Whole Genome Sequencing Research Articles

Whole Genome Sequencing (WGS) Analysis of Antimicrobial Resistance (AMR) Milk and Dairy-Derived Pathogens from Anand, Gujarat, India

This study aimed to evaluate the antimicrobial resistance (AMR) patterns and genomic characteristics of Enterococcus faecalis, Enterococcus faecium, and Staphylococcus aureus strains isolated from dairy products, including buttermilk, curd, ice cream, and sweets, in the Anand region of Gujarat, India. A total of 205 isolates were analyzed, with the highest contamination levels found in buttermilk and curd. The bacterial isolates were identified using biochemical tests and advanced Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) mass spectrometry. Antimicrobial susceptibility testing (AST) was performed using the Kirby-Bauer disk diffusion method, following CLSI guidelines, focusing on common antibiotics used for treating dairy-related bacterial infections. Resistance profiles were analyzed using WHONET software.s The findings revealed significant multidrug resistance (MDR), particularly among E. faecium and E. faecalis, with over 95% resistance to key antibiotics, including linezolid, ciprofloxacin, cefpodoxime, and carbapenems. Many strains were classified as MDR, XDR, and PDR. Staphylococcus aureus also exhibited substantial resistance to penicillin and enrofloxacin. whole-genome sequencing (WGS) and phylogenetic analysis to identify AMR determinants and conduct nucleotide sequence alignment. The study identified several antibiotic resistance genes, including LiaF, which regulates the expression of LiaR and LiaS genes. WGS revealed that genes such as GdpD, MprF, and PgsA encode intrinsic resistance determinants, contributing to antibiotic resistance. Additional AMR mechanisms were identified, including ABC transporter efflux pumps and the regulation of resistance genes by LiaR and LiaS. Phylogenetic analysis indicates a close relationship between Enterococcus faecium 640 1352.18624 and Enterococcus durans FB129-CNAB-4 883162.3.

Genome characteristics of a MDR Pseudomonas monteilii carrying a novel VIM-type β-lactamase, blaVIM-84

ObjectivesThis study aimed to determine the genetic environment and characterize plasmid carrying a novel VIM-type β-lactamase (VIM-84) in a multidrug-resistant Pseudomonas monteilii isolate obtained from the human gut through whole-genome sequencing. MethodsDNA extraction of P. monteilii L2757hy was performed using the Genomic DNA Isolation Kit (QIAGEN, Hilden, Germany). Whole-genome sequencing was performed by Illumina NovaSeq 6000 and Oxford Nanopore platforms. The transferability of resistance genes was screened single clonal on MHA plates containing rifampicin and meropenem. Verification was performed using MALDI/TOF-MS and PCR with Pseudomonas aeruginosa PAO1Ri as the recipient strain. ResultsL2757hy was identified as P. monteilii through sequencing and ANI analysis. The genome was assigned as ST147 and comprised a 6,130,057 bp chromosome with a GC content of 61.8% and a 49,704 bp plasmid. Several resistance genes, including blaIMP-1, aac(6′)-IIa and tmexCD-toprJ, as well as virulence genes such as iroN, and wzaJ, were identified on the chromosome. A novel VIM-type blaVIM-84 was found on the plasmid, which was previously identified in Pseudomonas aeruginosa. Plasmid harboring blaVIM-84 was untypable, and it could be transferred to P. aeruginosa PAO1Ri and was associated with a class I integron with the genetic environment intI1-blaVIM-84-tniR-tniQ-tniB-tniA, likely derived from Tn402. ConclusionsOur study revealed that the novel blaVIM-84 gene was harbored by P. monteilii rather than P. aeruginosa. We suggested that P. monteilii may serve as a reservoir for resistance genes.

Predicting antimicrobial resistance in E. coli with discriminative position fused deep learning classifier

Escherichia coli (E. coli) has become a particular concern due to the increasing incidence of antimicrobial resistance (AMR) observed worldwide. Using machine learning (ML) to predict E. coli AMR is a more efficient method than traditional laboratory testing. However, further improvement in the predictive performance of existing models remains challenging. In this study, we collected 1,937 high-quality whole genome sequencing (WGS) data from public databases with an antimicrobial resistance phenotype and modified the existing workflow by adding an attention mechanism to enable the modified workflow to focus more on core single nucleotide polymorphisms (SNPs) that may significantly lead to the development of AMR in E. coli. While comparing the model performance before and after adding the attention mechanism, we also performed a cross-comparison among the published models using random forest (RF), support vector machine (SVM), logistic regression (LR), and convolutional neural network (CNN). Our study demonstrates that the discriminative positional colors of Chaos Game Representation (CGR) images can selectively influence and highlight genome regions without prior knowledge, enhancing prediction accuracy. Furthermore, we developed an online tool (https://github.com/tjiaa/E.coli-ML/tree/main) for assisting clinicians in the rapid prediction of the AMR phenotype of E. coli and accelerating clinical decision-making.

The first report of submandibular lymph node infection with Flavobacterium ceti in a horse and its complete genome sequence.

This is the first report describing the isolation and genome sequence of Flavobacterium ceti (F. ceti IJFC025) from a draining submandibular lymph node abscess in a 6-year-old mixed-breed mare in Korea. Since F. ceti is not a usual infectious pathogen that causes equine health problems, this study presents the complete genome sequence of F. ceti isolated from a submandibular lymph node abscess sample in Jeju, South Korea, in 2022. The entire genome sequence of F. ceti IJFC025 consisted of a 3,144,426-bp chromosome with 35.6% G+C content. Gene prediction revealed that this strain possesses 2909 coding sequences (CDSs), 74 tRNAs, and five rRNAs. Average nucleotide identity analysis of the whole-genome sequence revealed 98.6%-98.8% nucleotide identity between F. ceti IJFC025 and F. ceti CECT7184, which was previously isolated from beaked whales. PathogenFinder revealed four different virulence genes in the genome that could potentially cause infections in horses. F. ceti CECT7184 contained genes encoding propionyl-CoA carboxylase, 3-oxoacid CoA-transferase A subunit, and the translation initiation factor IF-1, whereas F. ceti IJFC025 does not. Instead, F. ceti IJFC025 harbors genes encoding deoxyhypusine synthase, whereas F. ceti CECT7184 does not. Antimicrobial susceptibility tests revealed that F. ceti IJFC025 is a multidrug-resistant strain that is resistant to gentamicin, ceftazidime, nalidixic acid, ciprofloxacin, and colistin. However, no resistance genes were detected in the whole-genome sequence. Because of the similarity of the clinical signs with those of strangles, this pathogen needs to be included in the differential diagnosis of submandibular lymphadenopathy in horses.

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.

Microbiome shifts elicited by ornamental lighting of granite facades identified by MinION sequencing

Night-time outdoor illumination in combination with natural sunlight can influence the visible phototrophic colonizers (mainly algae) growing on stone facades; however, the effects on the microbiome (invisible to the naked eye) are not clear. The presence of stone-dwelling microbes, such as bacteria, diatoms, fungi, viruses and archaea, drives further biological colonization, which may exacerbate the biodeterioration of substrates. Considering the microbiome is therefore important for conservation of the built heritage. The impact of the following types of lighting on the relative abundance and diversity of the microbiome on granite ashlars was evaluated in a year-long outdoor pilot study: no lighting; lighting with a metal halide lamp (a traditional lighting system currently used to illuminate monuments); and lighting with a novel LED lamp (an environmentally sound prototype lamp with a biostatic effect, halting biological colonization by phototrophs, currently under trial). Culturable fractions of microbiome and whole-genome sequencing by metabarcoding with Oxford Nanopore Sequencing (MinION) was conducted for bacteria and fungi in order to complement both community characterization strategies. In addition, the possible biodeteriorative profiles of the isolated strains, relative to calcium carbonate precipitation/solubilisation and iron oxidation/reduction, were investigated by plate assays. Alpha and beta diversity indexes were also determined, along with the abundance of biocide and antibiotic resistance genes. Culture-dependent microbiological analysis failed to properly show changes in community composition, for which metagenomic approaches like MinION are better suited. Thus, MinION analysis identified shifts in the granite microbiome elicited by ornamental lighting. The novel LED lamp with the biostatic effect on phototrophs caused an increase in the diversity of bacteria and fungi. In this case, the microbiome was more similar to that in the unlit samples. In the samples illuminated by the metal halide lamp, dominance of bacteria was favoured and the presence of fungi was negligible.

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.

Comparative Genomic Analysis Reveals Distinct Virulence and Resistance Mechanisms in 21 Bacterial Fish Pathogens

The rising bacterial infections threaten world aquaculture and wild fish populations, making it imperative to increase the understanding of the mechanisms of pathogenic virulence and resistance. This study applies comparative genomic analysis to 21 bacterial fish pathogens, using whole-genome sequences from public genomic resources and sophisticated bioinformatics tools for screening of virulence factors, mobile genetic elements, antibiotic resistance genes, anti-phage defense mechanisms and secretory systems. We have seen that the different pathogens depict a wide range of variability regarding virulence and resistance potential, which may be attributed to species-specific adaptation. Notably, Streptococcus agalactiae and Mycobacterium salmoniphilum were found to possess high offensive and defensive virulence potential, but at different regulative controls. We also found diverse secretion systems and intricate mechanisms for antibiotic resistance, which have provided very important insights into how pathogens adapt to their environments. By categorizing functional genes and finding anti-phage systems, our analysis has revealed new insights into the complex interactions among bacterial virulence, resistance, and host defense mechanisms. These findings not only shed new light on the bacterial pathogenesis process in aquaculture but also provide the bases for focused, therapeutically-based strategies and genomic surveillance programs able to improve disease management and sustainability in aquaculture environments.

Whole genome-based characterization of extended-spectrum β-lactamase-producing Enterobacter cloacae from orthopedic patients and environment of a tertiary referral hospital in Tanzania

ObjectivesWe investigated the genomic epidemiology of extended-spectrum β-lactamase-producing Enterobacter cloacae (ESBL-Ec) isolates from patients and hospital environment to better understand their distribution to help devising effective strategies for infection prevention and control. MethodsWe screened ESBL-Ec at Bugando Medical Center (BMC) in Mwanza, Tanzania. Rectal swabs from orthopedic patients on admission and swabs from the neighboring inanimate environment were collected. Following microbial culture, DNA was extracted from pure ESBL-Ec, and whole-genome sequencing was done. Sequence typing (ST), plasmid replicons, drug resistance, and virulence genes were deciphered using the Rapid Microbial Analysis Pipeline (rMAP). ResultsWe obtained 209 ESBL isolates, of which 15 (7.2%) were ESBL-Ec [8 (53.30%) from patients and 7 (46.7%) from the environment]. Seven isolates were novel and eight were diverse each with a unique ST. All isolates harbored two to five β-lactamase genes, with the predominance of blaCTX-M-15(15/15), blaOXA-1(14/15), blaTEM(14/15) and blaACT(12/15). The most common non β-lactam drug resistance genes were aac(3)-IIa (14/15), aac(6’)-Ib-cr (14/15), fosA (14/15), and qnrB1 (12/15), aph(3’’)-Ib (10/15) and aph(6)-Id (10/15). Eleven different types of plasmid replicons were identified in 14/15 of the isolates, harboring one to five plasmids, with the most common plasmids being IncFII (11/15) and IncFIB (10/15). All isolates harbored the outer membrane protein (ompA), and curli protein (csg) was in 14/15 isolates. ConclusionAdmitted orthopedic patients and the hospital environment act as a reservoir of ESBL-Ec with diverse STs and endowed with drug resistance and arsenals of virulence genes, calling for their routine screening on admission for mitigation of potential subsequent infections.

Whole genome sequencing reveals antibiotic resistance pattern and virulence factors in Klebsiella quasipneumoniae subsp. similipneumoniae from Hospital wastewater in South-West, Nigeria.

Klebsiella quasipneumoniae is a distinct species from K. pneumoniae, even though it is sometimes mistaken phenotypically for the latter in clinical situations. K. quasipneumoniae is a pathogen and this study aims at understanding the genomic antibiotic resistance and virulence characteristics of Klebsiella quasipneumoniae subsp. similipneumoniae (B105 strain) isolated from tertiary hospital wastewater and the potential risks associated with its environmental spread.The Illumina platform was used for whole-genome sequencing (WGS), the generated raw reads (de novo) was assembled using RAPT NCBI, while other standardized bioinformatics tools were utilized to validate and examine the landscape of the genome’s antibiotic resistance and virulence factors. The K. quasipneumoniae subsp. similipneumoniae (B105 strain), belonged to sequence type 1422 and was resistant to ampicillin, amoxicillin-clavulanic acid, ceftazidime, cefepime, meropenem, tetracycline, but susceptible to gentamicin. The annotated genome acknowledged the presence of blaOKP-B-2, ompK 36, fosA5, oqxAB, virulence genes responsible for capsule formation, lipopolysaccharide, iron uptake aerobactin (iutA), salmochelins (iroE, iroN), enterobactin siderophore, efllux pump (acrA, acrB) adherence, (mrkC, mrkD, and fimD) and two plasmids replicon IncFIB(K) and IncR. The study resonates the inadequacy of conventional microbiological identification methods to distinguish K. pneumoniae and K. quasipneumoniae and at the same time heightens the importance of using a genomic platform to extol the identity of K. quasipneumoniae subsp. similipneumoniae strain. Furthermore, the peculiarities of the acquired antimicrobial resistance and virulence genes, in this strain, are a potential risk to the environment.

Whole-Genome Analysis of Stress Resistance-Related Genes in Listeria monocytogenes

Listeria monocytogenes is a crucial foodborne pathogen with significant public health implications. This study analyzed whole-genome sequences (WGS) of L. monocytogenes strains from public databases, examining associations between resistance genes, lineage, strain type, isolation source, and geography. Results revealed that after eliminating duplicates and strains with incomplete WGS, a total of 316 strains were deemed suitable for subsequent analyses. Within these strains, lineages I and II were extensively distributed, predominantly isolated from clinical and food sources. 56.65% of these strains fell into seven major Clonal Complexes (CC), identified by Multilocus Sequence Typing (MLST), correlating significantly with isolation information. Analysis of 46 resistance-related genes showed a high consistency of resistance genes in the same type of strains, hinting at a potential causal chain of ‘food-food environment-evolution of L. monocytogenes’. Moreover, the standard strains exhibit similar gene carriage rates as the sample strains, with multiple variations observed in acid-resistance genes. In conclusion, through a comprehensive analysis of the L. monocytogenes genome sequences, this study deepens our understanding of the differences and associations between its lineage, strain typing, isolation sources, geographical distribution, and resistance genes, thereby providing a scientific basis for formulating more effective prevention and control strategies.

Constructing phylogenetic trees for microbiome data analysis: A mini-review

As next-generation sequencing technologies advance rapidly and the cost of metagenomic sequencing continues to decrease, researchers now face an unprecedented volume of microbiome data. This surge has stimulated the development of scalable microbiome data analysis methods and necessitated the incorporation of phylogenetic information into microbiome analysis for improved accuracy. Tools for constructing phylogenetic trees from 16S rRNA sequencing data are well-established, as the highly conserved regions of the 16S gene are limited, simplifying the identification of marker genes. In contrast, metagenomic and whole genome shotgun (WGS) sequencing involve sequencing from random fragments of the entire gene, making identification of consistent marker genes challenging owing to the vast diversity of genomic regions, resulting in a scarcity of robust tools for constructing phylogenetic trees. Although bacterial sequence tree construction tools exist for upstream bioinformatics, many downstream researchers—those integrating these trees into statistical models or machine learning—are either unaware of these tools or find them difficult to use due to the steep learning curve of processing raw sequences. This is compounded by the fact that public datasets often lack phylogenetic trees, providing only abundance tables and taxonomic classifications. To address this, we present a comprehensive review of phylogenetic tree construction techniques for microbiome data (16S rRNA or whole-genome shotgun sequencing). We outline the strengths and limitations of current methods, offering expert insights and step-by-step guidance to make these tools more accessible and widely applicable in quantitative microbiome data analysis.

Molecular characterization and comparison of tomato zonate spot virus isolated in Japan and China.

The complete genome sequence of Orthotospovirus tomatozonae (tomato zonate spot virus, TZSV) isolated in Japan was determined and compared with that of Chinese isolates. The lengths of the S, M, and L segments of the RNA genomes of the Japanese isolate (TZSV-TZ1-3) were 3194, 4675, and 8916 nucleotides, respectively, which were similar to the Chinese isolates. Moreover, the eight motifs on the RNA-dependent RNA polymerase (RdRp) gene were conserved in both TZSV-TZ1-3 and Chinese TZSV isolates (TZSV-Bidens and TZSV-Tomato-YN). The nucleotide identity of the genes among the TZSV isolates was more than 94%, indicating low diversity among viruses. The phylogenetic analysis and the prediction of the cleavage sites in the glycoprotein showed that the TZSV-TZ1-3 isolate was closely related to TZSV-Tomato-YN isolated from China. However, there were unique frameshifts and deletions on the RdRp and glycoprotein genes of the TZSV-Tomato-YN isolate, suggesting that both isolates were genetically distinct. The findings of this study indicate that the TZSV-TZ1-3 isolate originated in China and show the sequence diversity among TZSV isolates.

Identification of SNPs and INDELS associated with duck egg quality traits through a genome-wide association analysis

Egg quality traits are economically important in the poultry industry. To explore the genetic architecture and identify potential candidate genes, a genome-wide association study (GWAS) was performed for 13 egg quality traits using data from whole-genome sequencing of 299 Longyan Shan-ma female ducks, including 12 quantitative traits and one qualitative trait, eggshell color (ESC; white, light green, green). From estimation of pedigree genetic parameters, heritability (h2) ranged from 0.022 to 0.996 for the 12 quantitative traits, with the highest h2 (0.996) for eggshell color a* value (ESCA) and the lowest h2 (0.022) for egg yolk percentage relative to EW. A total of 8,874 single nucleotide polymorphism (SNP)-based significant associations (1.0 × 10-6) and 247 insertion-deletion (indel)-based significant associations (1.00 × 10-5) were identified, including 5,980 SNPs and 159 indel markers. From 5,924 SNPs and 143 indels associated with ESC traits, 181 potential candidate genes were identified, and most significant SNPs and indels (P < 1.0 × 10-20) were located at 1.86 Mb (44.29-46.15 Mb) on chromosome 4. The top SNP (chr4:45325309:C>A; P = 7.97 × 10-43) and the top indel (chr4:45299595:delTTCCACTCCAC; P = 4.20 × 10-36) for the ESC a* value were within two known ESC candidate genes; ATP-binding cassette subfamily G member 2 (ABCG2) and protein kinase cGMP-dependent 2 (PRKG2). Of 56 SNPs and 16 indels associated with other egg quality traits, 46 potential candidate genes were identified including synapse differentiation-inducing 1-like (SYNDIG1L) for EW, and core histone macro-H2A.1 (LOC101795967) and neurogenin 1 (NEUROG1) for egg shape index; and four genes including collagen type VI alpha 3 chain (COL6A3), lysine demethylase 7A (KDM7A), LOC101802169, and sperm-associated antigen 16 (SPAG16) for egg yolk weight and the percentage of yolk to total egg weight. Of the 46 genes, the molecular functions of 22 are related to protein binding, indicating important roles in the formation of egg quality traits. Our findings provide new insight into the genetic basis of egg quality traits in ducks.

Phenotypic and genomic characterization of ST11-K1 CR-hvKP with highly homologous bla KPC-2 -bearing plasmids in China

ABSTRACT Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) strains present a significant global public health threat due to their high mortality rates. This study investigated the genomic characteristics of seven ST11-K1 CR-hvKP isolates harboring highly homologous KPC-2-encoding multidrug-resistance plasmids. The strains were isolated from a Chinese tertiary hospital between 2017 and 2020. Whole-genome sequencing and bioinformatic analysis revealed various antibiotic resistance genes (ARGs) and virulence determinants. The bla KPC-2 -bearing plasmids that contain multiple antibiotic-resistance genes were also identified in these strains. ISfinder and Orifinder were applied to identify insertion sequences (IS) and conjugation-related factors among these bla KPC-2 -bearing plasmids. The bla KPC-2 was highly consistent in seven bla KPC-2 -bearing plasmids (IS Kpn6-bla KPC-2 -IS Kpn27 -IS Yps3 -IS 26 ). In addition, we found a region composed of IS IR , Tn 5393 , and IS 26 . It was located upstream of the bla CTX-M-15 gene and presented in six bla KPC-2 -bearing plasmids, with pCR-hvKP221-KPC-P3 as an exception. Conjugation experiments demonstrated the horizontal transfer of resistance plasmids pCR-hvKP128-KPC-P1 and pCR-hvKP132-KPC-P1 across species. Notably, pLVPK-like virulence plasmids carrying virulence gene clusters pCR-hvKP173-Vir-P1, and pCR-hvKP221-Vir-P1 were also detected. A fusional plasmid pCR-hvKP221-Vir-P2, which carries virulence gene clusters and ARGs, was also identified. Five CR-hvKP strains displayed enhanced biofilm formation and high virulence in vivo infection models. Phylogenetic and single nucleotide polymorphism (SNP) analyses indicated a close genetic relationship among the isolates, suggesting a subclade. These findings highlight the complex genetic profiles and potential transmission mechanisms of CR-hvKP strains. IMPORTANCE We reported seven CR-hvKP strains all carried a highly homologous bla KPC-2 integrated IncFⅡ-resistant plasmid, and two strains harbored virulence plasmids. Conjugation experiments confirmed the transferability of these plasmids, indicating a potential for resistance spread. Phylogenetic analysis clarified the relationship among the CR-hvKP isolates. This study provides insights into the phenotypic and genomic characteristics of seven ST11-K1 CR-hvKP strains. The high prevalence and potential for local outbreaks emphasize the need for effective control measures.

De Novo Missense Mutation in FREM1 Identified in a Chinese Patient with Comorbid Congenital Microtia and Pulmonary Hypoplasia

Cases of microtia combined with pulmonary hypoplasia are occasionally in clinics, and its genetic etiology has so far proved inconclusive. Here, aiming to contribute to a better understanding of microtia-related comorbid respiratory anomalies, the authors provide a clinical and genetic description of a rare trio family of which the son suffers combined deformities of right microtia, left pulmonary hypoplasia, and dextrocardia using whole-genome sequence (WGS). A novel potential pathologic compound heterozygosity in the FREM1 gene was identified and validated by the trio and bioinformatics analysis. This case expands the FREM1 mutation genotype and phenotype spectrum, sheds light on the congenital deformities of microtia and pulmonary hypoplasia, and emphasizes the importance of the FREM1 gene in pulmonary and craniofacial development.

C-C motif chemokine receptor-2 blockade ameliorates pulmonary hypertension in rats and synergizes with a pulmonary vasodilator

Abstract Aims We investigated whether the disruption of C-C motif chemokine receptor (CCR) 2 may attenuate the development of pulmonary arterial hypertension (PAH) in any rat models with the reversal of the associated pro-inflammatory state and vascular dysfunction, and synergize with a conventional pulmonary vasodilator. Methods and Results Using Ccr2(-/-) rats generated by CRISPR/Cas9, we investigated pulmonary hypertension (PH) in Ccr2(+/+) or Ccr2(-/-) rats treated with monocrotaline (MCT), SU5416/hypoxia (SuHx) and chronic hypoxia (CH). Ccr2(-/-) decreased the right ventricular systolic pressure, an index of right ventricular hypertrophy and mortality rate, and reversed increased expression of inflammatory cytokines/chemokines (interleukin-6, tumor necrosis factor-α, C-C motif chemokine receptor (CCL)-2, interleukin-1β, transforming growth factor-β) in rats 3weeks after MCT injection, but not in SuHx or CH models. Consistently, Ccr2(-/-) decreased indices of pulmonary vascular diseases (PVD) and perivascular macrophage infiltration, as well as reversed impaired bone morphogenetic protein receptor type 2 signaling, increased endothelial apoptosis and impaired nitric oxide signaling and decreased phosphodiesterase-5 (PDE5) expression in lungs in MCT-treated rats. Gene expression of receptors for prostaglandin I2 and endothelin was not changed by Ccr2(-/-) in MCT-treated rats. In cultured pulmonary arterial smooth muscle cells (PASMCs), Ccr2(-/-) suppressed CCL2-induced hyperproliferation and dedifferentiation as well as reversed CCL2-induced decrease in PDE5 expression. The whole-genome RNA sequencing analysis identified differentially expressed genes in CCL2-stimulated Ccr2(-/-) PASMCs, which are related to regulation of cellular differentiation and contraction. Based on studies in rats and cultured PASMCs, we investigated whether a PDE5 inhibitor, tadalafil, synergizes with Ccr2(-/-). Tadalafil administration ameliorated PH and PVDs in MCT-treated Ccr2(-/-) rats but not in Ccr2(+/+) rats. Tadalafil further improved survival in MCT-treated Ccr2(-/-) rats. Conclusion The present findings demonstrated that CCR2 disruption ameliorated PAH in MCT-treated rats, which was associated with the reversal of dysregulated inflammatory pathways and vascular dysfunction and synergized with tadalafil. These findings suggest that CCR2 may be a therapeutic target in intractable PAH patients with a certain CCR2-related inflammatory phenotype and refractory to conventional pulmonary vasodilators.

Prognostic value of circulating tumor DNA in different cancer types detected by ultra-low-pass whole-genome sequencing. A systematic review and patient-level survival data meta-analysis.

Ultra-low-pass whole-genome sequencing (ULP-WGS) (≤0.5× coverage) of plasma cell-free DNA (cfDNA) has emerged as a low-cost promising tool to assess circulating tumor DNA (ctDNA) fraction. This meta-analysis aims to summarize the current findings and comprehensively investigate the prognostic value of baseline ctDNA detected by ULP-WGS in solid tumors. A systematic review was carried out by searching PubMed/MEDLINE and Scopus databases to identify eligible studies conducted between January 2014 and January 2024. Inclusion criteria comprised studies with reported overall survival (OS) and progression-free survival (PFS) outcomes across therapy-naïve patients with different solid tumors. All patients underwent baseline ULP-WGS of plasma cfDNA and were categorized as ctDNA positive (tumor fraction ≥10%) or negative (tumor fraction <10%). A one-stage meta-analysis was performed using patient-level survival data reconstructed from published articles. A Cox proportional hazards model with shared frailty was used to assess the difference in survival between arms. A total of six studies, comprising 620 patients (367 negative ctDNA and 253 positive ctDNA), were included in the OS analysis, while five studies, involving 349 patients (212 negative ctDNA and 137 positive ctDNA), were included in the PFS analysis. The meta-analysis showed that patients with baseline positive ctDNA had a significantly higher risk of death (HR = 2.60, 95% CI: 2.01-3.36) and disease progression (HR = 2.28, 95% CI: 1.71-3.05) compared to those with negative ctDNA. The presence of a positive ctDNA at baseline is associated with increased risk of death and progression in patients with same stage cancer.

Whole-genome sequencing confirms multiple species of Galapagos giant tortoises.

Galapagos giant tortoises are endemic to the Galapagos Archipelago, where they are found in isolated populations. While these populations are widely considered distinguishable in morphology, behavior, and genetics, the recent divergence of these taxa has made their status as species controversial. Here, we apply multispecies coalescent methods for species delimitation to whole genome resequencing data from 38 tortoises across all 13 extant taxa to assess support for delimiting these taxa as species. In contrast to previous studies based solely on divergence time, we find strong evidence to reject the hypothesis that all Galapagos giant tortoises belong to a single species. Instead, a conservative interpretation of model-based and divergence-based results indicates that these taxa form a species complex consisting of a minimum of 9 species, with most analyses supporting 13 species. There is mixed support for the species status of taxa living on the same island, with some methods delimiting them as separate species and others suggesting multiple populations of a single species per island. These results make clear that Galapagos giant tortoise taxa represent different stages in the process of speciation, with some taxa further along in that evolutionary process than others. Our study provides insight into the complex process of speciation on islands, which is urgently needed given the threatened status of island species around the world. Las tortugas gigantes de las Galápagos son endémicas del Archipiélago de Galápagos, donde se encuentran como poblaciones aisladas. Aunque estas poblaciones se consideran distinguibles en cuanto a morfología, comportamiento y genética, la divergencia reciente de estos taxones hace que su estatus como especies sea controvertido. Aquí aplicamos métodos de coalescencia de especies múltiples con datos de resecuenciación de genomas completos de 38 tortugas de los 13 taxones existentes para evaluar el sustento de la delimitación de estos taxones como especies. En contraste con estudios previos basados únicamente en el tiempo de divergencia, encontramos evidencia sólida para rechazar la hipótesis de que todas las tortugas gigantes de las Galápagos pertenecen a una sola especie. En cambio, una interpretación conservadora de los resultados basados en modelos y divergencia indica que estos taxones forman un complejo de especies que consiste de un mínimo de 9 especies, con la mayoría de los análisis respaldando la existencia de 13 especies. Hay sustento mixto para designar como especies a los taxones que habitan la misma isla, con algunos métodos delimitándolos como especies y otros sugiriendo la existencia de poblaciones múltiples de una sola especie por isla. Estos resultados dejan en claro que los taxones de tortugas gigantes de las Galápagos representan diferentes etapas del proceso de especiación, con algunos taxones más avanzados en ese proceso evolutivo que otros. Nuestro estudio ofrece una perspectiva sobre el complejo proceso de especiación en islas, la cual es urgentemente necesaria dado lo amenazadas que se encuentran las especies insulares en todo el mundo.

Genomic Prediction Using Imputed Whole-Genome Sequence Data in Australian Angus Cattle.

Whole-genome sequence (WGS) data was used to estimate genomic breeding values for growth and carcass traits in Australian Angus cattle. The study aimed to compare the accuracy and bias of genomic predictions with three marker densities, including 50K, high-density (HD) and WGS. The dataset used in this study consisted of animals born between 2013 and 2022. Body weight traits included birthweight, weight at 400 days and weight at 600 days of age. The carcass traits were carcass weight, carcass intramuscular fat and carcass marbling score. The accuracy and bias of prediction were assessed using the cross-validation. Further, for the growth traits, animals in the validation group were subdivided into two subgroups, which were moderately or highly related to the reference. Genomic best linear unbiased prediction (GBLUP) was used to compare genomic predictions with the three marker densities. The prediction accuracies were generally similar across the marker densities, ranging between 0.61 and 0.68 for the body weight traits and between 0.40 and 0.52 for the carcass traits. However, the accuracies marginally decreased as the marker density increased for all the traits studied. A similar lack of difference was found when considering the accuracy by the relatedness subgroups. The results indicated that no meaningful difference in prediction accuracy was estimated when comparing the three marker densities due to the population structure. In conclusion, there was no substantial improvement in genomic prediction when using the WGS in this study.