Journal of microbiological methods最新文献

{"title":"Validation of pleural fluid group A Streptococcus and Staphylococcus aureus PCR assays and their potential clinical impact in children with complicated pneumonia","authors":"Erin C. Ho ,&nbsp;Molly Butler ,&nbsp;Kaitlin E. Olson ,&nbsp;Dennis Simmons ,&nbsp;Colsen Beveridge ,&nbsp;Kristen Miller ,&nbsp;Meghan Birkholz ,&nbsp;Sarah A. Jung ,&nbsp;Samuel R. Dominguez","doi":"10.1016/j.mimet.2025.107192","DOIUrl":"10.1016/j.mimet.2025.107192","url":null,"abstract":"<div><h3>Background</h3><div>Better diagnostic tools are needed to increase the yield and speed of pathogen identification in pediatric complicated community-acquired pneumonia (cCAP), and thereby, improve patient care through optimization of antibiotic therapy.</div></div><div><h3>Methods</h3><div>We performed analytical and clinical validations of a laboratory-developed group A <em>Streptococcus</em> (GAS) PCR and a commercially available <em>Staphylococcus aureus</em> (<em>S. aureus</em>), including methicillin-resistant <em>S. aureus</em> (MRSA), PCR for pleural fluid specimens and studied the potential clinical impact of pleural fluid GAS and <em>S. aureus</em> PCR testing in children with cCAP.</div></div><div><h3>Results</h3><div>Both assays demonstrated high analytical sensitivity, specificity, reproducibility, and accuracy in detection of their target pathogens in pleural fluid. In potential clinical impact analysis for 62 children with cCAP requiring pleural fluid drainage, the addition of GAS and <em>S. aureus</em> PCR testing to existing diagnostic testing increased pathogen yield from 71.0 % to 83.2 % (<em>p</em> = 0.023), decreased theoretical median time from hospitalization to optimal therapy from 5.1 days (95 % CI: 2.4–7.2) to 3.7 days (95 % CI: 1.9–6.9, <em>p</em> = 0.001) and theoretical median time from start to stop of unwarranted MRSA therapy from 1.5 days (95 % CI: 1.0–3.7) to 0.8 days (95 % CI: 0.5–1.2, <em>p</em> &lt; 0.001).</div></div><div><h3>Conclusion</h3><div>PCR testing is a sensitive and specific method for detecting GAS and <em>S. aureus</em> in pleural fluid. Clinical implementation of these targeted pleural fluid PCR assays has the potential to significantly increase pathogen yield, facilitate faster de-escalation of MRSA therapy and transition to narrow, targeted antibiotics, suggesting their utility for pediatric complicated pneumonia.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"236 ","pages":"Article 107192"},"PeriodicalIF":1.7,"publicationDate":"2025-07-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144626589","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"styMdtM gene deletion with modified λ Red recombineering approach: Implications for multidrug resistance in Salmonella Typhi","authors":"Anam Tariq ,&nbsp;Aqsa Shaheen ,&nbsp;Mazhar Iqbal ,&nbsp;Moazur Rahman","doi":"10.1016/j.mimet.2025.107191","DOIUrl":"10.1016/j.mimet.2025.107191","url":null,"abstract":"<div><div>styMdtM is a multidrug efflux transporter of <em>Salmonella Typhi</em>. In the present study, a modified λ Red recombineering approach using shorter flanking arms (ca. 50 bp) was used to generate an <em>styMdtM</em> gene deletion in the <em>S. Typhi</em> genome. This approach worked efficiently to knockout the gene and the ensuing <em>S. Typhi</em> strain was considerably more sensitive to antibiotics.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"236 ","pages":"Article 107191"},"PeriodicalIF":1.7,"publicationDate":"2025-07-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144605956","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Diagnostic utility of multiplex PCR for direct detection and serotyping of group B streptococci from clinical specimens","authors":"Muhammed Basith Hafeez Ismail,&nbsp;Geetha Nagaraj,&nbsp;Kadahalli Lingegowda Ravikumar","doi":"10.1016/j.mimet.2025.107190","DOIUrl":"10.1016/j.mimet.2025.107190","url":null,"abstract":"<div><div>Multiplex PCR analysis of 348 recto-vaginal swabs from non-pregnant women revealed 33 % GBS colonization, with serotype V predominant. The study highlights the utility of multiplex PCR as a rapid and accurate diagnostic tool for detecting and serotyping Group B Streptococci directly from clinical specimens, supporting its role in surveillance, infection control, and guiding vaccine strategies in this underrepresented population.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"236 ","pages":"Article 107190"},"PeriodicalIF":1.7,"publicationDate":"2025-07-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144571096","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Nanotechnology-based electrochemical approach for effective root canal irrigation","authors":"Kaajal Rengaraj ,&nbsp;Mareeswari Paramasivan ,&nbsp;Govindaraj Perumal ,&nbsp;Gitika Thakur ,&nbsp;Wei Li ,&nbsp;Kari Severson ,&nbsp;Qian Xie ,&nbsp;Nikita B.Ruparel ,&nbsp;Christine D.Wu ,&nbsp;Xue-Jun Li ,&nbsp;Mathew T. Mathew","doi":"10.1016/j.mimet.2025.107189","DOIUrl":"10.1016/j.mimet.2025.107189","url":null,"abstract":"<div><div>Root canal treatment (RCT) is an endodontic procedure to preserve an infected/inflamed tooth and retain its function. Root canal irrigation is the most crucial step in RCT for cleaning the root canal space. Achieving total disinfection in root canal space is still a significant challenge in dentistry. We developed a novel irrigation approach combining electrochemistry and nanotechnology to eliminate root canal bacteria effectively. Cytotoxicity of the optimized irrigation technique was evaluated using <em>Enterococcus faecalis,</em> and cytocompatibility was evaluated with three different human cell lines (MG63, gingival fibroblast and osteoblasts differentiated from stem cells). A potential of -5 V was applied using an electrochemical set-up to these cells with two different solutions, 0.9 % saline (control) and 5 μg/mL ZnO NPs in 0.9 % saline over various periods (60, 120, and 180 s). <em>E. faecalis</em> biofilms were treated <em>in vitro</em> and viable colony-forming units (CFU) were determined. The morphology of pre- and post-treated cells was examined using SEM and TEM. Morphological changes such as cell membrane rupture or cell lysis were observed in SEM and TEM images of all treatment groups. The optimal ZnO NPs concentration was determined to be 5 μg/mL based on - viability assays of test human cell lines. More than 90 % cell viability was noted with 60s treatment time in all cell lines. Significantly reduced bacterial viability was observed in treatment groups when compared to the non-electrochemically treated groups, indicating effective bacterial eradication.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"236 ","pages":"Article 107189"},"PeriodicalIF":1.7,"publicationDate":"2025-07-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144575674","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Development of double-antibody sandwich ELISA for detecting PEDV N protein","authors":"Yao Zhao ,&nbsp;Fang Wei ,&nbsp;Zhongyin Liu ,&nbsp;Tianai Zhang ,&nbsp;Jiamin Qin ,&nbsp;Wenke Ruan ,&nbsp;Zhen Wang ,&nbsp;Dengjin Chen ,&nbsp;Dongjie Chen ,&nbsp;Shengkui Xu","doi":"10.1016/j.mimet.2025.107186","DOIUrl":"10.1016/j.mimet.2025.107186","url":null,"abstract":"<div><div>Porcine epidemic diarrhea virus (PEDV) severely impacts piglets with high contagion and lethality, leading to significant economic losses in the swine industry. In this study, two anti-PEDV N protein monoclonal antibodies (4F2 and 2G4) were successfully prepared using the HEK-293i cell-expressed recombinant N protein as an immunogen. Functional analysis revealed that both 4F2 and 2G4 were applicable to immunofluorescence assay (IFA) and Western blot. Epitope screening showed that these mAbs recognized distinct epitopes: 4F2 bound a conserved linear epitope in the N-terminal domain, while 2G4 targeted the linear epitope “SGKNTPKKNKSRATSKE” in the C-terminal domain, which is highly conserved across different virus strains. Using the obtained antibodies, we developed a double-antibody sandwich quantitative ELISA (DAS-ELISA) for PEDV detection. The assay demonstrated remarkable sensitivity, capable of detecting the recombinant N protein at a concentration as low as 0.05 ng/mL and the virus at a titer of 10^2.59 TCID₅₀ (50 % tissue culture infectious dose). It also exhibited specificity, with no cross-reactivity to PRV, PRRSV, ASFV, or PCV2. This ELISA should enable convenient PEDV detection in piglet excreta and aid in PEDV eradication efforts.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"236 ","pages":"Article 107186"},"PeriodicalIF":1.7,"publicationDate":"2025-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144556678","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Hepcidin vs Procalcitonin: Which biomarker best predicts bacteremia in the medical ICU?","authors":"Jayasree S,&nbsp;Jagan V,&nbsp;Leela K.V","doi":"10.1016/j.mimet.2025.107187","DOIUrl":"10.1016/j.mimet.2025.107187","url":null,"abstract":"<div><h3>Background</h3><div>Sepsis is a life-threatening condition caused by a dysregulated host response to infection, necessitating early diagnosis and intervention. While traditional diagnostics like blood cultures are crucial, they often delay treatment decisions. This study evaluates and compares the diagnostic and prognostic efficacy of Procalcitonin (PCT) and Hepcidin—two emerging biomarkers—in patients with bacteremia admitted to the Medical Intensive Care Unit (MICU) of a tertiary care centre.</div></div><div><h3>Methods</h3><div>A prospective, analytical study was conducted on 86 blood culture-positive MICU patients at SRM Medical College Hospital and Research Centre, Chennai. Serum PCT and Hepcidin levels were measured using Chemiluminescent Microparticle Immunoassay (CMIA) and sandwich ELISA techniques, respectively. The diagnostic value of each biomarker was assessed, and statistical analysis was performed using Chi-square tests with <em>p</em> &lt; 0.05 considered significant.</div></div><div><h3>Results</h3><div>Serum Hepcidin and PCT levels were elevated in 84.8 % and 82.6 % of sepsis patients, respectively, with statistically significant associations (<em>p</em> &lt; 0.05). Hepcidin showed a more consistent and gradual decline during recovery (96 % normalization) compared to PCT (77 % normalization). Both biomarkers correlated strongly with microbial profiles, particularly in Gram-negative infections, and demonstrated no significant variation with patient demographics or comorbidities.</div></div><div><h3>Conclusion</h3><div>Hepcidin emerges as a promising biomarker, demonstrating diagnostic and prognostic potential comparable to PCT, with a more consistent response during sepsis resolution. When used in conjunction, these markers can enhance early detection, guide treatment, and improve clinical outcomes in sepsis management.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"236 ","pages":"Article 107187"},"PeriodicalIF":1.7,"publicationDate":"2025-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144560420","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Coconut water: A novel inducer of germ tube formation in Candida albicans - A preliminary report","authors":"Smija Kakkuth Puthenvitil,&nbsp;Surya Sokkalingam,&nbsp;Palani Perumal","doi":"10.1016/j.mimet.2025.107188","DOIUrl":"10.1016/j.mimet.2025.107188","url":null,"abstract":"<div><div>The evolving epidemiology of candidiasis necessitates accurate methods for distinguishing <em>Candida</em> species. This study explores coconut water as a substitute for fetal bovine serum in inducing germ tube formation, offering a cost-effective and ethically sound solution for diagnosing and studying <em>Candida</em> infections.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"236 ","pages":"Article 107188"},"PeriodicalIF":1.7,"publicationDate":"2025-06-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144553776","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"MFDF-UNet: Multiscale feature depth-enhanced fusion network for colony adhesion image segmentation","authors":"Liu Hui ,&nbsp;Wang Zhiyi ,&nbsp;Li Xue ,&nbsp;Ge Peng ,&nbsp;Tuo Yanfeng ,&nbsp;Xie Xufen","doi":"10.1016/j.mimet.2025.107185","DOIUrl":"10.1016/j.mimet.2025.107185","url":null,"abstract":"<div><div>Colony counting plays a crucial role in evaluating food quality and safety. The segmentation of colony adhesion images can significantly enhance the accuracy of food safety assessments. To achieve high-precision segmentation of colony adhesion images, this paper presents a novel multi-scale feature deep-enhanced fusion network MFDF-UNet, specifically designed for colony adhesion image segmentation. The core of the network lies in the design of a self-similar fusion fractal structure, which recursively integrates layers to enhance the network's ability to extract, integrate, and transfer multi-scale feature information. The DEC (depth-enhanced connectivity) units and PF (progressive fusion) modules in each stage progressively accumulate detailed features, thus improving the network's capacity to handle complex structures. Additionally, the design strengthens the information transfer between different layers, ensuring consistency of features across multiple layers. This reduces the imbalance in feature information transfer that can occur when certain regions of the image contain prominent edges, textures, or structural features, while other areas are relatively blurred or lack distinct features.The MFDF-UNet model achieved an average segmentation accuracy of 77.95 %, precision of 97.55 %, and a mean intersection-over-union (mIoU) of 57.94 % on the AGAR-based hybrid colony adhesion segmentation test dataset. Compared to other deep learning methods, such as PSPNet, DeepLabv3+, SegFormer, YOLOv8, U-Net, and ResNet, MFDF-UNet outperforms the highest-performing ResUNet by 7.53 % in segmentation accuracy, improves precision by 1.5 %, and surpasses ResUNet by 4.82 % in mIoU.Although our model requires slightly more parameters and training time, the improvements in segmentation accuracy and image quality sufficiently justify the additional cost, demonstrating its potential for practical applications in colony adhesion segmentation.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"236 ","pages":"Article 107185"},"PeriodicalIF":1.7,"publicationDate":"2025-06-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144490715","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Strain-level typing of Wickerhamomyces anomalus using Fourier transform infrared spectroscopy and whole-genome sequencing","authors":"Asuka Kashiwaba ,&nbsp;Asako Mitani ,&nbsp;Takumi Sonoda ,&nbsp;Naofumi Shigemune ,&nbsp;Hiroki Takahashi","doi":"10.1016/j.mimet.2025.107184","DOIUrl":"10.1016/j.mimet.2025.107184","url":null,"abstract":"<div><div>In industrial settings, identifying the source of microbial contamination is crucial for effective microbiological risk assessment. While various strain identification technologies exist, many struggle with practicality, accuracy, and reproducibility. Fourier Transform Infrared Spectroscopy (FT-IR) has emerged as a rapid method, demonstrating a strong correlation with whole-genome sequencing (WGS) for certain bacteria. However, its accuracy for identifying yeast strains has been limited.</div><div>This study focuses on improving the accuracy of FT-IR for yeast strain identification by optimizing pretreatment conditions. We conducted phylogenetic analyses on <em>Wickerhamomyces anomalus</em> using both WGS single-nucleotide polymorphisms (SNPs) and FT-IR. Although initial FT-IR results were less accurate than WGS, refining the culture and sample preparation conditions led to significant improvements. We tested 16 different conditions, using Euclidean Distances (EDs) and dendrogram comparisons to evaluate discrimination ability, including metrics like the F-measure and adjusted Rand index (ARI).</div><div>The most accurate and reproducible FT-IR results were achieved with incubation in Sabouraud dextrose (SD) broth aligning closely with WGS results. This optimized FT-IR protocol now allows for rapid and precise strain-level discrimination of <em>W. anomalus</em>, offering a practical tool for tracking contamination sources in industrial environments.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"236 ","pages":"Article 107184"},"PeriodicalIF":1.7,"publicationDate":"2025-06-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144505999","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Streamlined E. coli expression and purification of amyloid beta peptides via inclusion body formation and fluorescence monitoring","authors":"Steven David ,&nbsp;Dayana Salazar ,&nbsp;Paula Puente Cantillo,&nbsp;Alvaro Barrera-Ocampo","doi":"10.1016/j.mimet.2025.107182","DOIUrl":"10.1016/j.mimet.2025.107182","url":null,"abstract":"<div><div>The recombinant production of amyloid beta peptides poses significant challenges due to their high aggregation propensity and cytotoxicity in bacterial hosts. In this study, we present a streamlined and reproducible method for the expression and purification of methionine-modified Aβ40 and Aβ42 peptides in <em>Escherichia coli</em> BL21 (DE3) pLysS. By leveraging inclusion body formation, the protocol enhances yield while simplifying purification. A key feature of this approach is the incorporation of real-time fluorescence spectroscopy and microscopy using Thioflavin-S and propidium iodide, enabling non-invasive monitoring of IB formation and expression dynamics. Purification was achieved through pH modulation, anion exchange chromatography, and ultrafiltration, yielding average peptide concentrations of 3.2 ± 1.3 mg/L for Aβ40 and 4.8 ± 3.2 mg/L for Aβ42. High-performance liquid chromatography confirmed average purities of 90.2 % ± 0.8 % for Aβ40 and 84.0 % ± 17.4 % for Aβ42. The structural integrity, aggregation kinetics, and neurotoxicity of the peptides were validated by PICUP, Thioflavin-T fluorescence assays, and cytotoxicity tests in primary hippocampal neurons. This tag-free, cost-effective platform provides a scalable solution for producing biologically active amyloid beta peptides, facilitating their use in structural biology, neurodegenerative disease research, and high-throughput drug screening.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"236 ","pages":"Article 107182"},"PeriodicalIF":1.7,"publicationDate":"2025-06-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144506000","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}