Journal of microbiological methods最新文献

{"title":"Identification of Legionella anisa, Legionella longbeachae and Legionella pneumophila using MALDI-TOF MS: A method validation study with environmental isolates","authors":"Ellinoora Savonen ,&nbsp;Silja Mentula ,&nbsp;Jenni Ikonen ,&nbsp;Ilkka T. Miettinen ,&nbsp;Pekka M. Rossi ,&nbsp;Marjo Niittynen","doi":"10.1016/j.mimet.2025.107330","DOIUrl":"10.1016/j.mimet.2025.107330","url":null,"abstract":"<div><div>Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) allows species-level microbial identification quickly and cost-effectively. The aim of this study was to validate MALDI-TOF MS for the identification of <em>Legionella anisa</em>, <em>Legionella longbeachae</em> and <em>Legionella pneumophila</em> and to examine whether incubation time or sample preparation method affect identification results.</div><div>Environmental <em>Legionella</em> strains and ATCC reference strains were used in the study. The strains underwent 16S rRNA sequencing and latex agglutination and fluorescence tests to which MALDI-TOF MS results were compared. The strains, incubated for 24 and 48 h, were analysed with MALDI-TOF MS using direct transfer and extraction methods. Following performance characteristics were calculated from the results: sensitivity, specificity, relative accuracy, predictive values, repeatability and reproducibility.</div><div>Species-level identification was achieved for 63 out of the 69 environmental and reference strains regardless of incubation time or preparation method. MALDI-TOF MS proved to be very specific (100 %) and sensitive (94.6–100 %) to the <em>Legionella</em> species analysed. With the extraction method, incubation time had a statistically significant, but species-dependent effect on the interpretative criteria, i.e. score values, in all species studied. No significant difference in any of the species' score values was found between the direct transfer and extraction methods with 24 h incubation. MALDI-TOF MS score values showed high repeatability (std 0.01–0.23) and reproducibility (std 0.02–0.18) for all <em>Legionella</em> species analysed. Based on the results, MALDI-TOF MS suits well for the analysis of environmental <em>Legionella</em> strains already after 24 h of incubation and using direct transfer method.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"240 ","pages":"Article 107330"},"PeriodicalIF":1.9,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145530611","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":"First report on ATR-FTIR and machine learning-based sputum profiling for TB detection: A compendium of potential biomarkers","authors":"Zubia Shahid ,&nbsp;Muhammad Tahir Hayat ,&nbsp;Irshad Hussain ,&nbsp;Javaria Qazi","doi":"10.1016/j.mimet.2025.107356","DOIUrl":"10.1016/j.mimet.2025.107356","url":null,"abstract":"<div><div>Tuberculosis (TB) remains a significant global health issue, with millions of new cases reported annually and persistent limitations in conventional diagnostic approaches. This study is the first report that explores the use of Attenuated Total Reflection Fourier-Transform Infrared (ATR-FTIR) spectroscopy for identifying biochemical signatures associated with TB in lyophilized sputum. Spectra were acquired across the full mid-infrared range (400–4000 cm⁻¹) from 100 samples (50 TB-positive, 50 healthy controls) from Pakistan Institute of Medical Sciences, Islamabad, followed by pre-processing and multivariate analysis. Principal Component Analysis (PCA) combined with Linear Discriminant Analysis (LDA) and Support Vector Machine (SVM) classification achieved strong separation between groups. Spectral differences were most pronounced in two regions: 2800–3500 cm⁻¹ and 900–1800 cm⁻¹. Key wavenumbers including 3290 cm⁻¹ (polysaccharides), 2954 cm⁻¹ (mycolic acid), and 1636 cm⁻¹ (β-sheets) have promising potential in establishing disease status. Peaks corresponding to carbohydrates, lipids, and nucleic acids further show the biochemical divergence between the two groups. The observed spectral variations demonstrate a clear potential for sputum-based differentiation of TB using vibrational spectroscopy. These findings highlight the potential for reagent-free, rapid TB screening in clinical and resource-limited settings and contribute to the growing body of research. Future studies on larger data sets can help validate our findings.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"240 ","pages":"Article 107356"},"PeriodicalIF":1.9,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145696165","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":"Exploration of the optimum reaction conditions for the reverse transcription of mycoviral RNA from Lentinula edodes","authors":"Zhifeng Zhang ,&nbsp;Yingjun Zhang ,&nbsp;Xiaoli Wang ,&nbsp;Nini Duan ,&nbsp;Zhaoyu Wang ,&nbsp;Yankui Li ,&nbsp;Jiayao Yan","doi":"10.1016/j.mimet.2025.107360","DOIUrl":"10.1016/j.mimet.2025.107360","url":null,"abstract":"<div><div>Reverse transcription (RT) of viral RNA to complementary DNA (cDNA) is essential for mycovirus detection; however, the stable secondary structures of double-stranded RNA (dsRNA) and the genomic diversity of viruses complicate this process. This study systematically optimized RT conditions for 15 mycoviruses infecting <em>Lentinula edodes</em> using RT-qPCR, focusing on thermal denaturation temperatures (no heat denaturation [CK], 65 °C [T65], 95 °C [T95], and 98 °C [T98]) and dimethyl sulfoxide (DMSO) concentrations (0–20 %). For purified LeV-HKB dsRNA, T95 and T98 increased cDNA yields by over 50,000- and 100,000-fold, respectively, compared to T65. In total RNA extracts from infected L. <em>edodes</em>, T98 increased cDNA yields for dsRNA mycoviruses by approximately 5-fold compared to CK. Conversely, T95/T98 did not improve cDNA yields for ssRNA viruses, and even resulted in slightly reduced yields for certain viruses. DMSO exerted virus-specific and temperature-dependent effects on RT efficiency: moderate concentrations (5–15 %) enhanced cDNA yields at CK or T65, whereas high concentrations (e.g., 20 %) reduced yields at T95 or T98. No universal DMSO concentration was optimal across viruses, necessitating tailored approaches. Overall, T98 is recommended for dsRNA viruses, CK/T65 for ssRNA viruses, and T98 as a practical compromise for mixed RNA types. This protocol improves detection sensitivity and reliability, thereby facilitating diagnosis and research on fungal RNA viruses.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"240 ","pages":"Article 107360"},"PeriodicalIF":1.9,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145743135","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":"Construction of an mKate-based fluorescent protein reporting system in Limosilactobacillus reuteri","authors":"Yihang Wang ,&nbsp;Xinxin Wang ,&nbsp;Daoyan Wu ,&nbsp;Wei Hong ,&nbsp;Yingqian Kang ,&nbsp;Renmin Li ,&nbsp;Guang Yang ,&nbsp;Haoqin Yan ,&nbsp;Zhenghong Chen ,&nbsp;Guzhen Cui","doi":"10.1016/j.mimet.2025.107347","DOIUrl":"10.1016/j.mimet.2025.107347","url":null,"abstract":"<div><div>An efficient genetic transformation system was established for <em>Limosilactobacillus reuteri</em> through protocol optimization. Subsequently, an mKate-based red fluorescent protein reporting system was constructed. This platform enables real-time monitoring of bacterial localization and gene expression, providing a robust tool for synthetic biology applications.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"240 ","pages":"Article 107347"},"PeriodicalIF":1.9,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145616466","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 IgG anti-L. tropica antibody response as a biomarker for cutaneous leishmaniasis using ELISA in endemic regions of Pakistan","authors":"Bashair Sheikh Pervez ,&nbsp;Arshad Islam ,&nbsp;Azhar Minhas ,&nbsp;Yusuf Ozbel ,&nbsp;Seray Toz ,&nbsp;Shahid Waseem ,&nbsp;Obaid Hayat ,&nbsp;Shumaila Naz","doi":"10.1016/j.mimet.2025.107340","DOIUrl":"10.1016/j.mimet.2025.107340","url":null,"abstract":"<div><div>Cutaneous leishmaniasis (CL) has diverse clinical manifestations that overlap with other skin conditions. Conventional diagnostic methods, such as microscopy, often lack sensitivity and make diagnosis a challenge due to varying sensitivity and specificity, highlighting the need for sensitive and cost-effective immunodiagnostic alternatives. The present study aimed to evaluate anti-leishmanial immunoglobulin G (IgG anti-<em>L. tropica</em>) antibody responses as a biomarker of CL. The lesional aspirate and serum samples from CL-infected individuals (<em>n</em> = 216) and healthy controls (<em>n</em> = 157) were collected from CL endemic regions of Punjab, Khyber Pakhtunkhwa (KPK) and Balochistan in Pakistan. <em>Leishmania tropica</em> (<em>L. tropica</em>) was identified as a dominant species via real-time ITS-1 PCR. The soluble <em>Leishmania</em> antigen was prepared from cultured L. <em>tropica</em> and utilized to detect levels of <em>Leishmania-</em>specific IgG anti-<em>L. tropica</em> antibodies in sera employing enzyme-linked immunosorbent assay (ELISA). The ELISA demonstrated significantly higher IgG anti-<em>L. tropica</em> levels in CL patients (<em>P</em> &lt; 0.0001) as compared to healthy controls. A cut-off value of 0.22 yielded a sensitivity of 79.35 % and specificity of 84.40 %, with an Area Under Curve (AUC) of 0.79. IgG anti-<em>L. tropica</em> levels. A significant difference in IgG anti-<em>L. tropica</em> levels of antibody was observed among CL patients with different lesion sizes and numbers. The developed ELISA highlighted the importance of employing reliable diagnostic techniques for CL. The serological approach may complement conventional diagnostics and contribute to better disease management in endemic settings.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"240 ","pages":"Article 107340"},"PeriodicalIF":1.9,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145596663","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":"CRISPR technology for diagnosis and treatment of human brucellosis","authors":"Cia-Hin Lau ,&nbsp;Xiaojun Li ,&nbsp;Qing-Le Liang ,&nbsp;Rui Guo ,&nbsp;Xiangyang Ren ,&nbsp;Zhenna Xu ,&nbsp;Yanqiu Zhu ,&nbsp;Ying Lai ,&nbsp;Guoqing Liu ,&nbsp;Yumei Huang ,&nbsp;Weidong Wu ,&nbsp;Haibao Zhu ,&nbsp;Jinhui Chen ,&nbsp;Xianpeng Zhang","doi":"10.1016/j.mimet.2025.107339","DOIUrl":"10.1016/j.mimet.2025.107339","url":null,"abstract":"<div><div>The global burden of <em>Brucella</em> infection in livestock and human health is substantial, particularly in developing countries or rural areas. Currently, brucellosis diagnosis primarily relies on PCR, microbiological culture, and serological tests. However, these approaches have several drawbacks such as long experimental duration, lengthy procedure, low positive detection rates, high variability in results, interspecies cross-reactivity, and require expensive equipment and professional operators. Herein, we review how recent emerging CRISPR/Dx technology can address some of these shortcomings to realize field-deployable detection of <em>Brucella</em> in domestic animals and point-of-care testing (POCT) for human brucellosis. CRISPR technology has been successfully used to treat brucellosis by deleting or inactivating the genes associated with the <em>Brucella</em> replication or survival. Therefore, we also discuss how CRISPR technology can be potentially used to treat brucellosis, as antibiotic therapy may lose efficacy when encountering multidrug-resistant <em>Brucella</em> strains and the treatment is long-lasting in infected individuals to prevent relapse. Lastly, we critically discuss the advances, pitfalls, and future perspectives of CRISPR technology for the diagnosis and treatment of brucellosis in humans and livestock. Ultimately, the continued refinement of CRISPR technology will pave the road for field-deployable pathogen diagnostics and home self-tests of brucellosis to mitigate global <em>Brucella</em> infections.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"240 ","pages":"Article 107339"},"PeriodicalIF":1.9,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145571179","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":"Vaginal microbe media (VMM): a novel medium to culture Lactobacillus iners and other fastidious vaginal bacteria","authors":"Lauren A. Roberts ,&nbsp;David C.A. Gaboriau ,&nbsp;David A. MacIntyre ,&nbsp;Julian R. Marchesi","doi":"10.1016/j.mimet.2025.107358","DOIUrl":"10.1016/j.mimet.2025.107358","url":null,"abstract":"<div><div>A more comprehensive understanding of the bacterial species in the vaginal microbiome and their roles requires their cultivation as pure isolates. However, difficulty in culturing fastidious species, particularly in liquid media, has limited certain types of experimental approaches for studying these organisms. To address this challenge, Vaginal Microbe Medium (VMM) was developed to enable robust growth of <em>Lactobacillus iners</em> within a relatively short period (36 h). A simplified version with fewer components, named VMM2, was subsequently developed. <em>L. iners</em> strains grown in VMM and VMM2 reached higher optical densities in a shorter period compared to NYCIII, supplemented MRS and Columbia broth. Bacterial cells cultured in VMM and VMM2 were significantly longer than those grown in NYCIII. These results indicate that VMM and VMM2 provide superior growth conditions for <em>L. iners</em> as compared to currently available media. Both media also support the growth of a range of other vaginal bacterial species. The development of these media facilitates liquid culture of fastidious bacteria from the vaginal niche, enabling broader experimental capabilities and deeper insights into causal relationships within the microbiome.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"240 ","pages":"Article 107358"},"PeriodicalIF":1.9,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145751813","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 concordance of cPCR and qPCR in Helicobacter pylori detection and association with gastric diseases","authors":"Bruno Mari Fredi ,&nbsp;Mikaela Nagahara ,&nbsp;Fabricio Abdala Brandt ,&nbsp;Eduardo Federighi Baisi Chagas ,&nbsp;Mônica Santiago Barbosa ,&nbsp;Spencer Luiz Marques Payao ,&nbsp;José Celso Rocha ,&nbsp;Lucas Trevizani Rasmussen","doi":"10.1016/j.mimet.2025.107363","DOIUrl":"10.1016/j.mimet.2025.107363","url":null,"abstract":"<div><div>This study compared cPCR and qPCR for <em>Helicobacter pylori</em> detection in 302 gastric biopsies and correlated the findings with gastric diseases. qPCR detected more positive cases, mainly among healthy individuals. Despite significant discordance (<em>p</em> &lt; 0.001), both methods showed substantial agreement (κ = 0.75), confirming the reliability of qPCR and its diagnostic equivalence to cPCR.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"240 ","pages":"Article 107363"},"PeriodicalIF":1.9,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145756819","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 MALDI-TOF systems (autof MS 1000) in identification of pharmaceutical environmental bacteria","authors":"Yunting Ma ,&nbsp;Bin Yang ,&nbsp;Chenshui Lin ,&nbsp;Meixia Wang ,&nbsp;Yang Che ,&nbsp;Huanying Wang ,&nbsp;Xiaoling Zheng ,&nbsp;Tingzhang Wang ,&nbsp;Hong Li ,&nbsp;Hongxia Zhao","doi":"10.1016/j.mimet.2025.107309","DOIUrl":"10.1016/j.mimet.2025.107309","url":null,"abstract":"<div><div>This study evaluated the performance of the Autof MS 1000 matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry system in identifying pharmaceutical environmental bacteria, with 16S rDNA sequencing as the gold standard. A total of 1041 non-repetitive isolates collected from various pharmaceutical environments from May 2022 to July 2022 were analyzed. The Autof MS 1000 system successfully identified 806 (77.42 %) of the 1041 isolates. The unsuccessful identifications were attributable to either ‘not reliable’ scores below 6.0 (<em>n</em> = 229, 22.00 %) or a complete failure to obtain a mass spectrum (<em>n</em> = 6, 0.58 %). Using 16S rDNA sequencing as the reference standard, the Autof MS 1000 demonstrated a species-level identification accuracy of 73.10 % (587/803) and a genus-level accuracy of 91.53 % (735/803). The Autof MS 1000 shows considerable potential for the high-throughput identification of pharmaceutical environmental bacteria. Future efforts to expand the mass spectrometry database and optimize pretreatment protocols are needed to further enhance its identification accuracy.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"239 ","pages":"Article 107309"},"PeriodicalIF":1.9,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145418896","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":"Kinetic analysis of dye reduction profiles provide deeper insights into the electron transfer activity of bacterial cultures","authors":"S.K. Shakthi Thangavel ,&nbsp;Sahashransu S. Mahapatra ,&nbsp;Prajal Chettri ,&nbsp;Shailesh Srivastava ,&nbsp;A.S. Vishwanathan","doi":"10.1016/j.mimet.2025.107313","DOIUrl":"10.1016/j.mimet.2025.107313","url":null,"abstract":"<div><div>The Dye Reduction-based Electron-transfer Activity Monitoring (DREAM) assay offers a rapid, scalable, and culture-independent method for assessing bacterial respiratory activity. In this study, we have developed and applied a kinetic framework to extract six quantitative metrics from DREAM assay absorbance-time profiles: two derived from raw absorbance data – Dye-reduction Quotient (DQ) and Maximal Slope Interval (MSI) – and four from inverse-sigmoidal curve fitting – Baseline Absorbance (BA), Reduction Amplitude (RA), Time of Maximum Kinetic Shift (TMKS), and Redox Completion Index (RCI). These metrics complement traditional bacterial count methods by offering functional insights into metabolic activity, though they must be interpreted in a comparative rather than absolute context. We have applied this framework to two experimental conditions with mixed bacterial cultures, viz., varying bacterial concentrations and different substrate environments, to demonstrate how these metrics collectively capture the magnitude, timing, and dynamics of electron transfer processes in practical situations. This comprehensive kinetic quantification enhances the interpretive power of the DREAM assay and broadens its applicability for evaluating microbial performance in the bioprocessing and biotechnology industry and in environmental samples.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"239 ","pages":"Article 107313"},"PeriodicalIF":1.9,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145409225","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}