Whole Genome Sequencing Analysis Research Articles

Genomic insights into drug resistance and virulence determinants in rare pyomelanin-producing clinical isolates of Acinetobacter baumannii.

Clinical isolates of multi-drug resistant Acinetobacter baumannii are a major cause of nosocomial infections, often attributed to the highly adaptable genome that helps it tothrive under environmental selection pressure. Here, we aim to provide genotypic-based surveys and comparative whole genome sequencing (WGS) analysis to explore the genomics of the rare pyomelanin-forming clinical isolates of A. baumannii from India. A total of 54 clinical isolates of A. baumannii obtained from two tertiary care hospitals were genotyped using repetitive sequence-based PCR(REP-PCR) for elucidating their molecular epidemiology, followed by their resistance profiling through the determination of minimum inhibitory concentration using the micro broth dilution method. The isolates' virulence and antibiotic-resistant determinants were detected by PCR screening, followed by biofilm quantification. Pyomelanin pigment produced by A. baumannii isolates was isolated and chemically characterized. Finally, WGS of three pigment-producing and one non-producing A. baumannii strains was performed to explore the factors contributing to their variability. REP-PCR genotyping identified around 8 clusters, with all isolates being multidrug-resistant (MDR). Pyomelanin-producing isolates were strong biofilm formers, characterized by the concurrent presence of 'pgaB, BfmR, BfmS, ompA, and cusE' biofilm-related genes. These pigmented strains belonged to ST2Pas and co-harbored blaOXA-23, blaADC-25, aph (3')-VIa, armA, aph (6)-Id, tet(B) and msr(E) genes. Thirteen common IS elements and biosynthetic gene clusters of arylpolyene, NI-siderophore, and NRP-metallophore were identified. Notably, genomic islands containing aminoglycoside 3'-phosphotransferase, oxidative stress, two-component response regulators, efflux pump-related, toxin-antitoxin protein, and virulence-related genes were also mapped by WGS. The pyomelanin-forming isolates were MDR and virulent. The elucidation of WGS analysis provided critical insights for understanding the epidemiology, virulome, and mobilome of rare pigment-producing A. baumannii strains.

Antimicrobial resistance and virulence factors analysis of a multidrug-resistant Acinetobacter baumannii isolated from chickens using whole-genome sequencing

Multidrug-resistant (MDR) Acinetobacter baumannii (A. baumannii) is currently recognized not only as a significant nosocomial pathogen but also is an emerging bacterial infection in food-producing animals, posing a critical threat to global health. However, this is a hindrance to detailed bioinformatic studies of MDR A. baumannii of chicken origin due to the lack of its complete genome sequence. Here, we report whole-genome sequencing analysis of MDR A. baumannii Y03 isolated from chickens. The Y03 genome consists of 1 circular chromosome and 4 circular plasmids, The Y03 chromosome harbors 41 antimicrobial resistance genes conferring resistance to major classes of antibiotics, including β-lactams, phenicols, macrolides, lincosamides, aminoglycosides, and nitrofurans, as well as 135 virulence factors involved in effector delivery system, immune modulation, adherence, stress survival, biofilm, exotoxin, and nutritional/metabolic factor. The in vivo infection experiments certificated that Y03 was virulent to chickens. Meanwhile, we used PCR amplification method to detect 10 antimicrobial resistance genes including abeM, adeB, adeH, adeK, blaapmC, blaOXA−90, catB9, macB, folP, and parE, as well as 14 virulence genes including lpxC, pilO, fimT, ompA, basA, bauA, gspL, csu, pgaC, plc2, tssA, tviB, bap, and vgrG. Whole-genome sequencing analysis revealed that Y03 contained 46 horizontal gene transfer elements, including 11 genomic islands, 30 transposons, and 5 prophages, as well as 518 mutations associated with reduced virulence and 44 mutations resulting in loss of pathogenicity. Furthermore, there were 22 antibiotic targets and 28 lethal mutations on the Y03 chromosome that could be used as potential targets to prevent, control, and treat infections caused by MDR A. baumannii Y03. Therefore, this study contributes to the development of strategies for the prevention, control, and treatment of A. baumannii infections and their spread in chickens.

Emergence and clonal dissemination of KPC-2- and NDM-1-coharboring Citrobacter freundii in China with an IncR plasmid.

The rise of carbapenem-resistant Enterobacteriaceae coharboring KPC-2 and NDM-1 poses a significant public health threat. KPC-2-NDM-1-Citrobacter freundii is rarely reported in clinical settings. In this study, we report the largest cohort of eight KPC-2-NDM-1-C. freundii isolated from children with urinary tract infections. Comprehensive characterization, including antimicrobial susceptibility testing, plasmid conjugation, whole-genome sequencing, and comparative genomics, was conducted for these clinical strains. Antimicrobial susceptibility testing indicated that all strains were resistant to meropenem [minimal inhibitory concentration (MICs) ≥ 8 µg/mL] and imipenem (MICs > 8 µg/mL). Genotyping and comparative genomics analyses identified the eight KPC-2-NDM-1- C. freundii belonging to ST523, exhibiting a close relationship characterized by 45 single nucleotide polymorphisms. Whole-genome sequencing and plasmid analysis confirmed the presence of blaKPC-2 and blaNDM-1 on an IncR plasmid named pC275-2 with 46,050 bp. The blaNDM-1 was integrated within the blaNDM-1 related region, which includes ΔISAba125, blaNDM-1, ble-MBL, trpF, dsbD, and ISCR1, while the blaKPC-2 gene was located on a novel non-Tn4401 genetic element. The blaKPC-2 genetic architectures containing blaKPC-2 differed from classical structures, underscoring the ongoing evolution of these genetic elements.IMPORTANCEOur study described the largest cohort to date of eight ST523 KPC-2-NDM-1-C. freundii isolated from children with urinary tract infections. The cooccurrence of KPC-2, a serine β-lactamases, and NDM-1, a metallo-β-lactamase on an IncR plasmid pC275-2 from a clinical carbapenem-resistant C. freundii. The conserved insertion structure mediated the blaNDM-1, and the propagation of blaKPC-2 gene with a new genetic background using IncR plasmid in clinical settings promotes the emergence of superbugs necessitating vigilant monitoring. Our research detected that ST523 KPC-2- NDM-1-C. freundii were disseminated in a children's hospital. The potential spread of an IncR plasmid within the hospital raises concerns about the pandemic potential of this clone that produces two carbapenemases: KPC-2 and NDM-1. Further investigations will be necessary to control and prevent the spread of KPC-2-NDM-1-C. freundiis.

Whole-genome sequencing and genomic analysis of four Akkermansia strains newly isolated from human feces

BackgroundNumerous studies have demonstrated that Akkermansia is closely associated with human health. These bacteria colonize the mucus layer of the gastrointestinal tract and utilize mucin as their sole source of carbon and nitrogen. Akkermansia spp. exhibit potential as probiotics under specific conditions. However, the gene accumulation curve derived from pan-genome analysis suggests that the genome of Akkermansia strains remains open. Consequently, current genome mining efforts are insufficient to fully capture the intraspecific and interspecific characteristics of Akkermansia, necessitating continuous exploration of the genomic and phenotypic diversity of new isolates.MethodsBased on this finding, we sequenced, assembled, and functionally annotated the whole genomes of four new human isolates from our laboratory: AKK-HX001, AKK-HX002, AKK-HX003, and AKK-HX004.ResultsPhylogenetic analysis revealed that all four isolates belonged to the AmII phylogroup, whereas the type strain DSM 22959 is classified within the AmI phylogroup. Moreover, 2,184 shared homologous genes were identified among the four isolates. Functional annotation using the COG, KEGG, and CAZy databases indicated that the functional genes of the four isolates were primarily associated with metabolism. Two antibiotic resistance genes were identified in AKK-HX001 and AKK-HX002, while three resistance genes were detected in AKK-HX003 and AKK-HX004. Additionally, each of the four isolates possessed two virulence genes and three pathogenicity genes, none of which were associated with pathogenicity. The prediction of mobile genetic elements indicated unequal distributions of GIs among the isolates, and a complete CRISPR system was identified in all isolates except AKK-HX003. Two annotated regions of secondary metabolite biosynthesis genes, both belonging to Terpene, were detected using the antiSMASH online tool.ConclusionThese findings indicate that the four Akkermansia isolates, which belong to a phylogroup distinct from the model strain DSM 22959, exhibit lower genetic risk and may serve as potential probiotic resources for future research.

Draft Whole-Genome Sequencing and Phenotypic Analysis of Salmonella from Retail Aquatic Products in Weifang.

Salmonella is one of the most common causative agents of infectious diarrhea in humans, but in China, there are very limited data on the presence of Salmonella in aquatic products. This study describes the isolation of Salmonella from aquatic products in Weifang, China, from April 2022 to April 2023. Seven out of 160 (4.38%) retail aquatic product samples were positive for Salmonella. Two distinct serotypes were identified: Salmonella enterica subsp. enterica serovar Senftenberg (n = 4) and S. enterica subsp. diarizonae serovar IIIb 59:z10:z57 (n = 3). The results of molecular typing of isolates with the same serotype were consistent. Only one of the isolates was resistant to ampicillin, while the other isolates were not resistant to the tested antibiotics, suggesting that Salmonella in aquatic products in this region are relatively susceptible to antibiotics. There were 17 resistance genes in the 7 strains, 13 of which were shared. golS, MdtK, mdsA, and mdtG were unique to S. Senftenberg. A total of 155 virulence genes were annotated in the S. Senftenberg isolates, and 136 virulence genes were annotated in the S. IIIb 59:z10:z57 isolates. The S. Senftenberg isolates harbored more adhesion-related genes than the S. IIIb 59:z10:z57 isolates. Multilocus sequence typing analysis revealed that ST34 has been the most prevalent type of Salmonella in China since 2020, followed by ST11. The predominant type of Salmonella in aquaculture is ST14. This study provided additional genetic information about Salmonella in aquatic sources, providing a basis for subsequent research related to risk assessment, antibiotic resistance mechanisms, and so forth.

Efficacy of colistin-based combinations against pandrug-resistant whole-genome-sequenced Klebsiella pneumoniae isolated from hospitalized patients in Egypt: an in vitro/vivo comparative study

BackgroundColistin resistance significantly constrains available treatment options and results in the emergence of pandrug-resistant (PDR) strains. Treating PDR infections is a major public health issue. A promising solution lies in using colistin-based combinations. Despite the availability of in vitro data evaluating these combinations, the in vivo studies remain limited.ResultsThirty colistin-resistant Klebsiella pneumoniae (ColRKp) isolates were collected from hospitalized patients. Colistin resistance was detected using broth microdilution, and antimicrobial susceptibility was tested using the Kirby-Bauer method against 18 antibiotics. Extremely high resistance levels were detected, with 17% of the isolates being PDR. Virulence profiling, assessed using Anthony capsule staining, the string test, and the crystal violet assay, indicated the predominance of non-biofilm formers and non-hypermucoid strains. The isolates were screened for mcr genes using polymerase chain reaction. Whole-genome sequencing (WGS) and bioinformatics analysis were performed to characterize the genomes of PDR isolates. No plasmid-borne mcr genes were detected, and WGS analysis revealed that PDR isolates belonged to the high-risk clones: ST14 (n = 1), ST147 (n = 2), and ST383 (n = 2). They carried genes encoding extended-spectrum β-lactamases and carbapenemases, blaCTX-M-15 and blaNDM-5, on conjugative IncHI1B/IncFIB plasmids, illustrating the convergence of virulence and resistance genes. The most common mechanism of colistin resistance involved alterations in mgrB. Furthermore, deleterious amino acid substitutions were also detected within PhoQ, PmrC, CrrB, ArnB, and ArnT. Seven colistin-containing combinations were compared using the checkerboard experiment. Synergy was observed when combining colistin with tigecycline, doxycycline, levofloxacin, ciprofloxacin, sulfamethoxazole/trimethoprim, imipenem, or meropenem. The efficacy of colistin combined with either doxycycline or levofloxacin was assessed in vitro using a resistance modulation assay, and in vivo, using a murine infection model. In vitro, doxycycline and levofloxacin reversed colistin resistance in 80% and 73.3% of the population, respectively. In vivo, the colistin + doxycycline combination demonstrated superiority over colistin + levofloxacin, rescuing 80% of infected animals, and reducing bacterial bioburden in the liver and kidneys while preserving nearly intact lung histology.ConclusionsThis study represents the first comparative in vitro and in vivo investigation of the efficacy of colistin + doxycycline and colistin + levofloxacin combinations in clinical PDR ColRKp isolates characterized at a genomic level.

Genomic Investigation and Comparative Analysis of European High-Risk Clone of Acinetobacter baumannii ST2

Multidrug-resistant Acinetobacter baumannii is a major concern in healthcare institutions worldwide. Several reports described the dissemination of A. baumannii high-risk clones that are responsible for a high number of difficult-to-treat infections. In our study, 19 multidrug-resistant A. baumannii strains from Budapest, Hungary, were investigated based on whole-genome sequencing (WGS). The obtained results were analysed together with data from 433 strains of A. baumannii from the Pathogenwatch database. WGS analysis of 19 A. baumannii strains detected that 12 belonged to ST2 and seven belonged to ST636. Among ST2 strains, 11 out of 12 carried either blaOXA-23 or blaOXA-58 genes; however, all strains of ST636 uniformly carried blaOXA-72 gene. All strains of ST2 and ST636 carried blaOXA-66 and blaADC-25 genes. Based on core genome multilocus sequence typing (cgMLST), 10 strains of ST2 belonged to cgMLST906, one strain to cgMLST458, and one strain to cgMLST1320; by contrast, all strains of ST636 belonged to cgMLST1178. Certain virulence determinants were present in all strains of both ST2 and ST636, namely, Ata, Bap, BfmRS, T2SS and PNAG. Interestingly, OmpA was present in all strains of ST2, but it was absent in all strains of ST636. Comparative analysis of 19 strains of this study and the collection of 433 isolates from Pathogenwatch database, proved a diverse clonal distribution of high-risk A. baumannii clones in Europe. The major clone in Europe is ST2, which is present all over the continent. However, ST636 has been mainly reported in Eastern Europe. Interestingly, cgMLSTs of ST2 correspond to the production of different beta-lactamases, namely, OXA-82 in cgMLST116, OXA-72 in cgMLST506, and cgMLST556, PER-1 in cgMLST456 and cgMLST1041. Our study demonstrates that the ST2 high-risk clone of A. baumannii is the most widespread in Europe; however, based on cgMLST analysis, a detailed detection of beta-lactamase production can be determined.

Biodegradation of different keratin waste by newly isolated thermophilic Brevibacillus gelatini LD5: Insights into the degradation mechanism based on genomic analysis and keratin structural changes.

Keratin is an abundant environmental solid waste. This work isolated a thermophilic strain from a hot spring with efficient keratinolytic ability. The strain was identified and named as Brevibacillus gelatini LD5 based on whole-genome sequence analysis. The strain has genes related to keratin degradation, including disulfide reduction, keratin denaturation, protein proteolysis and metabolism of amino acids. The keratinases derived from this strain were the endo-acting M4, M16 and S8 proteases, exo-acting S9 protease and oligo-acting M3 and M32 peptidases via Conserved Unique Peptide Patterns (CUPP) prediction. The LD5 can degrade different keratin biomass, e.g. chicken feathers (CF), goose feathers (GF), pig hair (PH), cat hair (CH) and dog hair (DH). The degradation rate of CF was 62.45% after 24-h fermentation. The hydrolysates from different keratin biomass have all shown keratinolytic activity, antioxidant and antiradical activities. The random structure of keratin was easier to be degraded by LD5 from Fourier transform infrared (FT-IR) analysis. The optimum temperature-pH conditions of the keratinases were 79.8°C and pH7.5, and thermal stability of the keratinases reached 71.5min at 70°C. These results demonstrated that B. gelatini LD5 has potential application in keratin wastes biodegradation and thermal stable keratinase production.

Co-existence of airborne SARS-CoV-2 infection and non-infection in three connected zones of a restaurant

The lack of knowledge on quanta generation rates presents a major obstacle to specifying the minimum ventilation required to prevent airborne infections. The expected largest quanta generation rate of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by a super-spreader remains unknown. Here we investigated a SARS-CoV-2 outbreak during lunch in a restaurant using epidemiological, whole-genome sequencing and environmental analyses. Both tracer gas and fine particles were used in field experiments to quantify aerosol dispersion and removal across three interconnected zones: Zone A, Zone B and Zone C. All 21 secondary patron infections occurred in Zone B. This unique infection feature and measured dilution flow rates allowed us to estimate the largest reported quanta generation rates to date, ranging from 1724 to 1968 quanta/h. These rates were sufficiently high to cause a high attack rate in Zone B but did not cause infections in Zones A and C, likely due to sufficient dilution and insignificant contaminated airflow from Zone B, respectively. Our finding of the largest quanta generation rate so far suggests that avoiding secondary infection by dilution alone in the presence of a super-emitter might not be possible in typical air-conditioned buildings and other prevention strategies need to be developed.

Cyclic Peptide MV6, an Aminoglycoside Efficacy Enhancer Against Acinetobacter baumannii

Background/Objectives: Acinetobacter baumannii is a globally emerging pathogen with widespread antimicrobial resistance driven by multiple mechanisms, such as altered expression of efflux pumps like AdeABC, placing it as a priority for research. Driven by the lack of new treatments, alternative approaches are being explored to combat its infections, among which efficacy-enhancing adjuvants can be found. This study presents and characterizes MV6, a synthetic cyclic peptide that boosts aminoglycoside efficacy. Methods: MV6’s activity was assessed through antimicrobial susceptibility testing in combination with different antibiotic classes against A. baumannii strains characterized by PCR and RT-qPCR. PAβN served as a reference efflux pump inhibitor. Synergy was evaluated using checkerboard assays, and spontaneous mutants were generated with netilmicin with/without MV6 (100 mg/L). Whole-genome sequencing and variant calling analysis were then performed. Results: MV6 presented low antimicrobial activity in A. baumannii with MICs higher than 2048 mg/L. MV6 showed a better boosting effect for aminoglycosides, especially netilmicin, exceeding that of PAβN. Checkerboard assays confirmed a strong synergy between netilmicin and MV6, and a significant correlation was found between netilmicin MIC and adeB overexpression, which was mitigated by the presence of MV6. MV6 reduced, by 16-fold, the mutant prevention concentration of netilmicin. Mutations in a TetR-family regulator and ABC-binding proteins were found in both groups, suggesting a direct or indirect implication of these proteins in the resistance acquisition process. Conclusions: MV6 lacks intrinsic antimicrobial activity, minimizing selective pressure, yet enhances netilmicin’s effectiveness except for strain 210, which lacks the AdeABC efflux pump. Resistant mutants indicate specific aminoglycoside resistance mechanisms involving efflux pump mutations, suggesting synergistic interactions. Further research, including transcriptomic analysis, is essential to elucidate MV6’s role in enhancing netilmicin efficacy and its resistance mechanisms.

Dairy farm waste: a potential reservoir of diverse antibiotic resistance and virulence genes in aminoglycoside- and beta-lactam-resistant Escherichia coli in Gansu Province, China

Aminoglycosides (AGs) and beta-lactams are the most commonly used antimicrobials in animal settings, particularly on dairy farms. Dairy farm waste is an important reservoir of antibiotic resistance genes (ARGs) and virulence genes (VGs) in environmental Escherichia coli, which is an important indicator of environmental contamination and foodborne pathogen that potentially threaten human and animal health. In the present study, we aimed to characterize the ARGs and VGs in AG- and beta-lactam-resistant E. coli from dairy farm waste in Gansu Province, China. The dairy farm waste consisted of fecal (n=265) and sewage (n=54) samples processed using standard microbiological techniques and the Clinical & Laboratory Standards Institute guidelines. The total DNA of AG- and beta-lactam-resistant E. coli was extracted, and whole-genome sequencing (WGS) was performed using the Illumina NovaSeq platform and analyzed using various bioinformatics tools. In this study, among 84.3% (269/319) of the E. coli strains, 23.8% (64/269) were identified as AG- and beta-lactam-resistant E. coli. WGS analysis revealed a large pool of ARGs belonging to multiple classes such as AGs, beta-lactams, aminocoumarins, fluoroquinolones, macrolides, phenicol, tetracyclines, phosphonic acid, disinfecting and antiseptic agents, elfamycin, rifamycin, and multidrug resistance genes. Furthermore, virulome analysis of 64 E. coli strains revealed clinically important virulence factors associated with adherence, biofilm, invasion, auto-transportation, siderophores, secretion systems, toxins, anti-phagocytosis, quorum sensing, regulation, metabolism, and motility. We identified dairy farm feces and sewage waste as important reservoirs of antimicrobial resistance and virulence determinants in E. coli in Gansu, China, which can threaten human and animal health through ecological exposure and ingestion of contaminated food and water. We recommend continuous large-scale surveillance in dairy farm settings to formulate protective guidelines for public health safety.

Bacillus velezensis SS-20 as a potential and efficient multifunctional agent in biocontrol, saline-alkaline tolerance, and plant-growth promotion

The versatile strain Bacillus velezensis SS-20 was studied to determine its agricultural benefits, including its ability to antagonize fungi, promote plant growth, and tolerate saline-alkaline conditions. Firstly, strain SS-20 was found to be an antagonist of plant pathogenic fungi such as Magnaporthe oryzae, Rhizoctonia solani, and Fusarium oxysporum, with control effects up to 82.07 %, 46.57 %, and 23.09 %, respectively. The hyphae of pathogenic fungi treated with SS-20 and its fermentation product were abnormal, swollen, deformed, and distorted. Meanwhile, strain SS-20 dissolved inorganic phosphorus (phosphate solubilization efficiency, 45.95 %), and produced siderophore (siderophore units, 54.18 ± 4.19 %), and poly-γ-glutamic acid (γ-PGA) (13.35 ± 2.61 g/L). Strain SS-20 significantly increased plant height, aboveground fresh weight, and root dehydrogenase activity compared to the control treatment under non-saline conditions (p < 0.05). In addition, strain SS-20 showed significant saline-alkaline tolerance, surviving in 10 % NaCl and pH 9.5, and enhanced plant growth under simulated saline-alkali conditions. Whole-genome sequencing analysis revealed that the chromosome of B. velezensis SS-20 consists of 3,929,792-bp with 46.5 % guanine-cytosine content and 4015 coding sequences. The antiSMASH analysis identified 12 gene clusters within strain SS-20 that encode antimicrobial compounds such as surfactin, fengycin, butirosin A/butirosin B, macrolactin H, bacillibactin, difficidin, and bacilysin, thereby corroborating the strain's biocontrol capabilities. Genes related to plant growth promotion, including siderophores, γ-PGA, indole acetic acid production, phosphate solubilization, glycine-betaine production, and Na+/H+ antiporters, were also annotated. In conclusion, B. velezensis SS-20 represents a promising biotic resource for enhancing plant growth and protecting against environmental stresses.

Whole-genome sequencing analysis of Japanese autism spectrum disorder trios.

Autism spectrum disorder (ASD) is a genetically and phenotypically heterogeneous neurodevelopmental disorder with a strong genetic basis. Conducting the first comprehensive whole-genome sequencing (WGS) analysis of Japanese ASD trios, this study aimed to elucidate the clinical significance of pathogenic variants and enhance the understanding of ASD pathogenesis. WGS was performed on 57 Japanese patients with ASD and their parents, investigating variants ranging from single-nucleotide variants to structural variants (SVs), short tandem repeats (STRs), mitochondrial variants, and polygenic risk score (PRS). Potentially pathogenic variants that could explain observed phenotypes were identified in 18 patients (31.6%) overall and in 10 of 23 patients (43.5%) with comorbid intellectual developmental disorder (IDD). De novo variants in PTEN, CHD7, and HNRNPH2 were identified in patients referred for genetic counseling who exhibited previously reported phenotypes, including one patient with ASD who had profound IDD and macrocephaly with PTEN L320S. Analysis of the AlphaFold3 protein structure indicated potential inhibition of intramolecular interactions within PTEN. SV analysis identified deletions in ARHGAP11B and TMLHE. A pathogenic de novo mitochondrial variant was identified in a patient with ASD who had a history of encephalitis and cognitive decline. GO enrichment analysis of genes with nonsense variants and missense variants (Missense badness, PolyPhen-2, and Constraint >1) showed associations with regulation of growth and ATP-dependent chromatin remodeler activity. No reportable results were obtained in the analysis of STR and PRS. Characterizing the comprehensive genetic architecture and phenotypes of ASD is a fundamental step towards unraveling its complex biology.

Degradation Kinetics and Mechanism of β-Cypermethrin and 3-Phenoxybenzoic Acid by Lysinibacillus pakistanensis VF-2 in Soil Remediation.

Pyrethroid pesticide residues have detrimental effects on soil ecology and crop growth during insecticidal operations in agriculture. In this study, a novel strain Lysinibacillus pakistanensis VF-2 was isolated from long-term pesticide-treated cropland and had a maximum degradation efficiency of 81.66% for synthetic pyrethroid β-cypermethrin (β-CY) under optimized conditions. The analysis of intermediate products revealed that the degradation pathway of β-CY mainly involves ester bond hydrolysis, diphenyl ether decomposition, and phthalate ester degradation. Whole-genome sequencing and RT-qPCR analysis revealed the involvement of carboxylesterases, dioxygenases, and aromatic compound degrading enzymes in the degradation of β-CY. In the soil bioaugmentation experiment, the strain VF-2 can synergistically interact with indigenous microorganisms, significantly enhancing the degradation efficiency of β-CY and its metabolite 3-phenoxybenzoic acid (3-PBA) from 17.08% and 7.62% to 73.46% and 62.29%, respectively. This study suggests that strain VF-2 is a promising candidate for in situ coremediation of pyrethroid and intermediate metabolite residues in soil.

Leveraging next-generation sequencing technology for the study of ginsenosides biosynthesis and exploring DNA markers in the endemic species Panax vietnamensis.

Panax species, particularly Panax ginseng, Panax quinquefolius, and Panax vietnamensis are renowned for their medicinal properties and economic value. Of these, the endemic P. vietnamensis species (native to Vietnam, Laos, and southern China) is currently receiving focused attention due to its special ginsenosides accumulation in comparison to the others. Recent advances in next-generation sequencing (NGS) technologies have accelerated the molecular genetic studies in this Panax species, providing deeper insights into the ginsenosides biosynthesis pathway as well as other aspects such as genetic diversity and molecular evolution. This work aims to systematically review all studies on the application of NGS in P. vietnamensis, particularly in whole-genome sequencing and transcriptome analysis. These key findings significantly contribute to identifying critical genes involved in ginsenosides biosynthesis, developing various DNA markers (such as SSR and SNP) for molecular genetic studies, and gaining insights into the species' molecular evolution. Based on these findings, future research can further expand to complete the full genomic database of this species and further investigate the underlying regulatory mechanisms of ginsenosides biosynthesis. These efforts will be crucial for enhancing the conservation, molecular breeding, and agricultural productivity of this valuable medicinal species.

Comprehensive Assessment of Initial Adaptation of ESBL Positive ST131 Escherichia coli to Carbapenem Exposure.

It remains unclear how high-risk Escherichia coli lineages, like sequence type (ST) 131, initially adapt to carbapenem exposure in their progression to carbapenem resistance. Carbapenem mutation frequency was measured in multiple subclades of extended-spectrum β-lactamase (ESBL) positive ST131 clinical isolates using a fluctuation assay followed by whole genome sequencing (WGS) characterization. Genomic, transcriptomic, and porin analyses of ST131 C2/H30Rx isolate, MB1860, under prolonged, increasing carbapenem exposure was performed using two experimental evolutionary platforms to measure fast vs. slow adaptation. All thirteen ESBL positive ST131 strains selected from a diverse (n=184) ST131 bacteremia cohort had detectable ertapenem (ETP) mutational frequencies with a positive correlation between initial ESBL gene copy number and mutation frequency (r = 0.87, P-value <1e-5). WGS analysis of mutants showed initial response to ETP exposure resulted in significant increases in ESBL gene copy numbers or mutations in outer membrane porin (Omp) genes in the absence of ESBL gene amplification with subclade specific associations. In both experimental evolutionary platforms, MB1860 responded to initial ETP exposure by increasing blaCTX-M-15 copy numbers via modular, insertion sequence 26 (IS26) mediated pseudocompound transposons (PCTns). Increased transcript levels of genes present within the PCTn was a conserved expression signal in both experimental evolutionary platforms. Stable mutations in Omp encoding genes were detected only after prolonged increasing carbapenem exposure consistent with clinical observations. ESBL gene amplification is a conserved response to initial carbapenem exposure, especially within the high-risk ST131 C2/H30Rx subclade. Targeting such amplification could assist with mitigating carbapenem resistance development.

Canine uropathogenic and avian pathogenic Escherichia coli harboring conjugative plasmids exhibit augmented growth and exopolysaccharide production in response to Enterococcus faecalis.

Uropathogenic Escherichia coli (UPEC) and avian pathogenic Escherichia coli (APEC) are extraintestinal pathogenic Escherichia coli (ExPEC) that infect dogs and poultry. These agents occur both as single-species infections and, commonly, in co-infection with Enterococcus faecalis (EF); however, it is unclear how EF co-infections modulate ExPEC virulence. Genetic drivers of interspecies interactions affecting virulence were identified using macrocolony co-culture, chicken embryo co-infection experiments, and whole-genome sequence analysis of ExPEC and EF clinical isolates. Ten of 11 UPEC strains originally co-isolated with EF exhibited a growth advantage when co-cultured with EF on iron-limited, semi-solid media in contrast to growing alone (P < 0.01). Phylogenetic analyses of these UPEC and 18 previously screened APEC indicated the growth-response phenotype was conserved in ExPEC despite strain diversity. When genomes of EF-responsive ExPEC were compared to non-responsive ExPEC genomes, EF-induced growth was associated with siderophore, exopolysaccharide (EPS), and plasmid conjugative transfer genes. Two matched pairs of EF-responsive and non-responsive ExPEC were selected for further characterization by macrocolony proximity and chicken embryo lethality assays. EF-responsive ExPEC produced 5 to 16 times more EPS in proximity to EF and were more lethal to embryos alone and during co-infection with EF compared to non-responsive ExPEC (P < 0.05). A responsive APEC strain cured of its conjugative plasmid lost the enhanced growth and EPS production response to EF. These data demonstrate that ExPEC growth augmentation by EF occurs in UPEC and APEC strains and is linked to conjugative virulence plasmids and EPS production, which are widely conserved ExPEC virulence determinants.

Targeted Variant Assessments of Human Endogenous Retroviral Regions in Whole Genome Sequencing Data Reveal Retroviral Variants Associated with Papillary Thyroid Cancer

Papillary thyroid cancer (PTC) is one of the fastest-growing cancers worldwide, lacking established causal factors or validated early diagnostics. Human endogenous retroviruses (HERVs), comprising 8% of human genomes, have potential as PTC biomarkers due to their comparably high baseline expression in healthy thyroid tissues, indicating homeostatic roles. However, HERV regions are often overlooked in genome-wide association studies because of their highly repetitive nature, low sequence coverage, and decreased sequencing quality. Using targeted whole-genome sequence analysis in conjunction with high sequencing depth to overcome methodological limitations, we identified associations of specific HERV variants with PTC. Analyzing WGS data from 138 patients with PTC generated through The Cancer Genome Atlas project and 2015 control samples from the 1000 Genomes Project, we examined the mutational variation in HERVs within a 20 kb radius of known cancer predisposition genes (CPGs) differentially expressed in PTC. We discovered 15 common and 13 rare germline HERV variants near or within 20 CPGs that distinguish patients with PTC from healthy controls. We identified intragenic–intronic HERV variants within RYR2, LRP1B, FN1, MET, TCRVB, UNC5D, TRPM3, CNTN5, CD70, RYR1, RUNX1, CRLF2, and PCDH1X, and three variants downstream of SERPINA1 and RUNX1T1. Sanger sequencing analyses of 20 thyroid and 5 non-thyroid cancer cell lines confirmed associations with PTC, particularly for MSTA HERV-L variant rs200077102 within the FN1 gene and HERV-L MLT1A LTR variant rs78588384 within the CNTN5 gene. Variant rs78588384, in particular, was shown in our analyses to be located within a POL2 binding site regulating an alternative transcript of CNTN5. In addition, we identified 16 variants that modified the poly(A) region in Alu elements, potentially altering the potential to retrotranspose. In conclusion, this study serves as a proof-of-concept for targeted variant analysis of HERV regions and establishes a basis for further exploration of HERVs in thyroid cancer development.

Andean Enriched NFKB1 Haplotype Reduces Inflammation and Improves Response to Ropeginterferon Alfa-2b in Polycythemia Vera (PV) and Essential Thrombocythemia (ET)

Chronic inflammation is a feature of polycythemia vera (PV), essential thrombocythemia (ET), and other myeloproliferative neoplasms (MPNs). NF-κB plays a key role in regulating pro-inflammatory cytokines. In PV and ET, activation of the NF-κB pathway is a primary driver of inflammation and augments hypoxia-inducible factors (HIFs) activity, increasing transcription of prothrombotic genes. Suppression of the NF-κB pathway has been shown to reduce disease burden in MPNs (Laranjeira, Blood, 2024).We investigated the genetic basis underlying elevated hemoglobin in Andean natives, Aymara, by using granulocyte transcriptome data from both Aymara and Europeans from La Paz, Bolivia. We found their differentially expressed and spliced genes are associated with inflammatory and HIFs pathways. We identified three novel, alternatively spliced NFKB1 transcripts (AS-NFKB1) in Aymara, with skipped exon 4, exon 5, or both. The AS-NFKB1 transcript with exon 5 skipped was translated into protein but showed a defect in nuclear translocation, while the other two AS-NFKB1 transcripts were not translated, indicating a partial or complete loss of NFKB1's function as an NF-κB suppressor. AS-NFKB1 also augmented activity of HIFs, explaining high Aymara hemoglobin. Using integrative analysis of whole-genome sequencing and granulocyte transcriptome, we identified splicing quantitative trait locus associated with AS-NFKB1; NFKB1 rs230511 is linked to high AS-NFKB1 expression. Its T allele (C/T or T/T genotype) is present in approximately 90% of Aymara but is also present in ~30% of Europeans, Hispanics, and Asians. Under TNF-induced inflammatory stress, unlike wild-type NFKB1, AS-NFKB1 suppressed inflammatory gene expression, suggesting a protective mechanism against inflammation (under review, Nature Communication).Thus, we examined NFKB1 rs230511 association with inflammatory markers and response to ropeginterferon alfa-2b (Ropeg) therapy in PV and ET. We genotyped 30 PV and 15 ET patients, assessing inflammatory gene expression (IL1B, CXCL8, IL6, IL15, and TNF) in neutrophils and platelets. In platelets, individuals with the C/T genotype showed lower levels of these inflammatory transcripts compared to those with the C/C genotype and IL1B, CXCL8, and TNF levels were also reduced in the T/T genotype. In neutrophils, IL1B and CXCL8 were decreased in the T/T genotype, and IL15 and TNF levels were lower in the C/T genotype.Given that AS-NFKB1 and NFKB1 rs230511 are linked to elevated transcriptional activity of HIFs, we analyzed the expression of HIF-target genes. LDHA, SLC2A1, and VEGFA transcripts were significantly lower in the C/T genotype in both PV and ET neutrophils and platelets. Since increased inflammation and HIF transcriptional activity are associated with thrombosis in PV and ET (Gangaraju, Blood Advances, 2020), we also measured expression levels of prothrombotic genes (F3, THBD, SELP, SERPINE1). In both platelets and granulocytes, F3 and THBD transcript levels were significantly lower in individuals with the C/T genotype compared to those with the C/C genotype.Ropeg modulates inflammation via interferon signaling and attacks PV and ET clones (Verger, Blood Cancer J., 2018). We found that the C/T genotype (58.6%) was more prevalent among patients who achieved complete hematological response (CHR) compared to the non-CHR cohort, which included patients who did not reach CHR or had cytopenia (33.3%) (p<0.0001). This finding also correlated with lower transcript levels of inflammatory, HIF-targeted, and thrombotic genes in the CHR group compared to non-CHR group.Our study indicates that presence of Aymara enriched NFKB1 rs230511 haplotype decreases PV and ET inflammatory, prothrombotic, and HIF's activity compared to NFKB1 wild allele. The NFKB1 rs230511 haplotype also modulates response to Ropeg in PV and ET. The presence of NFKB1 rs230511 T allele is associated with reduced expression of inflammatory and HIF-targeted genes, potentially decreasing inflammation and thrombosis risk in PV and ET. Patients with the C/T genotype were more likely to achieve CHR, suggesting this NFKB1 haplotype may impact Ropeg therapy efficacy and serve as a potential biomarker for response assessment in PV and ET patients. These novel insights offer valuable implications for both of our understanding of inflammatory and prothrombotic processes and may also lead to novel PV and ET therapies.

Insight into Antifungal Metabolites from Bacillus stercoris 92p Against Banana Cordana Leaf Spot Caused by Neocordana musae

Banana crop ranks among the most crucial fruit and food crops in tropical and subtropical areas. Despite advancements in production technology, diseases such as cordana leaf spot, caused by Neocordana musae, remain a significant challenge, reducing productivity and quality. Traditional chemical controls are becoming less effective due to the development of resistance in target pathogens, which pose significant environmental and health concerns. Consequently, there is growing attention toward the development of biocontrol strategies. Here, we identified a new bacterial strain, Bacillus stercoris 92p, from the rhizosphere soil of banana. We evaluated its ability to suppress the growth of N. musae and other fungal pathogens that cause leaf spot disease in bananas. The inhibitory effect of B. stercoris 92p were checked using dual culture assays, microscopic observations, and pot experiments. Furthermore, the biocontrol mechanisms were investigated using whole-genome sequencing and biochemical analyses. The results showed that B. stercoris 92p exhibited significant antifungal activity against N. musae and other fungal pathogens, with inhibition rates exceeding 70%. Microscopic examination revealed significant morphological alterations in the hyphae and conidia of the tested pathogens. In pot experiments, B. stercoris 92p effectively reduced the severity of cordana leaf spot, achieving a biocontrol efficacy of 61.55%. Genomic analysis and biochemical tests indicated that B. stercoris 92p produces various antifungal compounds, including lipopeptides (fengycins and surfactins), hydrolytic enzymes (proteases and amylases), and phosphate-solubilizing metabolites. In conclusion, the study highlights that B. stercoris could potentially be used as a potential biological control agent against cordana leaf spot.