Journal of microbiological methods最新文献

{"title":"Synthesis and evaluation of lipophilic fluorescent probes for the labelling of Listeria and their impact on biofilm formation","authors":"Nika Janež ,&nbsp;Márta Ladányi ,&nbsp;Nika Zaveršek ,&nbsp;Petra Čotar ,&nbsp;Aleksandar Sebastijanović ,&nbsp;Janez Štrancar ,&nbsp;Jerica Sabotič ,&nbsp;Stane Pajk","doi":"10.1016/j.mimet.2025.107206","DOIUrl":"10.1016/j.mimet.2025.107206","url":null,"abstract":"<div><div>Imaging bacterial biofilms using confocal fluorescence microscopy is used to study their structures, but its wider application is constrained by the limited availability of effective labelling tools. Small chemical fluorescent probes offer a versatile alternative to heterologous expression of fusion or reporter proteins, but data on their effects on biofilm formation are lacking. In this study, we synthesized a series of new lipophilic fluorescent probes based on Nile blue, Nile red and coumarin scaffold. We investigated them for the labelling of <em>Listeria</em> biofilms and determined their effects on the growth and biofilm biomass formation. The Nile red probe <strong>SP-AM 7</strong> and the coumarin probe <strong>PAG 31</strong> inhibited biofilm development and showed a strong bactericidal effect. The Nile blue probe <strong>PAG 19</strong> had the least effect on the tested parameters, but labelled slowly, while the fast-labelling Nile red probe <strong>SP-AM 8</strong> promoted biofilm formation. Both are suitable for use during biofilm growth, resulting in less variation in biomass-related measurements than probes added prior to imaging. In the 3D imaging-based measurements for selected probes, we found no difference in the total biomass formed compared to the control dye, but a redistribution of biomass in the 3D layers was observed. Other probes were found to be slow to label, leave traces of unused probes or interfere with attachment to the surface. Our results show that fluorescent probe labelling should be evaluated from chemical, physical and biological points of view to understand their reliability and credibility.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"236 ","pages":"Article 107206"},"PeriodicalIF":1.9,"publicationDate":"2025-07-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144758094","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 potential of recombinant Mycobacterium tuberculosis PcaA antigen and its enhancement of protective efficacy as a subunit vaccine booster following BCG priming","authors":"Yun Xu ,&nbsp;Xiaochun Wang ,&nbsp;Qiangsen Zhong","doi":"10.1016/j.mimet.2025.107202","DOIUrl":"10.1016/j.mimet.2025.107202","url":null,"abstract":"<div><div>Bacillus Calmette-Guérin (BCG) is the only vaccine currently used in clinical practice to prevent tuberculosis (TB), however, it remains limited in preventing latent infection, TB reactivation, and providing comprehensive protection. In this study, the <em>pcaA</em> gene, an antigen associated with persistent infection, was selected, and the recombinant plasmid pET28a-PcaA was successfully constructed for protein expression and purification. The specificity of the antigen was further verified using chemiluminescence, enzyme-linked immunosorbent assay (ELISA), flow cytometry, and mycobacterial growth inhibition assay (MGIA). Significant differences were observed in the expression levels of IFN-γ, IL-2, IL-8, and IgG in the peripheral blood of patients with <em>Mycobacterium tuberculosis</em> (<em>M. tb</em>) following stimulation with the PcaA antigen in the Active Tuberculosis (ATB) and Latent Tuberculosis Infection (LTBI) groups. An IL-8 combined diagnostic model could effectively distinguish between ATB and LTBI, while anti-PcaA IgG demonstrated strong performance in ruling out <em>M. tb</em> infection. Recombinant PcaA protein (rPcaA) was formulated with liposome dimethyl dioctadecylammonium bromide (DDA) / colloidal manganese salt (MnJ)/DM to immunize mice. Serum-specific antibody levels, cytokines secreted by splenocytes, and the number of multifunctional T cells in splenocytes were assessed. The results indicated that the BCG + rPcaA-DM vaccine group exhibited significantly elevated levels of Th1-type cytokines, antibody titers, and the frequencies of IFN-γ⁺/TNF-α⁺ single and double-positive CD4⁺ and CD8⁺ T cells compared to the BCG group. Furthermore, splenocytes and lung cells from immunized mice significantly inhibited mycobacterial growth. These findings suggest that the rPcaA-DM vaccine, as a BCG booster, significantly enhances Th1 polarization and provides robust protective efficacy, with potential to prevent LTBI progression to ATB.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"236 ","pages":"Article 107202"},"PeriodicalIF":1.9,"publicationDate":"2025-07-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144722982","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":"Improved pre-treatment for lipid staining of microalgae with a rigid cell wall","authors":"Samira Reuscher ,&nbsp;Gerd Klock ,&nbsp;Antje Kersten ,&nbsp;Samuel Schabel ,&nbsp;Rüdiger Graf","doi":"10.1016/j.mimet.2025.107205","DOIUrl":"10.1016/j.mimet.2025.107205","url":null,"abstract":"<div><div>The worldwide depletion of energy and feedstock resources is an important topic of the current decade. Microalgae are an interesting option, offering rapid biomass production in combination with CO₂-fixation. For ongoing research in microalgae applications, well-functioning laboratory methods are important. One major aspect in microalgae-related studies is the measurement of the biomass' lipid content. In order to avoid elaborate extraction protocols, lipid staining with fluorescent dyes such as Nile Red or BODIPY is currently preferred in small-scale screening trials. Although practical, staining was hindered for microalgae with a rigid cell wall. Additional pre-treatment using solvents has already been described, but this was not sufficient for all strains. Besides sonication, we show here that fluorescent staining can be successfully implemented following the disruption of microalgal cell walls by bead beating. The protocol functions with conventional laboratory equipment (ball mill, shaker and vortex mixer), all of them allowing for small-volume samples and processing of multiple samples in parallel. The bead beating method extends the fluorescence lipid staining approach to microalgae with an extremely rigid cell wall and can be applied for screening trials in biofuel or biomaterial related research.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"236 ","pages":"Article 107205"},"PeriodicalIF":1.9,"publicationDate":"2025-07-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144721285","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":"High-throughput screening assay for nitrification inhibitors and the discovery of goitrin as a biological nitrification inhibitor","authors":"Fabian Beeckman ,&nbsp;Andrzej Drozdzecki ,&nbsp;Alexa De Knijf ,&nbsp;Dominique Audenaert ,&nbsp;Tom Beeckman ,&nbsp;Hans Motte","doi":"10.1016/j.mimet.2025.107201","DOIUrl":"10.1016/j.mimet.2025.107201","url":null,"abstract":"<div><div>Nitrification inhibitors are valuable in mitigating nitrogen (N) losses from agricultural fertilizers, enhancing fertilizer use efficiency, and minimizing their environmental and climatic impacts. Currently, the portfolio of approved inhibitors is limited, creating a strong demand for new alternatives. Traditional nitrification inhibition assays rely on large soil systems, batch cultures, or, at best, microbial cultures in deep-well 96-well plates, none of which are suited for high-throughput screening. Here, we present a highly robust method that enables rapid and efficient screening of thousands of molecules on the soil-borne ammonia-oxidizing bacteria <em>Nitrosomonas europaea</em> and <em>Nitrosospira multiformis</em>. Our assay utilizes 384-well plates for both screening and read-out, requiring only 50 μL of culture per sample and low amounts of compound. The assay also allows for further characterization of nitrification inhibitors and differentiation between those targeting the ammonia monooxygenase (AMO) and hydroxylamine oxidoreductase (HAO) pathways. Finally, we applied the assay to test several oxazolidine variants and discovered goitrin as a novel biological nitrification inhibitor (BNI). Overall, this assay offers promising tools for the rapid identification of novel nitrification inhibitors, contributing to sustainable agriculture.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"236 ","pages":"Article 107201"},"PeriodicalIF":1.7,"publicationDate":"2025-07-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144702661","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":"Site-2 protease-like protein drives organic solvent tolerance in Rhodococcus ruber","authors":"Jinfang Wu,&nbsp;Zhonghao Wu,&nbsp;Ren Peng","doi":"10.1016/j.mimet.2025.107204","DOIUrl":"10.1016/j.mimet.2025.107204","url":null,"abstract":"<div><div>Engineered overexpression of a Site-2 protease-like protein (S2plp) in <em>Rhodococcus ruber</em> SD3 significantly enhanced organic solvent tolerance. Through electro-transformation with the recombinant plasmid pNV18-<em>s2plp</em>-<em>s2plp</em>, we generated a strain exhibiting superior growth under multiple organic solvent stresses. Transcriptomic analysis identified 13 significantly upregulated genes, including those encoding aminoglycoside O-phosphotransferase, replication initiation protein, two M50 family metallopeptidases, transposase, SMC family ATPase, ATP-dependent helicase, three hypothetical proteins, and three small RNAs (sRNAs). KEGG analysis linked S2plp overexpression to enhanced DNA repair mechanisms, directly contributing to organic solvent tolerance. These results establish S2plp as a novel molecular target for boosting solvent resilience in this important bacterium, advancing its potential in biocatalysis and bioremediation.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"236 ","pages":"Article 107204"},"PeriodicalIF":1.7,"publicationDate":"2025-07-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144707790","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":"Advancing innovative techniques in arbuscular mycorrhizal Fungi propagation: A key to sustainable agriculture and ecosystem management","authors":"Aditi Pandit ,&nbsp;Divya Gunsola ,&nbsp;Apaarna ,&nbsp;Rahul Kumar ,&nbsp;Periyasamy Panneerselvam ,&nbsp;Debasis Mitra","doi":"10.1016/j.mimet.2025.107200","DOIUrl":"10.1016/j.mimet.2025.107200","url":null,"abstract":"<div><div>Arbuscular mycorrhizal fungi (AMF) play pivotal roles in enhancing plant nutrient acquisition and overall ecosystem sustainability. The propagation of AMF involves several methodologies tailored to address specific research or practical objectives. So, enhancing methods for propagating AMF is pivotal for fostering sustainable agriculture and maintaining ecosystem balance at the application level. However, traditional substrate-based techniques focus on the isolation, purification, and cultivation of AMF spores, thereby enabling the establishment of pure cultures. Hydroponic and aeroponic systems have emerged as valuable platforms for AMF cultivation, offering controlled environments to study spore germination, hyphal growth, and mycorrhizal colonization. In addition, this review explores the significance of root-organ culture techniques in the propagation and study of AMF. As the demand for sustainable agriculture increases, the application of AMF in agroecosystems has gained prominence. This review discusses the implementation of these techniques in conventional and organic farming systems. In conclusion, this review synthesizes the diverse methodologies employed in AMF propagation, from traditional spore-based techniques to cutting-edge approaches. The integration of these techniques not only deepens our understanding of AMF biology but also holds promise for sustainable agricultural practices and ecosystem management.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"236 ","pages":"Article 107200"},"PeriodicalIF":1.7,"publicationDate":"2025-07-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144675002","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":"Assessing the diagnostic utility of routine liquid enriched media in the processing of microbiological samples","authors":"Victoria Jordan ,&nbsp;Hemalatha Varadhan","doi":"10.1016/j.mimet.2025.107196","DOIUrl":"10.1016/j.mimet.2025.107196","url":null,"abstract":"<div><div>A retrospective review of the utility of routinely inoculating thioglycollate broth for select sterile fluids was performed. The broth was contributory in ≤1 % of samples, prompting removal of broth inoculation from routine processing of these sample types. Laboratories should consider reviewing the routine use of enriched broth particularly in the context of modern ancillary methods of pathogen detection.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"236 ","pages":"Article 107196"},"PeriodicalIF":1.7,"publicationDate":"2025-07-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144667766","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":"Biphasic bactericidal activity of nitroxoline against Acinetobacter baumannii assessed by Raman-DIP","authors":"Xiaofei Yi ,&nbsp;Xin Chen ,&nbsp;Minggui Wang ,&nbsp;Jianfeng Zhang ,&nbsp;Xiaogang Xu","doi":"10.1016/j.mimet.2025.107199","DOIUrl":"10.1016/j.mimet.2025.107199","url":null,"abstract":"<div><div>The increasing antimicrobial resistance (AMR) of <em>Acinetobacter baumannii</em> presents a challenge to clinical management and underscores its role as a critical pathogen in refractory urinary tract infections (UTIs). Nitroxoline has gained renewed interest as a potential therapeutic option. Raman deuterium stable isotope probing (Raman-DIP), which allows for the analysis of bacterial metabolic activity, has shown promise as a tool for assessing antimicrobial efficacy. The aim of this study was to evaluate the bactericidal activity of nitroxoline against <em>A. baumannii</em> and the potential of Raman-DIP to assess this activity. Thirty-four <em>A. baumannii</em> isolates were collected from patients with UTI. Minimum inhibitory concentrations (MICs), minimum bactericidal concentrations (MBCs), optimal bactericidal concentrations (OBCs), and time–killing curves were determined. Raman-DIP evaluated nitroxoline's effects on bacterial metabolism and survival, using the C−D ratio (C−D to the sum of the C − H and C − D band intensities) as a metabolic activity metric. Nitroxoline demonstrated potent antimicrobial activity against <em>A. baumannii</em>, with MIC_50/90 and MBC_50/90 values of 2/2 and 2/4 mg/L, respectively. Notably, it showed biphasic bactericidal characteristics with an OBC_50/90 value of 4/8 mg/L. Raman-DIP revealed a decrease in the C−D ratio with an increase in nitroxoline concentration, indicating reduced metabolic activity. An inverse correlation was observed between bacterial survival and the C−D ratio at concentrations above the OBC. These findings underscore the necessity to optimize dosing regimens for enhancing the efficacy of nitroxoline. Raman-DIP may serve as an effective tool for investigating the effect of nitroxoline on bacterial metabolism, thereby informing its clinical applications.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"236 ","pages":"Article 107199"},"PeriodicalIF":1.7,"publicationDate":"2025-07-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144667767","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 digital PCR for the detection of bovine viral diarrhea virus in persistently infected cattle","authors":"Duan Sriyotee Loy ,&nbsp;Zachary Brown ,&nbsp;Enakshy Dutta ,&nbsp;John Dustin Loy","doi":"10.1016/j.mimet.2025.107198","DOIUrl":"10.1016/j.mimet.2025.107198","url":null,"abstract":"<div><div>Bovine Viral Diarrhea Virus (BVDV) is a significant pathogen of cattle, causing substantial economic losses. Identifying persistently infected (PI) animals is crucial for BVD control and prevention, with pooled ear-notch tissue testing being a standard screening method. Digital PCR (dPCR) is an emerging diagnostic tool that provides absolute quantification of animal pathogens in low abundant targets. This study evaluates the effectiveness of dPCR for BVD surveillance using field submissions of pooled ear-notch samples, comparing it to real-time quantitative reverse transcription PCR (qRT-PCR). The study compared performance using 107 pools of ear notches (4853 sample animals), including 76 BVD-positive pools (3543 sample animals) and 31 BVD-negative pools (1310 sample animals). Pools included those that contained PI animals detected by IHC (<em>n</em> = 25) and those with only virus detection that were negative by IHC (<em>n</em> = 51). Performance metrics evaluated included analytical and diagnostic sensitivity and specificity, limit of detection (LOD) and test agreement with qRT-PCR. Results showed that dPCR exhibited enhanced sensitivity and lower LOD compared to qRT-PCR, detecting as low as 0.6 viral copies/μL. dPCR achieved 100 % sensitivity (76/76) and 96.77 % specificity (30/31) compared to qRT-PCR and no detection of common bovine pathogens. The comparison between qRT-PCR and dPCR using Cohen's kappa coefficient in pooled samples was 0.98, indicating almost perfect agreement. In pooled ear-notch samples with qRT-PCR quantification cycle (Cq) values ranging from 30 to 33.99, the viral load ranged from 4.75 to 194.78 viral copies/μL. The study suggests that dPCR is a sensitive and specific method for detecting BVD in pooled ear-notches.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"236 ","pages":"Article 107198"},"PeriodicalIF":1.7,"publicationDate":"2025-07-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144655789","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":"Advances in molecular diagnostics of Neisseria gonorrhoeae","authors":"Choo Yee Yu ,&nbsp;Xin Rui Lim ,&nbsp;Thanh Van Phan ,&nbsp;Nurulfiza Mat Isa ,&nbsp;Chan Yean Yean ,&nbsp;Kok-Gan Chan ,&nbsp;Geik Yong Ang","doi":"10.1016/j.mimet.2025.107197","DOIUrl":"10.1016/j.mimet.2025.107197","url":null,"abstract":"<div><div><em>Neisseria gonorrhoeae</em> is a human obligate pathogen that causes the sexually transmitted infection (STI) gonorrhea. As the second most commonly reported STI of bacterial origin, gonorrhea is a growing global public health concern given that the causative pathogen has developed resistance to antibiotics that are currently used for treatment. A holistic approach is thus essential to reduce the incidence of gonorrhea and to control the spread of antimicrobial resistance <em>N. gonorrhoeae</em>. Improvement in diagnostics is one of the avenues to combat rising STI rates and this review aims to provide an overview of the strategies used for <em>N. gonorrhoeae</em> nucleic acid detection by examining nucleic acid amplification tests (NAATs) that are cleared by the United States Food and Drug Administration (FDA). The updated and comprehensive information of FDA-cleared NAATs presented in this review includes the recent clearance of NAATs for point-of-care testing and home-based specimen collection kit for gonorrhea testing that can potentially revolutionize STI services. Further progress in the molecular diagnostics for gonorrhea is anticipated along with vaccine development as the challenge remains to meet the World Health Organization goal of reducing the incidence of <em>N. gonorrhoeae</em> infection by 90 % by 2030.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"236 ","pages":"Article 107197"},"PeriodicalIF":1.7,"publicationDate":"2025-07-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144655788","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}