Journal of microbiological methods最新文献

{"title":"Microbial DNA sample preservation and possible artifacts for field-based research in remote tropical peatlands","authors":"Mark C. Reynolds ,&nbsp;Hinsby Cadillo-Quiroz","doi":"10.1016/j.mimet.2024.106997","DOIUrl":"10.1016/j.mimet.2024.106997","url":null,"abstract":"<div><p>Surveying bacterial and archaeal microbial communities in host and environmental studies requires the collection and storage of samples. Many studies are conducted in distant locations challenging these prerequisites. The use of preserving buffers is an important alternative when lacking access to cryopreservation, however, its effectivity for samples with challenging chemistry or samples that provide opportunities for fast bacterial or archaeal growth upon exposure to an aerobic environment, like peat samples, requires methodological assessment. Here, in combination with an identified optimal DNA extraction kit for peat soil samples, we test the application of several commercial and a homemade preservation buffer and make recommendations on the method that can most effectively preserve a microbiome reflective of the original state. In treatments with a non-optimal buffer or in the absence, we observed notable community shifts beginning as early as three days post-preservation lowering diversity and community evenness, with growth-driven artifacts from a few specific phyla. However other buffers retain a very close composition relative to the original state, and we described several metrics to understand some variation across them. Due to the chemical effects of preservation buffers, it is critical to test their compatibility and reliability to preserve the original bacterial and archaeal community in different environments.</p></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"224 ","pages":"Article 106997"},"PeriodicalIF":1.7,"publicationDate":"2024-07-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141620137","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":"Gene function characterization in Aspergillus niger using a dual resistance marker transformation system mediated by Agrobacterium tumefaciens","authors":"Hanh-Dung Thai ,&nbsp;Minh Thi Trinh ,&nbsp;Loc Thi Binh Xuan Do ,&nbsp;Thu-Hang Le ,&nbsp;Duc-Thanh Nguyen ,&nbsp;Que Thi Tran ,&nbsp;Van-Khanh Tong Tran ,&nbsp;Linh Thi Dam Mai ,&nbsp;Duc-Ngoc Pham ,&nbsp;Diep Hong Le ,&nbsp;Tao Xuan Vu ,&nbsp;Van-Tuan Tran","doi":"10.1016/j.mimet.2024.106989","DOIUrl":"10.1016/j.mimet.2024.106989","url":null,"abstract":"<div><p><em>Aspergillus niger</em> is a well-known workhorse for the industrial production of enzymes and organic acids. This fungus can also cause postharvest diseases in fruits. Although <em>Agrobacterium tumefaciens-</em>mediated transformation (ATMT) based on antibiotic resistance markers has been effectively exploited for inspecting functions of target genes in wild-type fungi, it still needs to be further improved in <em>A. niger</em>. In the present study, we re-examined the ATMT in the wild-type <em>A. niger</em> strains using the hygromycin resistance marker and introduced the nourseothricin resistance gene as a new selection marker for this fungus. Unexpectedly, our results revealed that the ATMT method using the resistance markers in <em>A. niger</em> led to numerous small colonies as false-positive transformants on transformation plates. Using the top agar overlay technique to restrict false positive colonies, a transformation efficiency of 87 ± 18 true transformants could be achieved for 10⁶ conidia. With two different selection markers, we could perform both the deletion and complementation of a target gene in a single wild-type <em>A. niger</em> strain. Our results also indicated that two key regulatory genes (<em>laeA</em> and <em>veA</em>) of the velvet complex are required for <em>A. niger</em> to infect apple fruits. Notably, we demonstrated for the first time that a <em>laeA</em> homologous gene from the citrus postharvest pathogen <em>Penicillium digitatum</em> was able to restore the acidification ability and pathogenicity of the <em>A. niger</em> Δ<em>laeA</em> mutant. The dual resistance marker ATMT system from our work represents an improved genetic tool for gene function characterization in <em>A. niger</em>.</p></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"224 ","pages":"Article 106989"},"PeriodicalIF":1.7,"publicationDate":"2024-07-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141600206","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 the diversity of anaerobic-cultured gut bacterial communities on different culture media using 16S rDNA sequencing","authors":"Anne Sophie Lichtenegger ,&nbsp;Sara Posadas-Cantera ,&nbsp;Mohamed Tarek Badr ,&nbsp;Georg Häcker","doi":"10.1016/j.mimet.2024.106988","DOIUrl":"10.1016/j.mimet.2024.106988","url":null,"abstract":"<div><p>The gut microbiome is a dense and diverse community of different microorganisms that deeply influence human physiology and that have important interactions with pathogens. For the correct antibiotic treatment of infections, with its twin goals of effective inhibition of the pathogen and limitation of collateral damage to the microbiome, the identification of infectious organisms is key. Microbiological culturing is still the mainstay of pathogen identification, and anaerobic species are among the most demanding bacterial communities to culture. This study aimed to evaluate the impact of growth media on the culture of an-aerobic bacteria from human stool samples. Stool samples from eight human subjects were cultured each on a yeast extract cysteine blood agar (HCB) and modified peptone-yeast extract-glucose (MPYG) plate and subjected to Illumina NGS analysis after DNA extraction and amplification. The results showed tight clustering of sequencing samples belonging to the same human subject. Various differences in bacterial richness and evenness could be observed between the two media, with HCB plates supporting the growth of a more diverse microbial community, and MPYG plates improving the growth rates of certain taxa. No statistical significance was observed between the groups. This study highlights the importance of choosing the appropriate growth media for anaerobic bacterial culture and adjusting culture conditions to target specific pathological conditions. HCB plates are suitable for standard microbiological diagnostics, while MPYG plates may be more appropriate for targeting specific conditions. This work emphasizes the role of next-generation sequencing in supporting future research in clinical microbiology.</p></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"224 ","pages":"Article 106988"},"PeriodicalIF":1.7,"publicationDate":"2024-07-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S0167701224001003/pdf?md5=edef7b67343c37d7bdd2fc361eed9178&pid=1-s2.0-S0167701224001003-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141558904","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":"Inhibitors of LAMP used to detect Tenacibaculum sp. strain Pbs-1 associated with black-spot shell disease in Akoya pearl oysters, and additives to reduce the effect of the inhibitors","authors":"Akihiro Sakatoku ,&nbsp;Takaya Suzuki ,&nbsp;Kaito Hatano ,&nbsp;Makoto Seki ,&nbsp;Daisuke Tanaka ,&nbsp;Shogo Nakamura ,&nbsp;Nobuo Suzuki ,&nbsp;Tadashi Isshiki","doi":"10.1016/j.mimet.2024.106986","DOIUrl":"10.1016/j.mimet.2024.106986","url":null,"abstract":"<div><p>Black-spot shell disease is an unresolved disease that decreases pearl quality and threatens pearl oyster survival. In previous studies, the bacterium <em>Tenacibaculum</em> sp. strain Pbs-1 was isolated from diseased Akoya pearl oysters <em>Pinctada fucata</em>, and a rapid, specific, and sensitive loop-mediated isothermal amplification (LAMP) assay for detecting this pathogen was established. This technology has considerable potential for routine diagnosis of strain Pbs-1 in oyster hatcheries and/or pearl farms; therefore, it is vital to identify substances in environmental samples that might inhibit LAMP and to find additives that can reduce the inhibition. In this study, we investigated the effects of six chemicals or proteins, otherwise known as conventional PCR inhibitors, on LAMP, using the DNA of strain Pbs-1 as template: humic acid, urea, iron (III) chloride hexahydrate, melanin, myoglobin, and Ethylenediamine-N,N,N′,N′-tetraacetic acid, disodium salt, dihydrate (EDTA; pH 6.5). Next, to reduce the effects of identified inhibitors, we tested the addition of bovine serum albumin (BSA) or T4 gene 32 protein (gp32) to the LAMP assay. When 50 ng of DNA template was used, 4 ng/μL of humic acid, 0.05% melanin, and 10 mM of EDTA (pH 6.5) inhibited the LAMP reaction, whereas myoglobin, urea, and FeCl₃ had no effect. When 50 pg of DNA template was used, 4 ng/μL of humic acid, 0.05% melanin, 4 μg/μL of myoglobin, 10 μg/μL of urea, and 10 mM of EDTA inhibited the LAMP reaction. Thus, it was shown that the gene-amplification inhibitory effect of melanin, humic acid, and urea could be reduced by adding BSA or gp32 to the LAMP reaction mixture. This technique could be applied as part of a protocol to prevent mass mortalities of pearl oysters; moreover, the results enhance our knowledge about substances that inhibit LAMP and methods to reduce the inhibition, which have rarely been reported.</p></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"223 ","pages":"Article 106986"},"PeriodicalIF":1.7,"publicationDate":"2024-07-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S0167701224000988/pdfft?md5=913c03ade9f3c54d6da2454f7a5f4e68&pid=1-s2.0-S0167701224000988-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141537900","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":"Interrelationship among substrate utilization, metabolic productions, and housekeeping-related gene expression levels in Mortierella alpine CBS 754.68","authors":"Hamid Reza Samadlouie ,&nbsp;Shahrokh Gharanjik ,&nbsp;Abdolah Vatandost ,&nbsp;Side Maryam Ghasemi Tarvigi","doi":"10.1016/j.mimet.2024.106987","DOIUrl":"10.1016/j.mimet.2024.106987","url":null,"abstract":"<div><p>The impacts of Magnesium oxide nanoparticles (MgONPs) on the expression of 10 potential housekeeping genes of <em>Mortierella alpine</em> were assayed. Actin emerged as the good candidate when <em>Mortierella alpine</em> entered the death phase subsequent to the growth phase while Dihydropteridine reductase and 28 s were identified as suitable candidates when <em>Mortierella alpine</em> remained in the growth phase.</p></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"223 ","pages":"Article 106987"},"PeriodicalIF":1.7,"publicationDate":"2024-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141498242","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":"Long-term storage stability of PGL-I coated ELISA plates for anti-Mycobacterium leprae IgM detection","authors":"Matheus Bernardes Torres Fogaça ,&nbsp;Djairo Pastor Saavedra ,&nbsp;Maria Beatris de Jesus Sousa ,&nbsp;Nicolle Kathlen Alves Belém de Oliveira ,&nbsp;Mariane Martins de Araújo Stefani ,&nbsp;Samira Buhrer-Sékula","doi":"10.1016/j.mimet.2024.106985","DOIUrl":"10.1016/j.mimet.2024.106985","url":null,"abstract":"<div><p>The assessment of ELISA plates coated with phenolic glycolipid-I/PGL-I revealed excellent stability during eight years of storage at room temperature, promoting consistent IgM antibody detection in multibacillary leprosy patients. These stable, standardized plates can significantly contribute to efficient leprosy serology research and support its widespread distribution and use in endemic countries.</p></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"224 ","pages":"Article 106985"},"PeriodicalIF":1.7,"publicationDate":"2024-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141498243","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":"Alternative protocol leading to rapid identification of Actinomycetes isolated from Algerian desertic soil by MALDI-TOF mass spectrometry","authors":"Sarra Benhasna ,&nbsp;Allaoueddine Boudemagh","doi":"10.1016/j.mimet.2024.106984","DOIUrl":"10.1016/j.mimet.2024.106984","url":null,"abstract":"<div><p>Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is the first-line method for the rapid identification of most cultured microorganisms. As for <em>Streptomyces</em> strains, MALDI-TOF MS identification is complicated by the characteristic incrustation of colonies in agar and the strong cell wall of <em>Actinomycetes</em> cells requiring the use of alternative protein extraction protocols. In this study, we developed a specific protocol to overcome these difficulties for the MALDI-TOF MS identification of <em>Actinomycetes</em> made on solid medium. This protocol includes incubation of colony removed from agar plate with the beta-agarase enzyme, followed by a mechanical lysis and two washes by phosphate buffer and ethanol. Twenty-four <em>Streptomyces</em> and two <em>Lentzea</em> strains isolated from Algerian desertic soils were first identified by 16S rRNA sequencing as gold standard method, <em>rpoB</em> gene was used as a secondary gene target when 16S rRNA did not allow species identification. In parallel the isolates were identified by using the MALDI-TOF MS protocol as reported. After the expansion of the database with the inclusion of this MSP_S, the strains were analyzed again in MALDI Biotyper, and all were identified. This work demonstrates that the rapid identification of <em>Actinomycetes</em> can be obtained without protein extraction step frequently used in MALDI-TOF mass spectrometry with this type of microorganisms.</p></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"223 ","pages":"Article 106984"},"PeriodicalIF":1.7,"publicationDate":"2024-06-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141492253","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":"Colorimetric strategies applicable for loop-mediated isothermal amplification","authors":"Syaidatul Akmal Saifuddin ,&nbsp;Roslina Rashid ,&nbsp;Nurin Jazlina Nor Azmi,&nbsp;Suharni Mohamad","doi":"10.1016/j.mimet.2024.106981","DOIUrl":"10.1016/j.mimet.2024.106981","url":null,"abstract":"<div><p>In recent years, loop-mediated isothermal amplification (LAMP) has gained popularity for detecting various pathogen-specific genes due to its superior sensitivity and specificity compared to conventional polymerase chain reaction (PCR). The simplicity and flexibility of naked-eye detection of the amplicon make LAMP an ideal rapid and straightforward diagnostic tool, especially in resource-limited laboratories. Colorimetric detection is one of the simplest and most straightforward among all detection methods. This review will explore various colorimetric dyes used in LAMP techniques, examining their reaction mechanisms, advantages, limitations and latest applications.</p></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"223 ","pages":"Article 106981"},"PeriodicalIF":1.7,"publicationDate":"2024-06-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141468688","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":"Applicability of non-invasive and live-cell holotomographic imaging on fungi","authors":"Susanne Fritsche ,&nbsp;Felix Fronek ,&nbsp;Robert L. Mach ,&nbsp;Matthias G. Steiger","doi":"10.1016/j.mimet.2024.106983","DOIUrl":"10.1016/j.mimet.2024.106983","url":null,"abstract":"<div><p>The ability to acquire three-dimensional (3D) information of cellular structures without the need for fluorescent tags or staining makes holotomographic imaging a powerful tool in cellular biology. It provides valuable insights by measuring the refractive index (RI), an optical parameter describing the phase delay of light that passes through the living cell.</p><p>Here, we demonstrate holotomographic imaging on industrial relevant ascomycete fungi and study their development and morphogenesis. This includes conidial germination, subcellular dynamics, and cytoplasmic flow during hyphal growth in <em>Aspergillus niger.</em> In addition, growth and budding of <em>Aureobasidium pullulans</em> cells are captured using holotomographic microscopy. Coupled to fluorescence imaging, lipid droplets, vacuoles, the mitochondrial network, and nuclei are targeted and analyzed in the 3D RI reconstructed images. While lipid droplets and vacuoles can be assigned to a specific RI pattern, mitochondria and nuclei were not pronounced. We show, that the lower sensitivity of RI measurements derives from the fungal cell wall that acts as an additional barrier for the illumination light of the microscope. After cell wall digest of hyphae and protoplast formation of <em>A. niger</em> expressing GFP-tagged histone H2A, location of nuclei could be determined by non-invasive RI measurements. Furthermore, we used coupled fluorescence microscopy to observe migration of nuclei in unperturbed hyphal segments and duplication during growth on a single-cell level. Detailed micromorphological studies in <em>Saccharomyces cerevisiae</em> and <em>Trichoderma reesei</em> are challenging due to cell size restrictions.</p><p>Overall, holotomography opens up new avenues for exploring dynamic cellular processes in real time and enables the visualization of fungi from a new perspective.</p></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"224 ","pages":"Article 106983"},"PeriodicalIF":1.7,"publicationDate":"2024-06-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141468687","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":"Quantification of early biofilm growth in microtiter plates through a novel image analysis software","authors":"Anderson J. Castilla-Sedano ,&nbsp;José Zapana-García ,&nbsp;Erika Valdivia-Del Águila ,&nbsp;Pierre G. Padilla-Huamantinco ,&nbsp;Daniel G. Guerra","doi":"10.1016/j.mimet.2024.106979","DOIUrl":"10.1016/j.mimet.2024.106979","url":null,"abstract":"<div><p>Given the significant impact of biofilms on human health and material corrosion, research in this field urgently needs more accessible techniques to facilitate the testing of new control agents and general understanding of biofilm biology. Microtiter plates offer a convenient format for standardized evaluations, including high-throughput assays of alternative treatments and molecular modulators. This study introduces a novel Biofilm Analysis Software (BAS) for quantifying biofilms from microtiter plate images. We focused on early biofilm growth stages and compared BAS quantification to common techniques: direct turbidity measurement, intrinsic fluorescence detection linked to pyoverdine production, and standard crystal violet staining which enables image analysis and optical density measurement. We also assessed their sensitivity for detecting subtle growth effects caused by cyclic AMP and gentamicin. Our results show that BAS image analysis is at least as sensitive as the standard method of spectrophotometrically quantifying the crystal violet retained by biofilms. Furthermore, we demonstrated that bacteria adhered after short incubations (from 10 min to 4 h), isolated from planktonic populations by a simple rinse, can be monitored until their growth is detectable by intrinsic fluorescence, BAS analysis, or resolubilized crystal violet. These procedures are widely accessible for many laboratories, including those with limited resources, as they do not require a spectrophotometer or other specialized equipment.</p></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"223 ","pages":"Article 106979"},"PeriodicalIF":1.7,"publicationDate":"2024-06-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141468693","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}