Journal of microbiological methods最新文献

{"title":"Cell-free synthesis of the Salmonella specific broad host range bacteriophage, felixO1","authors":"John A. McFarlane ,&nbsp;David Garenne ,&nbsp;Vincent Noireaux ,&nbsp;Steven D. Bowden","doi":"10.1016/j.mimet.2024.106920","DOIUrl":"10.1016/j.mimet.2024.106920","url":null,"abstract":"<div><p>Phage-based biocontrol of foodborne <em>Salmonella</em> is limited by the requisite use of <em>Salmonella</em> to propagate the phages. This limitation can be circumvented by producing <em>Salmonella</em> phages using a cell-free gene expression system (CFE) with a non-pathogenic chassis. Here, we produce the <em>Salmonella</em> phage felixO1 using an <em>E. coli</em>-based CFE system.</p></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":null,"pages":null},"PeriodicalIF":2.2,"publicationDate":"2024-03-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140131694","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":"Factors affecting the quality and reproducibility of MALDI-TOF MS identification for human Capnocytophaga species","authors":"Ahmed Algahawi ,&nbsp;Inka Harju ,&nbsp;Eija Könönen ,&nbsp;Kaisu Rantakokko-Jalava ,&nbsp;Mervi Gürsoy","doi":"10.1016/j.mimet.2024.106911","DOIUrl":"10.1016/j.mimet.2024.106911","url":null,"abstract":"<div><p>Reproducibility and quality of MALDI-TOF MS spectra are critical in the identification process, however, information on the factors affecting the identification scores are scarce. Here, we studied the influence of various factors during the identification process of human oral <em>Capnocytophaga</em> species. The influence of two incubation times, plate-spotting reproducibility of two examiners, extraction technique, storage period of plates, and different laser repetition rates on the quality of MALDI-TOF MS identification of 34 human <em>Capnocytophaga</em> strains (including <em>C. gingivalis, C. granulosa, C. haemolytica, C. leadbetteri, C. ochracea, C. sputigena,</em> and <em>Capnocytophaga</em> genospecies AHN8471) was examined. The identification rate did not show a significant difference (<em>P</em> = 0.05) between the two incubation times, except that <em>C. haemolytica</em> needed a longer incubation time to be recognized at the genus level. The reproducibility of spotting between two examiners was ensured by following the manufacturer's instructions. At the species level, formic acid extraction improved the identification of species with limited representation in the database, such as <em>C. haemolytica</em> and <em>C. granulosa</em>. The storage of plates for one week decreased the identification scores. No significant difference (<em>P</em> = 0.39) was observed between the 60 Hz and 120 Hz laser repetition rates for identifying <em>Capnocytophaga</em> species to the genus or species level. In conclusion, the MALDI TOF MS offers a reliable <em>Capnocytophaga</em> identification after following the universal protocol, while the formic acid extraction is restricted to species with a limited number of strains in the database.</p></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":null,"pages":null},"PeriodicalIF":2.2,"publicationDate":"2024-03-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S016770122400023X/pdfft?md5=d309baea60ab954aed5c2116e2589bc3&pid=1-s2.0-S016770122400023X-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140068399","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":"Host specific Eimeria genus diagnosis and qPCR development in Ovis aries and Capra hircus","authors":"Shijie Li ,&nbsp;Yichen Jian ,&nbsp;Kaihui Zhang ,&nbsp;Xiaoying Li ,&nbsp;Rongjun Wang ,&nbsp;Longxian Zhang ,&nbsp;Fuchun Jian","doi":"10.1016/j.mimet.2024.106910","DOIUrl":"10.1016/j.mimet.2024.106910","url":null,"abstract":"<div><p>The objective of the present study was to develop a real-time PCR (qPCR) technique for the diagnosis of <em>Eimeria</em> spp. in <em>Ovis aries</em> and <em>Capra hircus</em>. The qPCR technique was developed using SYBR Green, resulting in a PCR with high sensitivity, specificity, and reproducibility.</p></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":null,"pages":null},"PeriodicalIF":2.2,"publicationDate":"2024-03-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140059646","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":"The performance of a Fully Automated Urine Particle Analyzer, Sysmex UF-5000, in detecting fastidious bacteria in urine samples","authors":"Masako Kaido ,&nbsp;Mitsuru Yasuda ,&nbsp;Masahiro Hayashi ,&nbsp;Hazuki Ohashi ,&nbsp;Hirotoshi Ohta ,&nbsp;Yasumasa Akai ,&nbsp;Kaori Tanaka ,&nbsp;Takashi Deguchi","doi":"10.1016/j.mimet.2024.106913","DOIUrl":"10.1016/j.mimet.2024.106913","url":null,"abstract":"<div><p>Several types of fastidious bacteria can cause tract infections. We evaluated the performance of counting fastidious bacteria using a Fully Automated Urine Particle Analyzer UF-5000. The results showed that UF-5000 counts fastidious bacteria in urine without the need for culture using measurement principles based on flow cytometry.</p></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":null,"pages":null},"PeriodicalIF":2.2,"publicationDate":"2024-03-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S0167701224000253/pdfft?md5=667b35a571b34772d1cc34d22b9d917c&pid=1-s2.0-S0167701224000253-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140065266","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":"Optimisation of DNA electroporation protocols for different plant-associated bacteria","authors":"Edson Yu Sin Kim ,&nbsp;Emanuel Maltempi de Souza,&nbsp;Marcelo Müller-Santos","doi":"10.1016/j.mimet.2024.106912","DOIUrl":"10.1016/j.mimet.2024.106912","url":null,"abstract":"<div><p>Electroporation is a vital process that facilitates the use of modern recombineering and other high-throughput techniques in a wide array of microorganisms, including non-model bacteria like plant growth-promoting bacteria (PGPB). These microorganisms play a significant role in plant health by colonizing plants and promoting growth through nutrient exchange and hormonal regulation. In this study, we introduce a sequential Design of Experiments (DOE) approach to obtain highly competent cells swiftly and reliably for electroporation. Our method focuses on optimizing the three stages of the electroporation procedure—preparing competent cells, applying the electric pulse field, and recovering transformed cells—separately. We utilized a split-plot fractional design with five factors and a covariate to optimize the first step, response surface methodology (RSM) for the second step, and Plackett-Burman design for two categorical factors and one continuous factor for the final step. Following the experimental sequence with three bacterial models, we achieved efficiencies 10 to 100 times higher, reaching orders of 10⁵ to 10⁶ CFU/μg of circular plasmid DNA. These results highlight the significant potential for enhancing electroporation protocols for non-model bacteria.</p></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":null,"pages":null},"PeriodicalIF":2.2,"publicationDate":"2024-03-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140059647","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Development of a multiplex droplet digital PCR assay for simultaneous detection and quantification of Escherichia coli, E. marmotae, and E. ruysiae in water samples","authors":"Marie Moinet ,&nbsp;Rose M. Collis ,&nbsp;Lynn Rogers ,&nbsp;Megan L. Devane ,&nbsp;Patrick J. Biggs ,&nbsp;Rebecca Stott ,&nbsp;Jonathan Marshall ,&nbsp;Richard Muirhead ,&nbsp;Adrian L. Cookson","doi":"10.1016/j.mimet.2024.106909","DOIUrl":"10.1016/j.mimet.2024.106909","url":null,"abstract":"<div><p><em>Escherichia coli</em> are widely used by water quality managers as Fecal Indicator Bacteria, but current quantification methods do not differentiate them from benign, environmental <em>Escherichia</em> species such as <em>E. marmotae</em> (formerly named cryptic clade V) or <em>E. ruysiae</em> (cryptic clades III and IV). Reliable and specific techniques for their identification are required to avoid confounding microbial water quality assessments. To address this, a multiplex droplet digital PCR (ddPCR) assay targeting <em>lipB</em> (<em>E. coli</em> and <em>E. ruysiae</em>) and <em>bglC</em> (<em>E. marmotae</em>) was designed. The ddPCR performance was assessed using <em>in silico</em> analysis; genomic DNA from 40 local, international, and reference strains of target and non-target coliforms; and spiked water samples in a range relevant to water quality managers (1 to 1000 cells/100 mL). Results were compared to an analogous quantitative PCR (qPCR) and the Colilert method. Both PCR assays showed excellent sensitivity with a limit of detection of 0.05 pg/μL and 0.005 pg/μl for ddPCR and qPCR respectively, and of quantification of 0.5 pg/μL of genomic DNA. The ddPCR allowed differentiation and quantification of three <em>Escherichia</em> species per run by amplitude multiplexing and showed a high concordance with concentrations measured by Colilert once proportional bias was accounted for. <em>In silico</em> specificity testing underlined the possibility to further detect and distinguish <em>Escherichia</em> cryptic clade VI. Finally, the applicability of the ddPCR was successfully tested on environmental water samples where <em>E. marmotae</em> and <em>E. ruysiae</em> potentially confound <em>E. coli</em> counts based on the Most Probable Number method, highlighting the utility of this novel ddPCR as an efficient and rapid discriminatory test to improve water quality assessments.</p></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":null,"pages":null},"PeriodicalIF":2.2,"publicationDate":"2024-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S0167701224000216/pdfft?md5=3f2081dc3f0481b6e2ee9b11ed082e93&pid=1-s2.0-S0167701224000216-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140022007","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":"Occurrence of Rhodococcus sp. RR1 prmA and Rhodococcus jostii RHA1 prmA across microbial communities and their enumeration during 1,4-dioxane biodegradation","authors":"Zohre Eshghdoostkhatami,&nbsp;Alison M. Cupples","doi":"10.1016/j.mimet.2024.106908","DOIUrl":"10.1016/j.mimet.2024.106908","url":null,"abstract":"<div><p>1,4-Dioxane, a likely human carcinogen, is a co-contaminant at many chlorinated solvent contaminated sites. Conventional treatment technologies, such as carbon sorption or air stripping, are largely ineffective, and so many researchers have explored bioremediation for site clean-up. An important step towards this involves examining the occurrence of the functional genes associated with 1,4-dioxane biodegradation. The current research explored potential biomarkers for 1,4-dioxane in three mixed microbial communities (wetland sediment, agricultural soil, impacted site sediment) using monooxygenase targeted amplicon sequencing, followed by quantitative PCR (qPCR). A BLAST analysis of the sequencing data detected only two of the genes previously associated with 1,4-dioxane metabolism or co-metabolism, namely propane monooxygenase (<em>prmA</em>) from <em>Rhodococcus jostii</em> RHA1 and <em>Rhodococcus</em> sp. RR1. To investigate this further, qPCR primers and probes were designed, and the assays were used to enumerate <em>prmA</em> gene copies in the three communities. Gene copies of <em>Rhodococcus</em> RR1 <em>prmA</em> were detected in all three, while gene copies of <em>Rhodococcus jostii</em> RHA1 <em>prmA</em> were detected in two of the three sample types (except impacted site sediment). Further, there was a statistically significant increase in RR1 <em>prmA</em> gene copies in the microcosms inoculated with impacted site sediment following 1,4-dioxane biodegradation compared to the control microcosms (no 1,4-dioxane) or to the initial copy numbers before incubation. Overall, the results indicate the importance of <em>Rhodococcus</em> associated <em>prmA</em>, compared to other 1,4-dioxane degrading associated biomarkers, in three different microbial communities. Also, the newly designed qPCR assays provide a platform for others to investigate 1,4-dioxane biodegradation potential in mixed communities and should be of particular interest to those considering bioremediation as a potential 1,4-dioxane remediation approach.</p></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":null,"pages":null},"PeriodicalIF":2.2,"publicationDate":"2024-02-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139948404","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":"Bacteria as a source of biopigments and their potential applications","authors":"Moitrayee Devi ,&nbsp;Elancheran Ramakrishnan ,&nbsp;Suresh Deka ,&nbsp;Deep Prakash Parasar","doi":"10.1016/j.mimet.2024.106907","DOIUrl":"10.1016/j.mimet.2024.106907","url":null,"abstract":"<div><p>From the prehistoric period, the utilization of pigments as colouring agents was an integral part of human life. Early people may have utilized paint for aesthetic motives, according to archaeologists. The pigments are either naturally derived or synthesized in the laboratory. Different studies reported that certain synthetic colouring compounds were toxic and had adverse health and environmental effects. Therefore, knowing the drawbacks of these synthetic colouring agents now scientists are attracted towards the harmless natural pigments. The main sources of natural pigments are plants, animals or microorganisms. Out of these natural pigments, microorganisms are the most important source for the production and application of bioactive secondary metabolites. Among all kinds of microorganisms, bacteria have specific benefits due to their short life cycle, low sensitivity to seasonal and climatic variations, ease of scaling, and ability to create pigments of various colours. Based on these physical characteristics, bacterial pigments appear to be a promising sector for novel biotechnological applications, ranging from functional food production to the development of new pharmaceuticals and biomedical therapies. This review summarizes the need for bacterial pigments, biosynthetic pathways of carotenoids and different applications of bacterial pigments.</p></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":null,"pages":null},"PeriodicalIF":2.2,"publicationDate":"2024-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139922427","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":"Fluorine materials scavenge excess carbon dioxide and promote Escherichia coli growth","authors":"Yoshihisa Yamashige ,&nbsp;Shojiro Kikuchi ,&nbsp;Ryosuke Hosoki ,&nbsp;Koji Kawada ,&nbsp;Katsuaki Izawa ,&nbsp;Masahiko Harata ,&nbsp;Yuichi Ogawa","doi":"10.1016/j.mimet.2024.106898","DOIUrl":"10.1016/j.mimet.2024.106898","url":null,"abstract":"<div><p>Fluorinated solvents have been used as oxygen carriers in closed microbial cultures to sustain aerobic conditions. However, the growth-promoting effects of fluorinated solvents remain unclear. Therefore, this study aimed to elucidate the mechanism by which fluorinated solvents promote microbial growth and to explore alternative materials that can be easily isolated after culture. <em>Escherichia coli</em> and HFE-7200, a fluorinated solvent, were used to explore factors other than oxygen released by fluorinated solvents that promote microbial growth. <em>E. coli</em> growth was promoted in gas-permeable cultures, and HFE-7200 alleviated medium acidification. Gas chromatography confirmed that HFE-7200 functioned as a scavenger of carbon dioxide produced by <em>E. coli</em> metabolism. Because fluorinated solvents can dissolve various gases, they could scavenge metabolically produced toxic gases from microbial cultures. Furthermore, using polytetrafluoroethylene, a solid fluorine material, results in enhanced bacterial growth. Such solid materials can be easily isolated and reused for microbial culture, suggesting their potential as valuable technologies in food production and biotechnology.</p></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":null,"pages":null},"PeriodicalIF":2.2,"publicationDate":"2024-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139741224","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":"Enhancing sensitivity of qPCR assays targeting Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae by using a mutant Taq DNA polymerase","authors":"Selin Nar Otgun ,&nbsp;Canan Zohre Ketre Kolukirik ,&nbsp;Nuriye Unal Sahin ,&nbsp;Mustafa Kolukirik ,&nbsp;Gozde Girgin Ozgumus ,&nbsp;Meral Turan ,&nbsp;Mert Elmas ,&nbsp;Selcuk Kilic","doi":"10.1016/j.mimet.2024.106899","DOIUrl":"https://doi.org/10.1016/j.mimet.2024.106899","url":null,"abstract":"<div><h3>Aims</h3><p><em>Streptococcus pneumoniae</em>, <em>Neisseria meningitidis</em> and <em>Haemophilus influenzae</em> are important causes of bacterial meningitis. In this study, the DNA binding site of the wild type Taq DNA polymerase was modified to produce a mutant enzyme with enhanced DNA affinity and PCR performance. The engineered and the wild type enzymes were integrated into qPCR-based assays for molecular detection of <em>S. pneumoniae</em>, <em>N. meningitidis</em>, <em>H. influenzae</em>, and serogroups and serotypes of these three pathogens.</p></div><div><h3>Methods</h3><p>Bio-Speedy® Bacterial DNA Isolation Kit (Bioeksen R&amp;D Technologies, Turkiye) and 2× qPCR-Mix for hydrolysis probes (Bioeksen R&amp;D Technologies, Turkiye) and CFX96 Instrument (Biorad Inc., USA) were used for all molecular analyses. Spiked negative clinical specimens were tested using the developed qPCR assays and the culture-based conventional methods for the analytical performance evaluation.</p></div><div><h3>Results</h3><p>All qPCR assays did not produce any positive results for the samples spiked with potential cross-reacting bacteria. Limit of detection (LOD) of the assays containing the mutant enzyme was 1 genome/reaction (10 cfu/mL sample) which is at least 3 times lower than the previously reported LOD levels for DNA amplification based molecular assays. LODs for the spiked serum and cerebrospinal fluid (CSF) samples decreased 2.3–4.7 and 1.2–3.5 times respectively when the mutant enzyme was used instead of the wild type Taq DNA polymerase.</p></div><div><h3>Conclusions</h3><p>It is possible to enhance analytical sensitivity of qPCR assays targeting the bacterial agents of meningitis by using an engineered Taq DNA polymerase. These qPCR-based assays can be used for direct detection and serogrouping / serotyping of <em>S. pneumoniae</em>, <em>N. meningitidis</em> and <em>H. influenzae</em> at concentrations close to the lower limit of medical decision point.</p></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":null,"pages":null},"PeriodicalIF":2.2,"publicationDate":"2024-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139738556","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}