichia coli (n = 6), Klebsiella pneumoniae (n = 2), Klebsiella oxytoca (n = 2), and Citrobacter freundii (n = 1). Dual CP-CRE isolates exhibited resistance to all tested β-lactams except for cefiderocol. The blaKPC and blaNDM encoding genes were located on independent plasmids. Platforms harboring blaKPC were diverse and included IncN, IncF, and IncFIB plasmids. In contrast, blaNDM-7 was only found on fairly conserved IncX3 plasmids. We report that a rapid increase of CP-CRE in Chile, alongside with the emergence of multiple bacterial species of CP-CRE co-harboring blaKPC-2/3 and blaNDM-7, underscores a critical public health challenge. Our data suggest that the dissemination of blaNDM-7 was predominantly facilitated by IncX3 plasmids, whereas the spread of blaKPC involved multiple plasmid backbones. Active surveillance and genomic monitoring are critical to inform public policy and curtail the spread of these highly resistant pathogens.

Integrating genomic evidence for an updated taxonomy of the bacterial genus Spiribacter

The genus Spiribacter encompasses halophilic bacteria widely distributed in hypersaline environments worldwide. Despite their ecological significance, initially isolating Spiribacter species under laboratory settings was challenging due to the lack of knowledge of their growth and cultivation requirements. However, with improved understanding of their ecological niche and metabolic pathways, additional species of Spiribacter have been successfully isolated and identified from diverse locations around the globe. Enriched media with sodium pyruvate as carbon source facilitated the isolation of twelve new strains closely related to the genus Spiribacter from hypersaline environments in Spain. Genome sequencing and analysis of these new strains and previously described Spiribacter species provided insights into their genomic features and phylogenomic relationships, supporting the delineation of three distinct new species within this genus, designated as Spiribacter insolitus sp. nov., Spiribacter onubensis sp. nov., and Spiribacter pallidus sp. nov. In Spiribacter species, streamlined genomes enhance survival in hypersaline environments by reducing non-essential genes and optimizing resource utilization. Key genes involved in osmoprotectant mechanisms, including those for the metabolism of myo-inositol, hydroxyproline, and L-proline, were identified and numerous transporters were noted, ensuring efficient nutrient acquisition and osmotic balance. Notably, these new species, along with other Spiribacter strains, exhibit metabolic diversity in utilizing inorganic sulfur compounds, including thiosulfate and tetrathionate, for energy production and adaptation to hypersaline environments. The presence of thiosulfate dehydrogenase (TsdA) genes suggests their capability to oxidize thiosulfate to tetrathionate, potentially influencing both aerobic and anaerobic respiration. Furthermore, the prevalence of the sqr gene indicates a role for sulfide oxidation in Spiribacter metabolism, underlining their metabolic versatility in saline habitats. These adaptations allow Spiribacter to thrive in nutrient-limited, high-salinity habitats. Moreover, genome mining analysis and physiological disparities observed in the already described species Spiribacter halobius raise significant challenges to its classification within the genus Spiribacter.

Construction and Isolation of Recombinant Vaccinia Virus by Homologous Recombination Using Fluorescent Protein Markers.

Genetic modification of vaccinia virus (VACV) is a fundamental and valuable research technique in elucidating the function of VACV genes, as well as the development as vaccine vectors for other infectious diseases, oncolytic therapeutics for cancers, and protein expression systems in mammalian cells. Because of the large size of poxvirus genome and noninfectious feature of the naked viral DNA, construction of recombinant VACV relies on intracellular homologous recombination between transfected DNA and replicating viral DNA in infected cells occurred in VACV infected cells. The efficiency of homologous recombination event for vaccinia virus is relatively low, and recombinant viruses only account for 0.1% of progeny viruses. Therefore, fluorescent protein markers are often included in the transfected DNA to facilitate the selection and screening of recombined viruses. Here we provide a detailed procedure for the design, generation, isolation, and detection of recombinant VACV by homologous recombination using fluorescent protein markers.

Integrating genetic and morphological data to assess species delimitation of two Japanese large abalones: Haliotis discus discus and H. madaka

Abstract Two large abalone species prevalent in the Japanese archipelago, Haliotis discus discus and H. madaka, are considered to have different morphological characteristics, and previous population genomics analyses have suggested that they have distinct genomic features. However, integrated analyses comparing the genetic and morphological data from these species are lacking; thus, it remains unclear whether these two species are distinct biological species. In this study, we performed integrated genetic and morphological analyses on individuals of the two species captured in Tokushima Prefecture, Japan. Genetic analysis based on 118 species-diagnostic single-nucleotide polymorphism (SNP) loci showed that H. discus discus and H. madaka were clearly assigned to distinct genetic clusters corresponding to the two species, with the exception of one H. madaka individual showing evidence of genomic introgression from H. discus discus. Multiple statistical analyses based on quantitative characteristics of shell shape and foot muscle color suggested that this H. madaka individual with genomic introgression from H. discus discus had H. discus discus-like quantitative characteristics consistently. In summary, this study suggests that H. discus discus and H. madaka are distinct biological species reproductively isolated in natural environments, but genomic introgression possibly blurs the species boundary between H. discus discus and H. madaka.

Giant cell tumor of the tendon sheath: A critical review of current diagnostic and therapeutic approaches with treatment recommendations for hand and foot lesions

Giant cell tumor of the tendon sheath (GCTTS) is a common tumor affecting the hand and foot, often presenting diagnostic and therapeutic challenges due to its variable clinical presentations and biological behaviors. Recent evidence supports a neoplastic origin for this tumor, previously known by various names. GCTTS can be categorized as localized or diffuse, with distinct predilections for specific anatomical locations. While localized GCTTS is typically benign and more common in the hand and wrist, the diffuse form can be aggressive, also affecting the foot and ankle. This critical analysis review aims to provide a comprehensive overview of GCTTS in hand and foot, encompassing its clinical, radiological, histological, and genomic features. In addition, we summarize current management practices, discuss differential diagnoses, analyze recurrence rates following surgical excision, and offer evidence-based treatment recommendations for this diverse group of tumors.