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PubMed2025

Whole-genome sequencing, strain composition, and predicted antimicrobial resistance of Streptococcus pneumoniae causing invasive disease in England in 2017-20: a prospective national surveillance study.

BACKGROUND: Surveillance of the invasive disease burden caused by Streptococcus pneumoniae in England is performed by the UK Health Security Agency (UKHSA). In 2017, UKHSA switched from phenotypic methods to whole-genome sequencing (WGS) approaches for pneumococcal surveillance. Here, we present the first results of national WGS surveillance, up to the start of the COVID-19 pandemic, with the aim of describing the population genomics of this important pathogen. METHODS: We examined prospective national surveillance data from England, using bacterial isolates from cases of invasive pneumococcal disease (IPD) submitted to the national reference laboratory at UKHSA. A bioinformatic pipeline was developed to quality control WGS data and routinely report species and serotype. We assembled isolate data, assigned global pneumococcal sequencing clusters (GPSCs), and predicted antimicrobial resistance (AMR) profiles for isolates that passed further quality control. We collected additional data on patient outcomes and characteristics using enhanced surveillance questionnaires completed by patients' general practitioners. We used logistic regression analysis to assess the effects of various genomic and patient characteristics on the outcomes of IPD. FINDINGS: In England, between July 1, 2017, and Feb 29, 2020, there were 15 400 cases of IPD. From these cases, 13 749 (89·3%) isolates were sequenced, passed quality control, and were included in analyses. Serotype diversity was high during the study period, with 2751 (20%) isolates serotyped as 13-valent pneumococcal conjugate vaccine (PCV13) types, whereas serotype 8 was the most prevalent serotype (n=3074 [22·4%]) overall. There were 157 GPSCs within the collection, with GSPC3 the most common, encompassing 98·7% (3033 of 3074) of serotype 8 isolates. Most isolates (n=10 198 [74·2%]) did not contain AMR-associated genes. Resistance to co-trimoxazole was the most frequently predicted resistance (n=2331 [17%]), followed by resistance to tetracycline (n=1199 [8·7%]) and β-lactams (n=1149 [8·4%]). Logistic regression analysis found the presence of AMR-associated genes significantly increased the odds of patient death (odds ratio 1·18, 95% CI 1·01-1·38). Some GPSCs were also associated with a significant increase in the odds of patient death, such as GPSC12 (1·88, 1·48-2·38). Isolates from 2018 were associated with a significant increase in the odds of patient death (1·12, 1·00-1·25), whereas younger patient age was significantly associated with a reduction in the odds of patient death compared with being aged 85 years or older. INTERPRETATION: WGS-based surveillance has allowed us to interrogate country-wide population dynamics driving changes in pneumococcal serotype frequency. Here, we observe a stable but diverse population before the COVID-19 pandemic restrictions were enforced in England, with low rates of AMR. These findings will provide the baseline for pandemic and post-pandemic data, to collectively inform implementation and development of the vaccination programme within the country. FUNDING: None.

Streptococcus pneumoniae↗

PubMed2026

Microbiological analysis and whole-genome sequencing of Neisseria gonorrhoeae from the microbiological failures in the international, zoliflodacin, phase 3, clinical trial for treatment of uncomplicated urogenital gonorrhoea: a retrospective, genomic, observational study.

BACKGROUND: Zoliflodacin, a first-in-class oral bacterial, DNA gyrase (GyrB) inhibitor, showed non-inferiority to ceftriaxone combined with azithromycin in a recent large international, phase 3, randomised controlled trial for treatment of uncomplicated urogenital gonorrhoea. The aim of this study was to describe the microbiological and whole-genome sequencing (WGS) analyses of paired baseline (pre-treatment) and test-of-cure (TOC) gonococcal isolates from the zoliflodacin phase 3, randomised controlled trial to further characterise and evaluate the protocol-specified microbiological failures with zoliflodacin (n=22) or ceftriaxone and azithromycin (n=1). METHODS: In this retrospective, genomic, observational study, results from antimicrobial susceptibility testing (agar dilution method) of isolates (n=960; 936 baseline isolates from 763 participants and 24 TOC isolates [23 with a paired baseline isolate in the same anatomical site] from 20 participants) collected during the zoliflodacin phase 3, randomised controlled trial done in 16 outpatient clinics in Belgium, the Netherlands, South Africa, Thailand, and the USA (Nov 6, 2019-March 16, 2023) are described. WGS analysis was performed on paired baseline and TOC isolates from participants with microbiological failures (zoliflodacin 44 isolates [19 participants]; ceftriaxone and azithromycin two isolates [one participant]), and the three baseline isolates with highest zoliflodacin minimum inhibitory concentration (MIC 0·5 mg/L). FINDINGS: All isolates were inhibited by the same zoliflodacin concentrations (MICs ≤0·008 to 0·5 mg/L) as wild-type strains cultured internationally in 2013-23. In participants with a microbiological failure after zoliflodacin treatment (n=22, 19 participants), zoliflodacin MIC values for baseline and TOC isolates were similar, and resistance selection was lacking. WGS showed that five (23%) of 22 infections (95% CI 10-43 [in four participants]) of zoliflodacin microbiological failures had different strains at TOC versus baseline. In 17 zoliflodacin microbiological failures (15 participants), isolates at baseline and TOC were indistinguishable. 13 of these 17 microbiological failures, corresponding to 59% (95% CI 39-77; 13 of 22) of all zoliflodacin microbiological failures, were in urogenital or rectal sites in 11 participants and the isolates had zoliflodacin MICs less than or equal to 0·008 to 0·25 mg/L. The single microbiological failure after ceftriaxone and azithromycin treatment had different strains at TOC versus at baseline. No sequenced isolates had mutations associated with elevated zoliflodacin MICs. INTERPRETATION: In the zoliflodacin phase 3, randomised controlled trial, 23% of the zoliflodacin microbiological failures and the single ceftriaxone and azithromycin microbiological failure had different gonococcal strains at TOC versus baseline, which suggests reinfections and not treatment failures. In addition, 59% of the zoliflodacin microbiological failures, all in anogenital sites, had no obvious microbiological explanation based on the low zoliflodacin MICs, previous pharmacodynamic studies, and no evidence of resistance selection after zoliflodacin therapy. A reinfection as the cause for these microbiological failures could not be excluded. We recommend that WGS is implemented in future randomised controlled trials for gonorrhoea treatment to further evaluate possible microbiological failures, exclude reinfections (to avoid underestimating the cure rates), and characterise antimicrobial resistance determinants. FUNDING: GARDP through grants from Germany BMFTR (03KA1831), UK DHSC as part of GAMRIF, Japan MHLW, the Netherlands' Ministry of Health, Welfare and Sport and Directorate-General for International Cooperation, the Federal Office of Public Health of Switzerland, the Canton of Geneva, Switzerland, and Örebro University Hospital, Sweden.

Humans↗

PubMed2026

Development and evaluation of an ARTIC-based amplicon sequencing assay for whole-genome characterization of respiratory syncytial virus.

Respiratory syncytial virus (RSV), a ~15.2 kb negative-sense RNA virus, causes acute respiratory infections in infants and older adults. Its two subtypes, RSV-A and RSV-B, evolve rapidly, making ongoing monitoring of circulating strains essential. The Georgia Public Health Laboratory (GPHL) developed and evaluated an amplicon-based whole-genome sequencing (WGS) assay for RSV surveillance. A total of 214 de-identified remnant clinical specimens (102 RSV-A and 112 RSV-B) with RT-PCR Cq values <31 were included. RSV genomes were amplified using ARTIC-style and custom primer sets, with the ARTIC set showing superior performance. Libraries were prepared using a modified Illumina COVIDSeq protocol, sequenced on NextSeq 1000/2000 instruments, and analyzed using the GPHL-RSV-PIPE bioinformatics pipeline. Among genomes meeting validation criteria, sequencing depth was slightly higher for RSV-A (median 53,433×; mean 51,076×) than RSV-B (median 49,699×; mean 46,945×), whereas genomic coverage was slightly lower for RSV-A (median 97.5%; mean 96.6%) than RSV-B (median 98.3%; mean 97.6%). Predominant lineages were A.D.3.1 and A.D.5.2 for RSV-A and B.D.E.1 for RSV-B. For RSV-A, the assay showed 92.8% accuracy, 96.2% sensitivity, 87.2% specificity, 92.6% positive predictive value, and 93.2% negative predictive value. Intra- and inter-run precision assessed using 16 and 53-57 genomes, respectively, showed nearly 100% consensus genome identity with 0-5 nucleotide differences. Specificity testing of 31 non-RSV specimens produced no false-positive detections. Limits of detection were 4.4 TCID50/mL for RSV-A and 18.6 TCID50/mL for RSV-B. These results demonstrate that the ARTIC-based RSV WGS assay enables near real-time surveillance and strengthens data-driven public health responses to future outbreaks.IMPORTANCERSV, with two major subtypes, RSV-A and RSV-B, causes acute respiratory infections that can be severe in infants under 6 months and older adults. Current RSV surveillance at the GPHL relies on the Thermo Fisher TaqMan Gene Expression Capillary assay, which detects and subtypes RSV but lacks resolution for lineage classification and identification of emerging variants. To address this critical gap, GPHL developed and evaluated an amplicon-based WGS assay using 214 de-identified RSV clinical specimens. Genomes were amplified using ARTIC-style and custom-primer sets, with ARTIC primers showing superior performance. The assay demonstrated strong sequencing depth, genomic coverage, specificity, repeatability, reproducibility, and low limits of detection. RSV lineages were accurately determined based on genetic variation. These results establish that the ARTIC-based WGS assay enables near real-time genomic surveillance, supporting monitoring of circulating RSV strains and informing data-driven public health responses.

bioinformatics pipeline↗

PubMed2025

Use of IR Biotyper as a feasible methodology to type Klebsiella pneumoniae.

UNLABELLED: Klebsiella pneumoniae is one of the most frequently reported healthcare-associated pathogens. The current gold standard approach to perform the epidemiological typing of these bacteria is Whole Genome Sequencing (WGS), which is an expensive and challenging procedure. IR Biotyper (Bruker Daltonics, GmbH) is a new equipment based on Fourier transform infrared spectroscopy, which allows a rapid, low-cost, and user-friendly method to type bacterial isolates. However, there is a need for studies that evaluate the efficacy of the IR Biotyper. The aim of this study was to evaluate the capability of IR Biotyper to type K. pneumoniae according to sequence type (ST) and capsular type-using K locus (KL)-as well as to develop a classifier using machine learning. Seventy-three isolates of K. pneumoniae previously characterized by WGS were selected for IR Biotyper analysis using principal component analysis for dimensionality reduction, Euclidean, and unweighted pair group method with arithmetic mean (UPGMA) for clustering method, and spectra were analyzed in the 1,300-800 cm⁻¹ wavenumber range. Among these, 54 isolates were used to create a classifier, and 19 were used to validate the classifier. When considering the ST, ST307 was grouped in the same cluster as ST11. When KL was considered for the analysis, the clusters were 100% correctly grouped according to their KL type. Furthermore, the classifier developed was able to classify the isolates according to KL with a high concordance. This study showed that KL correlates well with KL for typing K. pneumoniae isolates using the IR Biotyper. Additionally, IR Biotyper demonstrated to be a cost-effective method and a promising tool to classify isolates within minutes. IMPORTANCE: Klebsiella pneumoniae is a major cause of severe hospital infections, and controlling its spread requires quick identification and comparison of bacterial strains. WGS is accurate but expensive, slow, and technically demanding. In this study, we evaluated the IR Biotyper, a device that uses infrared light to analyze bacteria and group them by capsule type-a key feature linked to their spread. The IR Biotyper matched WGS results with high accuracy, delivering results in minutes instead of days. This fast, affordable method can help hospitals detect outbreaks earlier and respond more effectively. Our findings suggest that the IR Biotyper is a valuable tool for routine use in microbiology laboratories, supporting epidemiological surveillance and outbreak control.

Klebsiella pneumoniae↗

PubMed2021

Whole-genome sequencing-based phylogeny, antibiotic resistance, and invasive phenotype of Escherichia coli strains colonizing the cervix of women in preterm labor.

BACKGROUND: Escherichia coli is a major neonatal pathogen and the leading cause of early-onset sepsis in preterm newborns. Maternal E. coli strains are transmitted to the newborn causing invasive neonatal disease. However, there is a lack of data regarding the phenotypic and genotypic characterization of E. coli strains colonizing pregnant women during labor. METHODS: This prospective study performed at the University of Oklahoma Medical Center (OUHSC) from March 2014 to December 2015, aimed to investigate the colonization rate, and the phylogeny, antibiotic resistance traits, and invasive properties of E. coli strains colonizing the cervix of fifty pregnant women diagnosed with preterm labor (PTL). Molecular analyses including bacterial whole-genome sequencing (WGS), were performed to examine phylogenetic relationships among the colonizing strains and compare them with WGS data of representative invasive neonatal E. coli isolates. Phenotypic and genotypic antibiotic resistance traits were investigated. The bacteria's ability to invade epithelial cells in vitro was determined. RESULTS: We recruited fifty women in PTL. Cervical samples yielded E. coli in 12 % (n=6). The mean gestational age was 32.5 (SD±3.19) weeks. None delivered an infant with E. coli disease. Phenotypic and genotypic antibiotic resistance testing did not overall demonstrate extensive drug resistance traits among the cervical E. coli isolates, however, one isolate was multi-drug resistant. The isolates belonged to five different phylogroups, and WGS analyses assigned each to individual multi-locus sequence types. Single nucleotide polymorphism-based comparisons of cervical E. coli strains with six representative neonatal E. coli bacteremia isolates demonstrated that only half of the cervical E. coli isolates were phylogenetically related to these neonatal invasive strains. Moreover, WGS comparisons showed that each cervical E. coli isolate had distinct genomic regions that were not shared with neonatal E. coli isolates. Cervical and neonatal E. coli isolates that were most closely related at the phylogenetic level had similar invasion capacity into intestinal epithelial cells. In contrast, phylogenetically dissimilar cervical E. coli strains were the least invasive among all isolates. CONCLUSIONS: This pilot study showed that a minority of women in PTL were colonized in the cervix with E. coli, and colonizing strains were not phylogenetically uniformly representative of E. coli strains that commonly cause invasive disease in newborns. Larger studies are needed to determine the molecular characteristics of E. coli strains colonizing pregnant women associated with an increased risk of neonatal septicemia.

Adult↗

PubMed2026

Paired Whole-Genome Sequencing of Scalp Angiosarcoma and Matched Lung Metastasis Reveals Common Clonal Origin and Lung-Specific Evolution.

Background and Clinical Significance: Cutaneous angiosarcoma frequently metastasizes to the lungs, where it may rarely present as diffuse cystic lung disease with recurrent pneumothorax, resulting in substantial diagnostic difficulty. We report a case of pulmonary metastatic cutaneous angiosarcoma in which paired whole-genome sequencing (WGS) of the primary and metastatic lesions was performed to clarify clonal origin and characterize metastatic evolution. Case Presentation: A 65-year-old man with recurrent right-sided pneumothorax and progressive bilateral cystic lung lesions underwent skin and lung biopsies. Histopathological examination and immunohistochemistry established the diagnosis of cutaneous angiosarcoma with pulmonary metastases. Paired WGS was performed on matched scalp and lung tumor specimens to evaluate shared and lesion-specific genomic alterations, pathway enrichment, and copy-number changes. Histopathology confirmed metastatic angiosarcoma involving the lungs. WGS identified 128 shared somatic alterations, supporting a common clonal origin, together with lung-specific and skin-specific mutations indicative of continued genomic divergence. Recurrent alterations involving POT1 and FLT4 were preserved in both lesions, whereas additional POT1 and TP53 alterations were detected only in the pulmonary metastasis. Pathway analysis demonstrated preferential enrichment of IGF1-mTOR, RAS, and WNT/LRP6 signaling in the metastatic lesion, while Gene Ontology analysis suggested functional divergence associated with metastatic progression. Conclusions: Pulmonary metastatic angiosarcoma should be considered in patients presenting with unexplained diffuse cystic lung disease and recurrent pneumothorax, particularly when pathological findings are inconclusive. Paired WGS complemented conventional histopathology by confirming the metastatic origin and providing insights into clonal evolution and lesion-specific molecular alterations, highlighting its potential value in the investigation of rare metastatic malignancies.

angiosarcoma↗

PubMed2025

Whole-genome sequencing, as a powerful diagnostic tool in hearing loss, reveals novel variants in PTPRQ missed by whole-exome sequencing.

BACKGROUND/OBJECTIVES: Hearing loss (HL) is one of the most common congenital disorders, affecting 1-2 in 1,000 newborns. Modern genetic diagnostics using large gene panels and/or whole exome analysis (WES) can identify disease-causing mutations in 25-50 % of patients, with higher solve rates in individuals with earlier onset. RESULTS: Here, we used whole-genome sequencing (WGS) to reanalyze 14 index patients/families who remained without genetic diagnosis by WES. We were able to identify the genetic cause of HL in 6 families (43 %). Two families were diagnosed with DFNB84A caused by compound heterozygous recessive mutations in PTPRQ. Three of the four underlying variants, including a structural variant, a deep intronic variant, and a splice variant, escaped detection by WES. Minigene assays confirmed the pathogenicity of the intronic and the splice variants. In addition, we used protein 3D structure prediction and rigid ligand docking to study the pathogenicity of variants that escape nonsense-mediated decay. CONCLUSION: In our study, we present four novel variants in PTPRQ, three of which were detected only by WGS. To our knowledge, we report here the first pathogenic deep intronic PTPRQ variant causing HL. Our results suggest that the mutational spectrum of PTPRQ is not well covered by standard WES and that PTPRQ-associated hearing loss may be more frequent than previously thought. WGS provides an additional layer of information in the diagnostics of HL.

Humans↗

PubMed2025

Founder Homozygous Nonsense CREB3 Variant and Variable-Onset Retinal Degeneration.

IMPORTANCE: Uncovering the genetic basis of inherited retinal diseases (IRDs) can enhance both diagnostic accuracy and the development of targeted treatment strategies. OBJECTIVE: To evaluate the association between a homozygous nonsense variant in CREB3 with IRDs. DESIGN, SETTING, AND PARTICIPANTS: Thirteen patients with a clinical diagnosis of retinitis pigmentosa or cone-rod degeneration were analyzed by whole-genome sequencing (WGS) and whole-exome sequencing (WES). Clinically, patients presented with 2 main phenotypes, rod-cone and cone-rod dystrophies, demonstrating variable electrophysiological and fundoscopic findings. Expression analysis was performed on patient-derived skin fibroblasts using the reverse transcription-polymerase chain reaction and Western blot analysis, and by interrogating previously published retinal single-cell RNA sequence data. Immunohistochemistry staining was performed on wild-type mouse retinal sections using an anti-CREB3 antibody. Patients with variable phenotypes of IRDs were recruited from 3 medical centers in Israel and Italy. Ophthalmologists clinically diagnosed patients at the relevant medical centers and referred them for genetic screening. WES and WGS were performed at different national and international centers, and the findings of the previously unreported gene were shared between investigators. EXPOSURES: CREB3 and IRDs. MAIN OUTCOMES AND MEASURES: The main outcome was evidence supporting an association between CREB3 and IRD. Measures included WES, WGS, and immunohistochemistry staining. RESULTS: A founder homozygous nonsense variant in CREB3 (c.881G>A, p.Trp294*) was identified in 13 patients from 4 unrelated families; 12 descendent from North-African Jewish origins and 1 from Italian origins. All patients manifested retinal degeneration with varying ages at onset. In patient-derived fibroblasts, the variant mRNA transcript generated a truncated CREB3 protein. Expression analysis and immunohistochemistry staining revealed CREB3 RNA and protein expression in various retinal cell types, indicating its vital role in photoreceptor function. CONCLUSIONS AND RELEVANCE: This study found an association between CREB3 and IRDs. CREB3 was previously shown to be upregulated following ultraviolet radiation. This might contribute to the extensive clinical variability observed in this relatively large cohort of homozygous patients with the same truncated variant.

Humans↗

PubMed2025

Development and Validation of Amplicon-Based Protocol for Sequencing of Respiratory Syncytial Virus Genome.

The most prevalent cause of severe respiratory infections in children is the human respiratory syncytial virus (RSV). The advent of next-generation sequencing (NGS) has made it possible to incorporate this technology into pathogen monitoring and surveillance. Whole-genome sequencing (WGS) of RSV has now become a relatively widely used method for tracking viral evolution. Here we report an improved high-throughput RSV-WGS assay performed directly on clinical samples that is suitable for short-read sequencing platforms. A total of 100 RSV-positive samples collected between November 2022 and March 2024 fulfilled the inclusion cycle quantification criteria and were randomly included in the validation process. The WGS protocol was designed to amplify three distinct amplicons to cover the entire RSV genome. The protocol described here can be successfully replicated in several instances (approximately 95%) in samples with a relatively low viral load, typically corresponding to cycle of quantification values of 27-32. The amplicon-based protocol produced meaningful sequencing results in terms of median depth of coverage (more than 12000×) and median of mapped reads (> 1 × 106 reads). The sequences that had passed the filters showed a coverage of at least 98% across the entire genome, with cycle quantification values of 32. Based on the obtained data resulting in an easy-to-perform protocol helpful for the molecular epidemiology surveillance of RSV.

Humans↗

PubMed2025

Assessment of genomic prediction capabilities of transcriptome data in a barley multi-parent RIL population.

Low-cost and high-throughput RNA sequencing data for barley RILs achieved GP performance comparable to or better than traditional SNP array datasets when combined with parental whole-genome sequencing SNP data. The field of genomic selection (GS) is advancing rapidly on many fronts including the utilization of multi-omics datasets with the goal of increasing prediction ability and becoming an integral part of an increasing number of breeding programs ensuring future food security. In this study, we used RNA sequencing (RNA-Seq) data to perform genomic prediction (GP) on three related barley RIL populations. We investigated the potential of increasing prediction ability by combining genomic and transcriptomic datasets, adding whole-genome sequencing (WGS) SNP data, functional annotation-based filtering, and empirical quality filtering. Our RNA-Seq data were generated cost-efficiently using small-footprint plant cultivation, high-throughput RNA extraction, and Library preparation miniaturization. We also examined sequencing depth reduction as an additional cost-saving measure. We used fivefold cross-validation to evaluate the prediction ability of the gene expression dataset, the RNA-Seq SNP dataset, and the consensus SNP dataset between the RNA-Seq and parental WGS data, resulting in prediction abilities between 0.73 and 0.78. The consensus SNP dataset performed best, with five out of eight traits performing significantly better compared to a 50K SNP array, which served as a benchmark. The advantage of the consensus SNP dataset was most prominent in the inter-population predictions, in which the training and validation sets originated from different RIL sub-populations. We were therefore able to not only show that RNA-Seq data alone are able to predict various complex traits in barley using RILs, but also that the performance can be further increased with WGS data for which the public availability will steadily increase.

Hordeum↗

PubMed2026

Clinical and Genomic Insights into the Allodiploid Hybrid Pathogen Aspergillus latus: A Retrospective Case Series.

Aspergillus latus is an emerging cryptic allodiploid hybrid pathogen within Aspergillus section Nidulantes that closely resembles related species and therefore prone to misidentification by routine diagnostic methods. Therefore, its true clinical burden is likely underestimated. In this study, we retrospectively characterized five patients with A. latus infections identified by metagenomic next-generation sequencing (mNGS) at a tertiary hospital in China. Clinical manifestations varied according to host immune status, ranging from a subclinical pulmonary lesion in an immunocompetent individual to aggressive disease in highly immunocompromised patients. Conventional microbiological methods showed limited sensitivity and consistently misidentified the isolates as A. nidulans, whereas mNGS enabled accurate detection of A. latus together with complex co-infections. Three viable clinical isolates were recovered for morphological characterization, antifungal susceptibility testing, and whole-genome sequencing (WGS). All tested isolates demonstrated reduced susceptibility to echinocandins but remained susceptible to mold-active triazoles and amphotericin B. Furthermore, WGS and macrosynteny analyses confirmed their allodiploid hybrid nature, revealing a mosaic genome derived from A. spinulosporus and an A. quadrilineatus-related lineage. Collectively, these findings highlight that A. latus may be missed by routine diagnostic methods and may exhibit a distinct antifungal susceptibility profile. Molecular approaches such as mNGS and WGS may therefore help achieve accurate species-level identification and support targeted antifungal therapy. Given this small retrospective case series, larger prospective and multicenter studies are needed to validate these observations and better define the epidemiology, clinical spectrum, and therapeutic implications of this emerging allodiploid hybrid pathogen.

Retrospective Studies↗

PubMed2026

Genomic and phenotypic characterization of mcr-9 carrying Enterobacter oligotrophicus recovered from bovine mastitis.

Bovine mastitis (BM) is a leading cause of economic loss in the dairy industry, driven by decreased milk yields, involuntary culling, and substantial veterinary costs. A single Gram-negative isolate recovered from BM was characterized in this study. For this, antimicrobial susceptibility testing (AST) was performed using the Neg-Urine-Combo 98 panel. Whole-genome sequencing (WGS) was employed to identify antimicrobial resistance genes (ARG), virulence factors (VF) genes, plasmid replicons and prophage sequences. Comparative genomic analysis was performed through phylogenetic analysis. The BM isolate was phenotypically identified as Citrobacter rodentium, however, WGS analysis reclassified the isolate as Enterobacter oligotrophicus. Phenotypic AST revealed a resistance profile of 12%, with the isolate exhibiting resistance to β-lactams antimicrobials, specifically amoxicillin-clavulanate, ampicillin and the cephalosporin-cefoxitin. Conversely, susceptibility was demonstrated for the remaining tested antimicrobials. Genomic profiling identified 31 ARG, 10 VF genes and 6 prophage sequences within the E. oligotrophicus genome. This study provides the first evidence of E. oligotrophicus as a causative agent of BM, expanding the known spectrum of pathogens affecting the dairy industry by delivering the second complete genome of E. oligotrophicus available globally. The identification of 31 ARG, 10 VF, and 6 prophage sequences underscore the potential pathogenic risk and environmental resilience of this isolate. These findings highlight the critical role of WGS-based surveillance in identifying non-conventional mastitis pathogens and underscore the need for targeted mitigation strategies in veterinary medicine.

Animals↗

PubMed2026

Routine methods misidentify Serratia spp.: Limitations of MALDI-TOF MS revealed by whole-genome sequencing.

Accurate species-level identification within the genus Serratia remains challenging due to extensive phenotypic overlap and high genomic relatedness among closely related and recently described taxa. This study presents an evaluation of routine and genome-based identification approaches applied to clinical Serratia isolates, integrating phenotypic assays, MALDI-TOF MS (Bruker Daltonics), 16S rRNA gene sequencing, and Whole-Genome Sequencing (WGS). A total of 103 isolates collected from a teaching hospital were analyzed. WGS was performed on a subset of isolates. Conventional biochemical methods classified all isolates as Serratia marcescens, whereas MALDI-TOF MS identified 60.1% as S. marcescens, 11.6% as S. ureilytica, and 28.1% just at the genus level. Peak analysis from MALDI-TOF MS revealed specific peaks associated with S. marcescens and S. ureilytica, but limited discriminatory power. WGS of six isolates initially identified as S. ureilytica by MALDI-TOF MS revealed reclassification as Serratia sarumanii (n = 5) and Serratia montpellierensis (n = 1), supported by Average Nucleotide Identity (ANI), Average Amino Acid Identity (AAI), and Digital DNA-DNA Hybridization (dDDH) thresholds. In contrast, 16S rRNA analysis showed limited species-level resolution. Phylogenomic and SNP-based analyses confirmed these classifications with strong support. Overall, this study underscores the critical role of high-resolution genomic approaches for precise species identification and highlights the need for continuous expansion and curation of MALDI-TOF MS reference databases to support reliable clinical diagnostics and epidemiological surveillance of emerging Serratia species.

Spectrometry, Mass, Matrix-Assisted Laser Desorpti↗

PubMed2026

Machine learning-based drug susceptibility prediction from Candida genomic data.

OBJECTIVES: Invasive Candida infection is an increasing clinical concern, with antifungal resistance rising across multiple species. However, rapid and accurate antifungal susceptibility testing (AFST) remains limited in routine practice. The study evaluated species distribution and antifungal susceptibility of invasive Candida isolates in China and assessed the feasibility of combining whole-genome sequencing (WGS) with machine learning to predict minimum inhibitory concentrations (MICs). METHODS: Consecutive non-repetitive isolates were collected from 20 hospitals in 13 provinces during 2022-2023. MICs of nine antifungal agents were determined by broth microdilution, and WGS was performed for species accounting for >5% of the total isolates. Genomic 11-mer features were extracted and used to train random forest (RF), support vector machine (SVM), and extreme gradient boosting (XGBoost) models, followed by optimization of the best-performing algorithm. RESULTS: A total of 337 isolates were obtained from blood (n = 232) and sterile body fluids (n = 105), comprising C. albicans (n = 103), C. tropicalis (n = 71), C. parapsilosis (n = 67), and C. glabrata (n = 63). Non-albicans Candida showed higher azole and echinocandin resistance, with C. tropicalis notably resistant to azoles and C. glabrata to echinocandins. Among the three models, RF demonstrated the best performance on 304 sequenced isolates. The optimized RF model was evaluated by the receiver operating characteristic (ROC) curve analysis and achieved an average area under the ROC curve (AUC) of 0.979 (95% CI: 0.974-0.984), essential agreement over 90.1%, and categorical agreement over 93.2% across species. CONCLUSIONS: These findings underscore the clinical challenge posed by non-albicans Candida resistance, and indicate that WGS-based MIC prediction may offer a highly accurate reference for earlier antifungal therapy.

Antifungal Agents↗

PubMed2026

Nanopore-based, long-range Parvovirus B19 amplicon sequencing for near-whole genome characterization.

BACKGROUND: Whole-Genome Sequencing (WGS) enables monitoring of genomic variation and evaluation of diagnostic PCR assays. However, WGS data for Parvovirus B19 (B19V) remains limited despite its relevance for clinical care and transfusion safety. To increase the availability of high-quality B19V genomic data, a near-WGS protocol was developed and validated. METHOD: The protocol combines long-range PCR to generate a 4.6-kb amplicon, covering ∼82% of the B19V genome, with Oxford Nanopore sequencing. Validation was performed using six reference samples and nineteen B19V-positive donor plasma samples. RESULTS: After quality control, samples achieved a median sequencing depth of 152x. Sequences generated from the six reference samples showed 100% concordance with previously published data. Genomic analysis of donor samples explained atypical amplification profiles observed during routine PCR screening. CONCLUSION: The newly developed protocol provides a scalable method for B19V genome characterization, enabling assessment of oligonucleotide-binding regions for PCR assay monitoring and facilitating the generation of genomic data for future epidemiological investigations.

PCR assay monitoring↗

PubMed2026

Characterisation of Carbapenem-Resistant Raoultella planticola and Structural Analysis of NDM Composite Plasmids.

OBJECTIVE: This study aimed to investigate the molecular characteristics, resistant plasmid structures and phylogeny of a carbapenem-resistant Raoultella planticola (CRRP) strain from a patient with pneumonia to inform antimicrobial resistance control strategies. METHODS: We performed strain identification using MALDI-TOF MS, the BD Phoenix 100 system and whole-genome sequencing (WGS). We assessed antimicrobial susceptibility and resistance gene transfer using PCR, conjugation and stability assays, plasmid structure using a bioinformatics tool and phylogeny using a core-genome phylogenetic tree. RESULTS: WGS confirmed the isolate as R. planticola (average nucleotide identity (ANI) > 98.9% with reference type strains), co-harbouring blaKPC-2 and blaNDM-1. It was resistant to 19 antimicrobial agents and susceptible to only polymyxin, amikacin and chloramphenicol. Resistance genes were present on two conjugative plasmids: pzwx_KPC (IncFIA) and pzwx_NDM (a novel repFIB/repHI5B hybrid assembled via non-homologous end joining). Both plasmids demonstrated efficient transfer and stable inheritance over 12 passages. pzwx_KPC was highly homologous to plasmids from Klebsiella pneumoniae. Phylogenetic analysis revealed the closest relationship with German R. planticola strains. CONCLUSION: CRRP carries highly transmissible and stable resistance plasmids. Strengthened monitoring in immunocompromised patients and improved environmental disinfection are recommended. The risk of misidentification by automated systems underscores the importance of WGS for accurate pathogen identification.

Carbapenem resistance↗

PubMed2026

Evaluation of one-step amplicon-based targeted enrichment for SARS-CoV-2 whole-genome sequencing using the Midnight amplicon scheme.

Genomic surveillance proved invaluable during the COVID-19 pandemic for tracking SARS-CoV-2 variants and guiding outbreak responses, underscoring the ongoing need to reduce whole-genome sequencing (WGS) costs and improve workflow efficiency to ensure accessibility in resource limited settings. Here, we evaluated a one-step reverse transcription polymerase chain reaction (RT-PCR) approach using the Midnight V2 primer scheme for targeted amplification of the SARS-CoV-2 genome, assessed its compatibility with Illumina sequencing, and compared its performance to a well-established two-step method. Initially, we determined optimal RT-PCR reaction conditions using the Midnight V2 primer panel for the one-step RT-PCR kit and scaled reaction volumes for both RT-PCR and library preparation. Clinical specimens (n = 53) that had undergone routine WGS for surveillance purposes using the established two-step RT-PCR method were compared using the one-step RT-PCR assay. For samples with genome completeness greater than 70%, both methods gave comparable results with similar sequence coverage and 100% concordance for lineage assignment. Further investigation revealed a higher percentage of reads aligning to the SARS-CoV-2 genome with a greater depth of coverage using the one-step method compared to the two-step method. Finally, analysis of scaled one-step and library reaction volumes revealed significant cost savings for samples undergoing WGS. Overall, the results presented here verify the accuracy and reproducibility of one-step targeted amplification and offer an efficient and cost-effective workflow for routine SARS-CoV-2 genomic surveillance.

Humans↗

PubMed2025

4CMenB vaccine coverage of invasive serogroup B meningococci collected in Belgium between 2016 and 2022.

Neisseria meningitidis infections can cause life-threatening meningitis and septicemia. In Europe, serogroup B (MenB) is the leading cause of invasive meningococcal disease (IMD), particularly in young children. Genomic surveillance of circulating MenB strains through whole genome sequencing (WGS) provides a powerful tool to assess the potential impact of vaccination strategies, including the 4CMenB vaccine, which is available for infants from 2 months of age. Here, we present a retrospective WGS-based analysis of clinical MenB IMD cases (n = 311) recovered in Belgium from 2016 to 2022 by the Belgian National Reference Center. High-quality WGS data were obtained for 281 of these strains, demonstrating high genetic diversity of the antigen targets included in the 4-component meningococcal serogroup B vaccine 4CMenB (fHbp, PorA, NHBA and NadA) and at the 4CMenB Antigen Sequence Types (BAST) level. Novel antigen combinations, not yet assigned a BAST ID, were detected in 23.5% of isolates. Vaccine coverage was predicted using the Genetic Meningococcal Antigen Typing System (gMATS) and the Meningococcal Deduced Vaccine Antigen Reactivity (MenDeVAR) index. Of the 281 strains, 79.5% (lower limit-upper limit: 68.0-91.5%) were predicted to be covered by the vaccine by gMATS, and 80.7% (lower limit-upper limit: 66.5-95.4%) by MenDeVAR. No evidence of variation in vaccine coverage was found throughout the study period nor between different age groups, demonstrating the broad applicability of 4CMenB. This study highlights the benefits of a pathogen surveillance program and the need for experimental characterization of continuously evolving antigenic subvariants of Neisseria meningitidis.

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