Journal of microbiological methods最新文献

{"title":"Protocols to enable fluorescence microscopy of microbial interactions on living maize silks (style tissue)","authors":"Michelle E.H. Thompson,&nbsp;Manish N. Raizada","doi":"10.1016/j.mimet.2024.107027","DOIUrl":"10.1016/j.mimet.2024.107027","url":null,"abstract":"<div><p>There is interest in studying microbes that colonize maize silks (style tissue, critical for reproduction) including the fungal pathogen <em>Fusarium graminearum</em> (<em>Fg</em>) and its interactions with the microbiome and biocontrol agents. <em>In planta</em> imaging of these interactions on living silks using confocal fluorescence microscopy would provide key insights. However, newly discovered microbes have unknown effects on human health, and there are regulatory requirements to prevent the release of fluorescently tagged microbes into the environment. Therefore, the microbe infection, colonization, and interaction stages on silks prior to microscopy must be contained. At the same time, silk viability must be maintained and experiments conducted that are biologically relevant (e.g. silks should remain attached to the cob), yet the silk tissue must be accessible to the researcher (i.e. not within husk leaves) and allow for multiple replicates. Here we present methods that meet these five contrasting criteria. We tested these methods using <em>Fg</em> and four silk-derived bacterial endophytes. The endophytes were previously known to have anti-<em>Fg</em> activity in vitro, but <em>in planta</em> observations were lacking. In Method 1, a portion of the tip of a cob was dissected, and silks remained attached to the cob in a Petri dish. The cob was placed on a water agar disc to maintain hydration. DsRed-tagged bacteria and GFP-tagged <em>Fg</em> were inoculated onto the silks and incubated, allowing the two microbes to grow towards one another before staining with propidium iodide for confocal microscopy. A variation of the protocol was presented in Method 2, where detached silk segments were placed directly on water agar where they were inoculated with bacteria and <em>Fg</em> to promote dense colonization, and to allow for many replicates and interventions such as silk wounding. The bacterial endophytes were successfully observed colonizing <em>Fg</em> hyphae, silk trichomes, and entering silks via cut ends and wounds. These protocols can be used to study other silk-associated microbes including several globally important fungal pathogens that enter maize grain through silks.</p></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"225 ","pages":"Article 107027"},"PeriodicalIF":1.7,"publicationDate":"2024-08-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S0167701224001398/pdfft?md5=ceb70988dd3da6a0a097114fe12f9c40&pid=1-s2.0-S0167701224001398-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142098211","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Establishment and methodological evaluation of a rapid detection method for Cryptococcus neoformans and Cryptococcus gattii species complexes based on CRISPR-Cas12a technology","authors":"Runde Liu ,&nbsp;Yuqing Xing ,&nbsp;Jilu Shen","doi":"10.1016/j.mimet.2024.107026","DOIUrl":"10.1016/j.mimet.2024.107026","url":null,"abstract":"<div><h3>Purpose</h3><p>The opportunistic pathogens causing Cryptococcal meningitis are <em>Cryptococcus neoformans</em> and <em>Cryptococcus gattii</em> species complexes<em>.</em> At present, clinical detection methods for this condition include culture, ink staining, and cryptococcal antigen detection. In addition, enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and real-time quantitative PCR (qPCR) can be applied for the detection of <em>Cryptococcus</em>. Nevertheless, these methods cannot achieve point-of-care detection (POCT); thus, there is a pressing need to establish a fast, sensitive, and effective detection method.</p></div><div><h3>Methods</h3><p>Recombinase polymerase amplification (RPA) and clustered regularly spaced short palindromic repeat (CRISPR) techniques are effective tools for achieving rapid POCT. In this study, RPA was combined with CRISPR-Cas12a to establish a fast, sensitive, and specific detection method for cryptococcal meningitis.</p></div><div><h3>Results</h3><p>This study included RPA-Cas12a fluorescence detection and RPA-Cas12a immunochromatographic detection, which can be performed within 50 min. Moreover, the detection limit was as low as 10² copies/μL. Interestingly, the developed method demonstrated satisfactory specificity and no cross-reactivity with other fungi and bacteria. 36 clinical samples were tested, and the consistency between the test results and those obtained using the commonly used clinical culture method was 100 %.</p></div><div><h3>Conclusion</h3><p>In this study, a rapid detection method for <em>Cryptococcus neoformans</em> and <em>Cryptococcus gattii</em> species complexes was developed based on CRISPR-Cas12a technology, characterized by its high sensitivity and specificity, ease of use, and cost-effectiveness, making it suitable for on-site detection.</p></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"225 ","pages":"Article 107026"},"PeriodicalIF":1.7,"publicationDate":"2024-08-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142055825","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":"Image cropping for malaria parasite detection on heterogeneous data","authors":"Ibrahim Mouazamou Laoualy Chaharou ,&nbsp;Ismail Lawani ,&nbsp;Theophile Dagba ,&nbsp;Jules Degila ,&nbsp;Habiboulaye Amadou Boubacar","doi":"10.1016/j.mimet.2024.107022","DOIUrl":"10.1016/j.mimet.2024.107022","url":null,"abstract":"<div><p>Malaria is a deadly disease of significant concern for the international community. It is an infectious disease caused by a <em>Plasmodium</em> spp. parasite and transmitted by the bite of an infected female <em>Anopheles</em> mosquito. The parasite multiplies in the liver and then destroys the person's red blood cells until it reaches the severe stage, leading to death. The most used tools for diagnosing this disease are the microscope and the rapid diagnostic test (RDT), which have limitations preventing control of the disease. Computer vision technologies present alternatives by providing the means for early detection of this disease before it reaches the severe stage, facilitating treatment and saving patients. In this article, we suggest deep learning methods for earlier and more accurate detection of malaria parasites with high generalization capabilities using microscopic images of blood smears from many heterogeneous patients. These techniques are based on an image preprocessing method that mitigates some of the challenges associated with the variety of red cell characteristics due to patient diversity and other artifacts present in the data.</p><p>For the study, we collected 65,970 microscopic images from 876 different patients to form a dataset of 33,007 images with a variety that enables us to create models with a high level of generalization. Three types of convolutional neural networks were used, namely Convolutional Neural Network (CNN), DenseNet, and LeNet-5, and the highest classification accuracy on the test data was 97.50% found with the DenseNet model.</p></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"225 ","pages":"Article 107022"},"PeriodicalIF":1.7,"publicationDate":"2024-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142036051","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":"Mass production of Isaria fumosorosea pfu5 for community approach management of rugose spiraling whitefly in oil palm","authors":"A.R.N.S. Subbanna,&nbsp;P. Kalidas","doi":"10.1016/j.mimet.2024.107023","DOIUrl":"10.1016/j.mimet.2024.107023","url":null,"abstract":"<div><p>The management of an alien Rugose spiraling whitefly (RSW) on oil palm using a native entomopathogenic fungus, <em>Isaria fumosorosea</em> pfu5 necessitated community approach for pest management. Moreover, coverage of huge leaf biomass warrants massive multiplication of biocontrol agent. In this communication, a two-step strategy, first including pure culture production and the second including ready-to-use culture production of the biocontrol agent is disclosed. The production costs and success of this technology in RSW management of oil palm are also discussed.</p></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"225 ","pages":"Article 107023"},"PeriodicalIF":1.7,"publicationDate":"2024-08-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142004480","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":"Application of the second-generation sequencing technology of metagenomics in the detection of pathogens in respiratory patients","authors":"Danfeng Zhang ,&nbsp;Ali Yang ,&nbsp;Kai Sheng ,&nbsp;Shuyu Fang ,&nbsp;Liang Zhou","doi":"10.1016/j.mimet.2024.107021","DOIUrl":"10.1016/j.mimet.2024.107021","url":null,"abstract":"<div><h3>Objective</h3><p>To explore the application value of the second-generation metagenomic next-generation sequencing (mNGS) in the detection of pathogens in patients with pulmonary infection.</p></div><div><h3>Methods</h3><p>We conducted a retrospective analysis of 65 pulmonary infection cases treated at our institution and the Fifth People's Hospital of Shanghai between January 2021 and May 2023. All subjects were subjected to mNGS, targeted next-generation sequencing (tNGS), and conventional microbiological culture. A comparative analysis was performed to evaluate the diversity and quantity of pathogens identified by these methodologies and to appraise their respective diagnostic capabilities in pulmonary infection diagnostics.</p></div><div><h3>Results</h3><p>The mNGS successfully identified etiological agents in 60 of the 65 cases, compared to tNGS, which yielded positive results in 42 cases, and conventional laboratory cultures, which detected pathogens in 24 cases. At the bacterial genus level, mNGS discerned 9 genera, 11 species, and 92 isolates of pathogenic bacteria, whereas tNGS identified 8 genera, 8 species, and 71 isolates. Conventional methods were less sensitive, detecting only 6 genera, 7 species, and 33 isolates. In terms of fungal detection, mNGS identified 4 fungal species, tNGS detected 4 isolates of the Candida genus, and conventional methods identified 2 isolates of the same genus. Viral detection at the species level revealed 10 species and 46 isolates by mNGS, whereas tNGS detected only 3 species and 7 isolates. The area under the receiver operating characteristic curve (AUC) with 95% confidence intervals for diagnosing pulmonary infections was 0.818 (0.671 to 0.966) for mNGS, 0.668 (0.475 to 0.860) for tNGS, and 0.721 (0.545 to 0.897) for conventional culture.The mNGS demonstrates superior diagnostic efficacy and pathogen detection breadth in critically ill patients with respiratory infections, offering a significant advantage by reducing the time to diagnosis. The enhanced sensitivity and comprehensive pathogen profiling of mNGS underscore its potential as a leading diagnostic tool in clinical microbiology.</p></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"225 ","pages":"Article 107021"},"PeriodicalIF":1.7,"publicationDate":"2024-08-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S0167701224001337/pdfft?md5=76785589bd399a508377d26423988ea4&pid=1-s2.0-S0167701224001337-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141988177","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Quantitative method for measuring the proportion of bacterial cells expressing phase 1 or 2 of flagellin of Salmonella enterica serovar Typhimurium","authors":"Momoko Nakayama,&nbsp;Nobuo Arai,&nbsp;Yohsuke Ogawa,&nbsp;Masahiro Kusumoto,&nbsp;Masahiro Eguchi","doi":"10.1016/j.mimet.2024.107013","DOIUrl":"10.1016/j.mimet.2024.107013","url":null,"abstract":"<div><p><em>Salmonella enterica</em> subsp. <em>enterica</em> is a major pathogen that causes zoonotic foodborne diseases worldwide. Some <em>Salmonella</em> serovars possess two antigenic phases for flagellin: phase 1 and 2. In <em>Salmonella enterica</em> serovar Typhimurium (<em>S.</em> Typhimurium), the flagellin is antigenically divided into “Hi” as phase 1 and “H1 or H2” as phase 2. Flagellin phase variation is regulated by inversion of <em>hin</em> gene. We focused on the inversion of <em>hin</em> and developed a real-time PCR system to quantitatively measure the proportion of bacterial cells expressing each phase of flagellin. In this study, we demonstrated that our newly developed real-time PCR system shows high quantitative accuracy and aligns with flagellin expression status. Furthermore, the newly developed real-time PCR system was applicable to various <em>S.</em> Typhimurium laboratory and field strains. This newly developed real-time PCR system has the potential to become a powerful tool for analyzing flagellin phase variation.</p></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"225 ","pages":"Article 107013"},"PeriodicalIF":1.7,"publicationDate":"2024-08-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141916978","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":"Comparison of in-house enzyme-linked immunosorbent assay (ELISA) and six commercial ELISAs for Detection of antibodies to Bordetella pertussis","authors":"Waldemar Rastawicki","doi":"10.1016/j.mimet.2024.107011","DOIUrl":"10.1016/j.mimet.2024.107011","url":null,"abstract":"<div><p>Enzyme-linked immunosorbent assays (ELISA) are currently the method of choice for the serodiagnosis of pertussis and play a key role in the diagnosis of pertussis in adolescents and adults, as well as in epidemiological studies. In the present study, the in-house developed indirect ELISA was comparatively evaluated with six commercial kits from various manufacturers. Antipertussis antibodies were measured in 40 serum samples from patients with clinical symptoms of respiratory tract infection, in two WHO standards, and in seven human ECDC control sera. IgA and IgG antibodies were detected at a diagnostically significant level by different ELISA kits of 5.0% to 27.0% and 12.0% to 70.0% of patients' sera, appropriately. The analysis of results carried out with six commercial kits showed only 17.5% consistent results in class IgG (either clearly positive or negative). The average percentage of errors in the level of antibodies determined in the control samples, reference serum samples, differed quite significantly and ranged from 9.5% to 35.4% depending on the kit. This poor correlation of the results obtained on various serological tests intended for the serodiagnosis of pertussis may cause very serious diagnostic problems, especially when examining a serum sample obtained once during the course of the disease.</p></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"224 ","pages":"Article 107011"},"PeriodicalIF":1.7,"publicationDate":"2024-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141901974","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":"A simplified laboratory approach for isolating M. oryzae spores from rice samples infected with multiple pathogens","authors":"S. Terensan ,&nbsp;H.N.S. Fernando ,&nbsp;J.N. Silva ,&nbsp;N.S. Kottearachchi ,&nbsp;O.V.D.S.J. Weerasena","doi":"10.1016/j.mimet.2024.107012","DOIUrl":"10.1016/j.mimet.2024.107012","url":null,"abstract":"<div><p>A method for separating <em>M. oryzae</em> from rice samples infected with multiple pathogens using basic laboratory equipment is described. We conducted a series of experiments to obtain a single spore of <em>M. oryzae</em>. This method can also be used to isolate spores from other fungal species.</p></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"224 ","pages":"Article 107012"},"PeriodicalIF":1.7,"publicationDate":"2024-08-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141897639","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":"A novel PCR-based genotyping method for Proteus mirabilis – Intergenic region polymorphism analysis","authors":"Nianqing Kong ,&nbsp;Yilin Hu ,&nbsp;Chenglu Lan ,&nbsp;Shuilian Bi","doi":"10.1016/j.mimet.2024.107008","DOIUrl":"10.1016/j.mimet.2024.107008","url":null,"abstract":"<div><p><em>Proteus mirabilis</em> is a predominant species in cases of food poisoning associated with meat products and is also an opportunistic pathogen causing numerous infections in humans. This study aimed to differentiate <em>P. mirabilis</em> isolates using intergenic region polymorphism analysis (IRPA). The IRPA typing scheme was developed to amplify polymorphic fragments in intergenic regions (IGRs). The presence, absence, or size change of amplified products were identified and utilized as genetic markers for rapid differentiation of strains. A total of 75 <em>P. mirabilis</em> isolates were isolated from 63 fresh poultry and pork samples were subtyped using the IRPA and ERIC-PCR methods, and their antibiotic resistance profiles were tested. The majority of <em>P. mirabilis</em> isolates showed resistance to tetracycline (85.3%), doxycycline (93.3%), chloramphenicol (82.7%), streptomycin (92.0%), spectinomycin (80.0%), trimethoprim (97.3%); trimethoprim-sulfalleth (82.7%), and erythromycin (100.0%). In contrast, resistance rates to ceftriaxon, cefoxitin, cefepime, and cefotaxim were lower at only 17.3%, 5.3%, 6.7%, and 13.3%, respectively, among <em>P. mirabilis</em> isolates. Eleven loci were selected for analysis of the genetic diversity of 75 <em>P. mirabilis</em> isolates. A combination of 4 loci was determined as the optimal combination. The results compared to those obtained using ERIC-PCR for the same isolates. The Simpson's index of diversity was 0.999 for IRPA and 0.923 for ERIC-PCR, indicating that IRPA has a higher discriminatory power than ERIC-PCR. The concordance between IRPA and ERIC-PCR methods was low, primarily because IRPA classified isolates from the same ERIC cluster into separate clusters due to its high resolution. The IRPA method presented in this study offers a rapid, simple, reproducible, and economical approach for genotyping <em>P. mirabilis</em>.</p></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"224 ","pages":"Article 107008"},"PeriodicalIF":1.7,"publicationDate":"2024-08-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141893624","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":"Application of MTT assay for probing metabolic activity in bacterial biofilm-forming cells on nanofibrous materials","authors":"Marta Stindlova ,&nbsp;Vaclav Peroutka ,&nbsp;Vera Jencova ,&nbsp;Kristyna Havlickova ,&nbsp;Simona Lencova","doi":"10.1016/j.mimet.2024.107010","DOIUrl":"10.1016/j.mimet.2024.107010","url":null,"abstract":"<div><p>The quantification of cellular metabolic activity via MTT assay has become a widespread practice in eukaryotic cell studies and is progressively extending to bacterial cell investigations. This study pioneers the application of MTT assay to evaluate the metabolic activity of biofilm-forming cells within bacterial biofilms on nanofibrous materials. The biofilm formation of <em>Staphylococcus aureus</em> and <em>Escherichia coli</em> on nanomaterials electrospun from polycaprolactone (PCL), polylactic acid (PLA), and polyamide (PA) was examined. Various parameters of the MTT assay were systematically investigated, including (i) the dissolution time of the formed formazan, (ii) the addition of glucose, and (iii) the optimal wavelength for spectrophotometric determination. Based on interim findings, a refined protocol suitable for application to nanofibrous materials was devised. We recommend 2 h of the dissolution, the application of glucose, and spectrophotometric measurement at 595 nm to obtain reliable data. Comparative analysis with the reference CFU counting protocol revealed similar trends for both tested bacteria and all tested nanomaterials. The proposed MTT protocol emerges as a suitable method for assessing the metabolic activity of bacterial biofilms on PCL, PLA, and PA nanofibrous materials.</p></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"224 ","pages":"Article 107010"},"PeriodicalIF":1.7,"publicationDate":"2024-08-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141889480","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}