Journal of microbiological methods最新文献

{"title":"Evaluation of nucleic acid extraction methods for recovery of Cyclospora cayetanensis, Salmonella enterica, and murine norovirus from water and sludge","authors":"Amy Kahler ,&nbsp;Jessica Hofstetter ,&nbsp;Camila Rodrigues ,&nbsp;Mia Mattioli","doi":"10.1016/j.mimet.2025.107195","DOIUrl":"10.1016/j.mimet.2025.107195","url":null,"abstract":"<div><div>The coccidian parasite <em>Cyclospora cayetanensis</em> is the causative agent for foodborne outbreaks of cyclosporiasis and multiple fresh produce recalls annually. In recent years, this organism has been reported in the water near produce growing operations during outbreak investigations, prompting a call for more research on its environmental prevalence in the United States. Currently, there is a lack of performance data available on methods for conducting this research, including the performance of DNA extraction methods for molecular testing. Extraction methods for environmental samples must be efficient due to the often-limited amount of target nucleic acid and the potential for molecular inhibitors present in an environmental sample. This study assessed the performance of <em>C. cayetanensis</em> nucleic acid extraction seeded into surface water, produce wash water, and tap water by two methods designed for use with environmental samples: the PowerViral and UNEX methods. The PowerSoil extraction method (2 g) was assessed for <em>C. cayetanensis</em> extraction from seeded sewage sludge – an environmental sample type used to evaluate parasite carriage within communities. Extraction performance of the PowerViral and UNEX methods were also assessed for the detection of the foodborne bacterial pathogen <em>Salmonella</em> and a surrogate for foodborne viruses, murine norovirus (MNV) seeded into surface water, produce wash water, and tap water. The PowerViral method resulted in consistent detection (83–100 %) of <em>C. cayetanensis</em>, <em>S. enterica</em>, and MNV across all water types. Detection rates for the UNEX method ranged from 56 to 100 % prevalence for tap water and wash water, but there were no detections for any microbe from surface water. The PowerSoil method resulted in poor recovery of <em>C. cayetanensis</em> from sludge (≤1 % recovery), while both the PowerViral and UNEX methods effectively recovered <em>C. cayetanensis</em> from sludge (4–36 % recovery).</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"236 ","pages":"Article 107195"},"PeriodicalIF":1.7,"publicationDate":"2025-07-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144649631","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":"Evaluation of a high-throughput method for processing sponge-stick samples to detect viable, non-spore-forming biothreat agents","authors":"Vanessa L. Brisson ,&nbsp;Staci R. Kane ,&nbsp;M. Worth Calfee ,&nbsp;Stephen Cendrowski ,&nbsp;Sanjiv R. Shah","doi":"10.1016/j.mimet.2025.107194","DOIUrl":"10.1016/j.mimet.2025.107194","url":null,"abstract":"<div><div>After a bioterrorism incident, surface sampling is often used to determine the extent of contamination and exposure, guiding decontamination efforts and decisions for re-occupancy of affected sites. The sponge-stick (SS) is a preferred and commonly used device for sample collection to detect both spore-forming and non-spore-forming biothreat agents from non-porous surfaces. Here, a recently developed high-throughput method (HTM) for processing SS samples to detect viable <em>Bacillus anthracis</em> spores was adapted for detection of non-spore-forming biothreat agents, <em>Yersinia pestis</em> and <em>Francisella tularensis</em>. The scalable HTM was used to process up to 20 SS samples simultaneously, compared to the current stomacher-based method which processes one SS at a time. Comparisons of the HTM and the stomacher-based method were statistically indistinguishable for most experiments (<em>P</em> &gt; 0.05) with HTM recoveries of 37–60 % for <em>Y. pestis</em> inoculated at 10²–10³ cells/SS and held 48 h at 4 °C to mimic sample transport/storage. The HTM was integrated with Rapid Viability-Polymerase Chain Reaction (RV-PCR) analysis to detect viable <em>Y. pestis</em> in the presence of particulate contamination (Arizona Test Dust, ATD). This approach detected <em>Y. pestis</em> inoculated at 20 cells/SS and ATD did not impact detection (<em>P</em> &gt; 0.05). <em>F. tularensis</em> showed significantly lower recoveries between no-hold time and 48-h hold time (4 °C, <em>P</em> &lt; 0.05) using the HTM, which further testing showed could be due to toxicity of the neutralizing buffer used for SS pre-wetting. With modifications, this method could enhance throughput capacity while maintaining similar recovery efficiencies to current methods for other non-spore-forming bacterial pathogens.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"236 ","pages":"Article 107194"},"PeriodicalIF":1.7,"publicationDate":"2025-07-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144626574","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":"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":"Effect of epinephrine on growth characteristics of Actinobacillus pleuropneumoniae field isolates","authors":"Elena Schreiber ,&nbsp;Fritjof Freise ,&nbsp;Nicole de Buhr ,&nbsp;Isabel Hennig-Pauka","doi":"10.1016/j.mimet.2025.107193","DOIUrl":"10.1016/j.mimet.2025.107193","url":null,"abstract":"<div><div>Based on empirical observations, stressors in swine populations may contribute to outbreaks due to <em>Actinobacillus pleuropneumoniae</em> (<em>App</em>) in colonized pigs. Stress hormones as catecholamines were found to impact immune mechanism but also bacterial protein expression <em>in vivo</em>, so it can be hypothesized that they are key molecules in disease pathogenesis. Culturing methods to isolate <em>App</em> from tonsillar tissue of carrier pigs for further typing are mostly not successful. In this study the effect of epinephrine in culturing media on <em>App</em> growth behavior was tested. Starting cultures of different age (24-h-old or 7-day-old cultures) of three <em>App</em> field strains per serotypes 2, 5 and 12 were compared in their growth behavior in medium supplemented with various epinephrine concentrations. The response of <em>App</em> strains to epinephrine varied between strains. Highest differences in CFU/mL and in growth curve characteristics between different epinephrine concentrations were found in serotype 5 strains. Inflection points of bacterial growth curves with 24-h-old starting cultures were delayed with 100 μM epinephrine with exception of one ST5 strain. In all ST2 and ST12 as well as one ST5 strain 100 μM epinephrine led to a growth advantage in 7-day old compared to 24-h-old starting cultures. Epinephrine did not consistently enhance growth of <em>App</em>, so that it cannot be recommended as a supplement for growth media to facilitate isolation from colonized tonsils of subclinically infected pigs.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"236 ","pages":"Article 107193"},"PeriodicalIF":1.7,"publicationDate":"2025-07-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144623648","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}