Search Results (258)

Treatment of Mycobacterium tuberculosis requires multi-drug regimens, and resistance to any individual antibiotic can compromise outcomes. For slow-growing organisms like M. tuberculosis, rapid detection of resistance-conferring mutations enables timely initiation of effective therapy. Conversely, confirming wild-type status in resistance-associated genes supports confidence in standard regimens. We developed an amplicon-based next generation sequencing (amplicon tNGS) assay on the Illumina platform targeting eight genes linked to resistance to isoniazid, rifampin, ethambutol, pyrazinamide, and fluoroquinolones. Sequencing results were analyzed using a custom bioinformatics pipeline. Forty-seven samples were used for assay development, and 37 additional samples underwent post-implementation clinical validation. Compared to whole genome sequencing (WGS), amplicon tNGS demonstrated 97.7% sensitivity, 98.9% specificity, and 98.7% overall accuracy for variant detection in targeted regions. Resistance prediction showed 79.3% concordance with WGS; discrepancies were primarily due to mutations outside of target regions. Among post-implementation samples, 27/37 passed quality metrics for all targets, with 95.7% concordance between amplicon tNGS results and final susceptibility results. This assay is now in use in our laboratory and offers significantly faster turnaround than both WGS and phenotypic methods on cultured isolates, enabling more rapid, informed treatment decisions for tuberculosis patients. Full article

Accurate quantification of Bifidobacterium animalis subsp. lactis HN019, a clinically validated probiotic strain conferring immune modulation, gastrointestinal health, and gut barrier integrity benefits, is essential for diverse applications. To address the critical need for strain-specific detection, we developed a quantitative PCR (qPCR) assay targeting a unique single-nucleotide polymorphism (SNP) within the galK gene, identified through comparative whole-genome sequencing (WGS) analysis of 31 B. animalis subsp. lactis strains. The assay exhibited exceptional specificity, distinguishing HN019 from 19 other Bifidobacterium strains. Sensitivity tests indicated a detection limit of 0.5 pg of DNA and 10³ CFU/mL of bacterial cells, making it suitable for industrial-scale applications. Additionally, the method exhibited strong repeatability, reproducibility across different qPCR platforms, and resistance to interference from high cell density of B. animalis subsp. lactis DSMZ 10140. Successful quantification of HN019 in complex multi-strain probiotic powders confirmed its practical reliability. This work establishes a rapid, robust, and scalable tool for precise probiotic strain tracking, addressing critical quality control and regulatory compliance needs within the rapidly expanding probiotic industry. Full article

Antimicrobial resistance (AMR) is a rapidly growing global concern resulting from the overuse of antibiotics in both agricultural and clinical settings, the lack of surveillance for resistant bacteria, and the low quality of some available antimicrobial agents. Resistant pathogens are no longer susceptible to common clinical antimicrobials, which decreases the effectiveness of medicines used to treat infections caused by these organisms. Carbapenems are an important class of antibiotics due to their broad-spectrum effectiveness in treating infections caused by Gram-positive and Gram-negative organisms. Carbapenem-resistant bacteria have been found not only in healthcare but also in the environment and food supply chain, where they have the potential to spread to pathogens and infect humans and animals. Current methods of detecting AMR genes are expensive and time-consuming. While these methods, like polymerase chain reactions or whole-genome sequencing, are considered the “gold standard” for diagnostics, the development of inexpensive, rapid diagnostic assays is necessary for effective AMR detection and management. Biosensors have shown potential for success in diagnostic testing due to their ease of use, inexpensive materials, rapid results, and portable nature. Biosensors can be combined with nanomaterials to produce sensitive and easily interpretable results. This review presents an overview of carbapenem resistance, current and emerging detection methods of antimicrobial resistance, and the application of biosensors for rapid diagnostic testing for bacterial resistance. Full article

Tuberculosis (TB), primarily caused by Mycobacterium tuberculosis complex (MTBC), remains a global health challenge and can lead to severe pulmonary and extrapulmonary complications. Multidrug-resistant TB (MDR-TB) poses additional challenges, requiring advanced diagnostic and treatment strategies. This study evaluates the BD MAX MDR-TB molecular test for a rapid diagnosis of MDR-TB, detecting resistance to rifampicin (RIF) and isoniazid (INH). The BD MAX MDR-TB test, utilizing real-time PCR, was used to analyze specimens collected from TB-suspected patients, identifying MTB DNA and mutations associated with rifampicin and isoniazid resistance. Results were compared with traditional drug susceptibility testing, and 79 out of 638 samples tested were positive for MTB DNA, with 65 showing a sufficient amount of genetic material for resistance gene identification. The BD MAX test showed a 100% correlation with phenotypic rifampicin resistance, though discrepancies were noted for isoniazid resistance, with a 93% concordance. The BD MAX MDR-TB test is an effective tool for a rapid diagnosis of MDR-TB, especially for rifampicin resistance. However, it may not detect certain mutations related to isoniazid resistance. Complementary tests like Xpert MTB/XDR or whole-genome sequencing could improve diagnostic accuracy and support more effective TB control strategies. Full article

Background/Objectives: Mango, which is known as the “King of Tropical Fruits”, is an evergreen plant belonging to the Anacardiaceae family. It belongs to the genus Mangifera, which comprises 69 species of plants found in tropical and subtropical regions, including India, Indonesia, the Malay Peninsula, Thailand, and South China. However, research on the structural information of complete chloroplast genomes of Mangifera is limited. Methods: The rapid advancement of high-throughput sequencing technology enables the acquisition of the entire chloroplast (cp) genome sequence, providing a molecular foundation for phylogenetic research. This work sequenced the chloroplast genomes of six Mangifera samples, performed a comparative analysis of the cp genomes, and investigated the evolutionary relationships within the Mangifera genus. Results: All six Mangifera samples showed a single circular molecule with a quadripartite structure, ranging from 157,604 bp to 158,889 bp in length. The number of RNA editing sites ranged from 60 to 61, with ndhB exhibiting the highest number of RNA editing sites across all species. Seven genes—namely, atpB, cemA, clpP, ndhD, petB, petD, and ycf15—exhibited a Ka/Ks value > 1, suggesting they may be under positive selection. Phylogenetic analysis revealed that Mangifera siamensis showed a close relationship between Mangifera indica and Mangifera sylvatica. Conclusions: Our comprehensive analysis of the whole cp genomes of the five Mangifera species offers significant insights regarding their phylogenetic reconstruction. Moreover, it elucidates the evolutionary processes of the cp genome within the Mangifera genus. Full article

Background/Objective:Staphylococcus aureus produces staphyloferrin A (Sfna) siderophores to sequester host iron during infection and rapid cell proliferation We examined drug susceptibility, siderophore production, and genome sequencing of clinical isolates of S. aureus. Methods: A total of 100 specimens, including pus, sputum, hemoculture, urine, tissue, fluid, and skin scrap specimens, were grown in iron-deprived Luria broth agar. The isolates were investigated for spectral signature using MALDI–TOF/MS, while antibiotic susceptibility and siderophore content were assessed using the chrome azurol S method. Whole genome and partial 16S rRNA DNA sequences were employed, and VITEK/MS revealed specific spectra. Results: Clindamycin, erythromycin, gentamicin, linezolid, moxifloxacin, oxacillin, trimethoprim/sulfamethoxazole, and vancomycin (100%) were the most common antibiotics to which the S. aureus isolates were susceptible. Sfna was not detectable in fluid and skin scrap isolates, which were encoded by sfnaB, sfnaD, and sfnaB/sfnaD genes. However, they were detectable in pus (73.8%), sputum (85.3%), hemoculture (50.0%), and urine (85.7%) isolates. The aureus subspecies, JKD6159, SA268, and MN8, were found to be 72.73% according to genome sequencing. Conclusion: most staphylococci in the isolates, including S. aureus JKD6159, SA268, and MN8, were sensitive to antibiotics and were detected by MALDI–TOF/MS, resulting in the production of Sfna encoded by sfna genes. Full article

Background: Previous evidence demonstrated DNA methylation changes in response to stress in plants, showing rapid changes within a limited time frame. Exposure to self-DNA inhibits seedling root elongation, and it was shown that it causes changes in CG DNA methylation in Lactuca sativa. We assessed cytosine methylation changes and associated gene expression patterns in roots of Arabidopsis thaliana Col-0 seedlings exposed to self-DNA for 6 and 24 h. Methods: We used whole genome bisulfite sequencing (WGBS) and RNA-seq analyses to assess genomic cytosine methylation and corresponding gene expression, respectively, on DNA and RNA extracted with commercial kits from roots exposed to self-DNA by an original setup. Fifteen hundred roots replicates, including the control in distilled water, were collected after exposure. Sequencing was performed on a NovaSeq 6000 platform and Ultralow Methyl-Seq System for RNA and DNA WGBS, respectively. Results: Gene expression in roots exposed to self-DNA differed from that of untreated controls, with a total of 305 genes differentially expressed and 87 ontologies enriched in at least one treatment vs. control comparison, and particularly after 24 h of exposure. DNA methylation, particularly in CHG and CHH contexts, was also different, with hyper- and hypomethylation prevailing in treatments vs. controls at 6 h and 24 h, respectively. Differentially expressed genes (DEGs) analysis, Gene Ontology (GO) enrichment analysis, and differentially methylated regions (DMRs) analysis, provided an integrated understanding of the changes associated with self-DNA exposure. Our results suggest differential gene expression associated with DNA methylation in response to self-DNA exposure in A. thaliana roots, enhanced after prolonged exposure. Conclusions: Main functional indications of association between DNA methylation and gene expression involved hypomethylation and downregulation of genes related to nucleotide/nucleoside metabolism (ATP synthase subunit) and cell wall structure (XyG synthase), consistent with previous observations from metabolomics and physiological studies. Further confirmation of these findings will contribute to improving our understanding of the plant molecular response to self-DNA and its implications in stress responses. Full article

Aquilaria sinensis (Lour.) Spreng. is an economically important tree specie that produces agarwood, a valuable medicinal and aromatic resin, when injured. However, its large-scale cultivation has led to confusion regarding its resources and genetic backgrounds, hindering the conservation and management of A. sinensis accessions. This study systematically developed and validated simple sequence repeat (SSR) molecular markers by using whole-genome resequencing (WGR) data from 60 A. sinensis accessions to elucidate their genetic diversity and population structure. A total of 56,657 SSR sequences (24,430 loci) were identified, which were dominated with dinucleotide repeat motifs (73.59%). After stringent quality control, 46 high-quality SSR loci were obtained, and 93 primer pairs were designed for amplification validation. Ultimately, 20 primer pairs with stable amplification and high polymorphism were selected, of which 11 exhibited high polymorphism (polymorphic information content: 0.554–0.688). These 20 primer pairs identified a total of 121 alleles, with an average of 6 alleles per locus. These primers successfully classified 149 A. sinensis accessions into three subpopulations, achieving a discrimination rate of 95.97%. The analysis of molecular variance revealed that genetic variation within the individuals accounted for 84% of the total variation. This study establishes a rapid and efficient SSR-based method by leveraging resequencing data for large-scale marker discovery in A. sinensis. It further provides a robust technical framework for the conservation and sustainable utilization of this valuable species. Full article

The COVID-19 pandemic has necessitated urgent measures to curb its spread, with vaccination emerging as a pivotal strategy. This prospective observational study evaluated breakthrough COVID-19 infections among healthcare workers (HCWs) vaccinated with Comirnaty (Pfizer-BioNTech) at a tertiary care hospital in Greece. Over an 8-month period, from February to September 2021, 1958 fully vaccinated HCWs were monitored. Rapid antigen tests and RT-PCR tests were conducted weekly for asymptomatic HCWs. Contact tracing and whole-genome sequencing were performed. Results showed that 2.75% (54 cases) of HCWs experienced breakthrough infections. Among these, 25 (45%) were asymptomatic, mild symptoms occurred in 21 (37%), while 7 (13%) had a fever (≥38 °C) alone and 3 (5%) developed high fever (≥39 °C) with respiratory symptoms. Physicians and nursing staff were the most affected groups. Dominant SARS-CoV-2 variants detected included Delta, British, and Wild type variants. Comparison with existing literature underscored the effectiveness of Comirnaty in reducing breakthrough infections. The findings emphasize the importance of continued booster vaccinations and ongoing surveillance to mitigate breakthrough infections among HCWs. Full article

The complexity of tracing foodborne pathogens in the food chain has increased significantly due to the long and complicated chain, the involvement of numerous links, and the presence of various types of pathogens at different stages and environments. Traditional typing techniques are not sufficient to meet the requirements of tracing pathogens in the food chain. Whole-Genome Sequencing (WGS) has gradually become an important technological tool for characterizing and tracing pathogens in the food chain due to comprehensive information, speed, and superior discriminatory power. This paper provides an overview of the advantages of WGS and its application in foodborne pathogen traceability. This paper focused on foodborne pathogen contamination pathways during the processing of animal foods in commercial restaurant kitchens and the potential contamination of milk, milk powder, and other dairy products by pathogens during processing in the dairy industry chain and environments. Improper handling practices during meat processing (i.e., using cloths, washing hands without soap, and cleaning boards with knives) were a critical point of foodborne pathogen cross-contamination in commercial kitchen premises. However, in dairy products, contamination of pathogens in raw milk was the main cause of foodborne disease outbreaks. Therefore, preventing the contamination of pathogens in food should not only be focused on hygiene measures during processing and in environments but also on the quality and hygiene of raw materials to prevent the spread of foodborne pathogens throughout the entire production chain. Further, Whole-Metagenome Sequencing and DNA sequence markers are considered to be the future direction of WGS. The purpose of this work is to promote the wider application of WGS during the processing of meat and dairy products and provide theoretical support for the rapid investigation and accurate traceability of foodborne pathogen outbreaks in food. Full article

In this study, we developed, validated and applied an NGS-based SNP genotyping protocol for the molecular identification of Vitis vinifera varieties, demonstrating a reliable and efficient approach for distinguishing grapevine cultivars. By utilizing a small but highly informative set of SNP loci, this method provides effective molecular genotyping while capturing the genetic diversity needed for accurate identification. This straightforward and accessible approach allows for the rapid generation of genetic profiles, which can be compared with the profiles in existing databases to precisely identify grapevine varieties, even in cases where traditional morphological methods fall short due to environmental variability or developmental differences. The process is designed to be both time-efficient and cost-effective, making it a practical tool for routine use in vineyard management, breeding programs, and conservation efforts. Furthermore, the workflow minimizes the need for whole-genome sequencing or other resource-intensive techniques, making molecular profiling accessible to a wider range of researchers, growers, and industry professionals. Analysis of the molecular profiles of known varieties validated the accuracy of the protocol. Moreover, 14 autochthonous Greek grapevine varieties that have not been previously identified were also genotyped and the data were compared with those of all Greek varieties in the Vitis International Variety Catalogue, revealing no matching multilocus genotypes across Greece. Full article

The completion of the Human Genome Project in 2003 has led to significant advances in patient care in medicine, particularly in diagnosing and managing genetic diseases and cancer. In the realm of genetic diseases, approximately 15% of critically ill infants born in the U.S.A. are diagnosed with genetic disorders, which comprise a significant cause of mortality in neonatal and pediatric intensive care units. The introduction of rapid whole-genome sequencing (rWGS) as a first-tier test in critically ill children with suspected, undiagnosed genetic diseases is a breakthrough in the diagnosis and subsequent clinical management of such infants and older children in intensive care units. Rapid genome sequencing is currently being used clinically in the USA, the UK, the Netherlands, Sweden, and Australia, among other countries. This review is intended for students and clinical practitioners, including non-experts in genetics, for whom it provides a historical background and a chronological review of the relevant published literature for the progression of pediatric diagnostic genomic sequencing leading to the development of pediatric rWGS in critically ill infants and older children with suspected but undiagnosed genetic diseases. Factors that will help to develop rWGS as a clinical test in critically ill infants and the limitations are briefly discussed, including an evaluation of the clinical utility and accessibility of genetic testing, education for parents and providers, cost-effectiveness, ethical challenges, consent issues, secondary findings, data privacy concerns, false-positive and false-negative results, challenges in variant interpretation, costs and reimbursement, the limited availability of genetic counselors, and the development of evidence-based guidelines, which would all need to be addressed to facilitate the implementation of pediatric genomic sequencing in an effective widespread manner in the era of precision medicine. Full article

Background/Objectives: Methicillin-resistant Staphylococcus aureus (MRSA) presents a major public health challenge due to its multidrug resistance and high virulence. Developing representative model strains is crucial for systematically assessing pathogenesis and antimicrobial therapies. Methods: The highly virulent MRSA strain T144, isolated from pigs, was characterized through whole-genome sequencing and antimicrobial susceptibility testing. Infection models were successfully established in Galleria mellonella and mice to evaluate virulence. A mouse lung infection model was specifically developed to assess bacterial load dynamics, immune responses, and the efficacy of vancomycin treatment. Results: MRSA T144 demonstrated broad-spectrum antibiotic resistance and high mortality rates in both Galleria mellonella and mouse models. Whole-genome sequencing identified multiple virulence-associated genes, including hemolysins and enterotoxins. The concentration of 7 × 10⁸ CFUs was optimized for establishing the mouse lung infection model. In the mouse lung infection model, MRSA T144 demonstrated rapid bacterial proliferation within the first 24 h, followed by a slower growth rate. Significant changes in immune markers were observed, with elevated levels of pro-inflammatory cytokines (IL-1β, IL-6, IL-8, IL-17a, TNF-α) and decreased IL-10 levels. Vancomycin treatment significantly improved survival rates and reduced bacterial load, confirming the model’s utility for antimicrobial efficacy studies. Conclusions: The successful establishment of MRSA T144 infection models provides a robust platform for investigating bacterial dynamics, immune responses, and antimicrobial efficacy against highly virulent MRSA strains. These findings highlight the potential of MRSA T144 as a valuable model for developing novel therapeutic strategies. Full article

Pokkah Boeng Disease (PBD) is a severe and devastating disease that causes significant damage and yield losses in China. The pathogenic fungus Fusarium oxysporum is responsible for the rapid onset of top rot symptoms in sugarcane. In this study, we selected a representative strain, BS2-6, to perform morphological observations of colonies and determine pathogenicity. We examined the effects of BS2-6 infestation on the ultrastructure of sugarcane leaves. Moreover, we sequenced the whole genome of BS2-6 and examined the effects of various nitrogen sources and chemical reagents on its growth and pathogenicity. Our results indicate that sugarcane leaves inoculated with BS2-6 quickly succumb to heart leaf and growing rot. Ultrastructural analysis revealed that the surface tissues of the diseased leaves were destroyed with mycelium, and conidia blocked leaf stomata, which ultimately led to the degradation of leaf tissues. Ammoniacal nitrogen significantly promoted mycelial growth, pigment secretion, and the expression of genes related to secondary metabolite synthesis, thereby accelerating the development of PBD. In addition, we found that carbendazim effectively inhibited the growth of BS2-6 at various concentrations. These findings provide important insights for the effective prevention and control of PBD during sugarcane production. Full article

The genus Alternaria, functioning as a saprobe, endophyte, and plant pathogen, is widely distributed across various natural and human-impacted environments. Leaf spot and black spot diseases, caused by Alternaria species, are the most prevalent plant diseases within this genus, leading to significant reductions in crop yields and substantial economic losses. To facilitate the timely detection of Alternaria species during the early stages of infection, enable targeted treatments, and mitigate associated damages, we employed a species identification method based on Analysis of whole-GEnome (AGE). We downloaded 148 genomes, including 31 Alternaria species, from the NCBI GenBank database. Through bioinformatics analysis, we constructed a specific-target sequence library and selected a representative sequence per species. The specific target sequences of the seven exemplary Alternaria species were subsequently used for validation and rapid detection, utilizing Sanger sequencing and CRISPR-Cas12a technology, respectively. The results demonstrated that our method accurately identified the target species. Additionally, by combining Enzymatic Recombinase Amplification (ERA) with CRISPR-Cas12a, we achieved rapid and precise identification of genomic DNA samples, with a detection limit as low as 0.01 ng/µL within 30 min. Therefore, AGE proves to be a highly robust and efficient method for the detection of Alternaria species, offering broad potential for various applications. Full article