Journal of microbiological methods最新文献

{"title":"Rapid detection of plasmid-mediated AmpC-producers by eazyplex® SuperBug AmpC assay compared to whole-genome sequencing","authors":"Vladimira Hinić ,&nbsp;Helena M.B. Seth-Smith ,&nbsp;Sabrina Stammler ,&nbsp;Adrian Egli","doi":"10.1016/j.mimet.2024.106938","DOIUrl":"https://doi.org/10.1016/j.mimet.2024.106938","url":null,"abstract":"<div><p>Current methods for plasmid-mediated AmpC β-lactamase (pAmpC) detection in routine microbiological laboratories are based on various phenotypic tests. Eazyplex®SuperBug AmpC assay is a molecular assay based on isothermal amplification for rapid detection of the most common pAmpC types from bacterial culture: CMY-2 group, DHA, ACC and MOX. Our aim was to evaluate the diagnostic performance of this assay. The assay was evaluated on 64 clinical isolates of <em>Enterobacterales</em> without chromosomal inducible AmpC, and with phenotypically confirmed AmpC production. The results were confirmed, and isolates further characterized by whole-genome sequencing (WGS). eazyplex®SuperBug AmpC assay correctly detected the two most common pAmpC types CMY-2 group (16/16) and DHA (19/19). Detection of ACC and MOX could not be evaluated on our set of isolates since there was only one isolate harbouring ACC and none with MOX. pAmpC encoding genes could be detected in only eight of 36 investigated <em>Escherichia coli</em> isolates. The remaining 28 <em>E. coli</em> isolates harboured previously described mutations in the <em>bla</em>_EC promoter, leading to the overexpression of chromosomally encoded <em>E. coli</em> specific AmpC β-lactamase. All results were 100% concordant with the results of WGS. eazyplex®SuperBug AmpC assay enabled rapid and reliable detection of pAmpC-encoding genes in <em>Enterobacterales</em> like <em>Klebsiella</em> spp. and <em>Proteus</em> spp. and the distinction between plasmid-mediated and chromosomally encoded AmpC in <em>E. coli</em>.</p></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":null,"pages":null},"PeriodicalIF":2.2,"publicationDate":"2024-04-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S0167701224000502/pdfft?md5=359ef7bb4baec30073f17ce68541c43e&pid=1-s2.0-S0167701224000502-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140632913","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":"Comparative evaluation of analytical pipelines for illumina short- and nanopore long-read 16S rRNA gene amplicon sequencing with mock microbial communities","authors":"Yusuke Ota ,&nbsp;Kei Yasunaga ,&nbsp;Samiratu Mahazu ,&nbsp;Isaac Prah ,&nbsp;Satoshi Nagai ,&nbsp;Takaya Hayashi ,&nbsp;Masato Suzuki ,&nbsp;Mitsunori Yoshida ,&nbsp;Yoshihiko Hoshino ,&nbsp;Yukihiro Akeda ,&nbsp;Toshihiko Suzuki ,&nbsp;Yoshiaki Gu ,&nbsp;Ryoichi Saito","doi":"10.1016/j.mimet.2024.106929","DOIUrl":"https://doi.org/10.1016/j.mimet.2024.106929","url":null,"abstract":"<div><p>Utility of a recently developed long-read pipeline, Emu, was assessed using an expectation-maximization algorithm for accurate read classification. We compared it to conventional short- and long-read pipelines, using well-characterized mock bacterial samples. Our findings highlight the necessity of appropriate data-processing for taxonomic descriptions, expanding our understanding of the precise microbiome.</p></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":null,"pages":null},"PeriodicalIF":2.2,"publicationDate":"2024-04-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140548973","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 flexible ‘plug and play’ bicistronic construct and its application in the screening of protein expression system in Escherichia coli","authors":"Binbin Feng,&nbsp;Weiwei Hu,&nbsp;Yingyi Duan,&nbsp;Zhaopeng Li","doi":"10.1016/j.mimet.2024.106928","DOIUrl":"https://doi.org/10.1016/j.mimet.2024.106928","url":null,"abstract":"<div><p>The bicistronic expression system that utilizes fluorescent reporters has been demonstrated to be a straightforward method for detecting recombinant protein expression levels, particularly when compared to polyacrylamide gel electrophoresis and immunoblot analysis, which are tedious and labor-intensive. However, existing bicistronic reporter systems are less capable of quantitative measurement due to the lag in reporter expression and its negative impact on target protein. In this work, a plug and play bicistronic construct using mCherry as reporter was applied in the screening of optimal replicon and promoter for Sortase expression in <em>Escherichia coli</em> (<em>E. coli</em>). The bicistronic construct allowed the reporter gene and target open reading frame (ORF) to be co-transcribed under the same promoter, resulting in a highly positive quantitative correlation between the expression titer of Sortase and the fluorescent intensity (R² &gt; 0.97). With the correlation model, the titer of target protein can be quantified by noninvasively measuring the fluorescent intensity. On top of this, the expression of reporter has no significant effect on the yield of target protein, thus favoring a plug and play design for removing reporter gene to generate a plain plasmid for industrial use.</p></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":null,"pages":null},"PeriodicalIF":2.2,"publicationDate":"2024-04-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140545955","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":"Proteus mirabilis biofilm expansion microscopy yields over 4-fold magnification for super-resolution of biofilm structure and subcellular DNA organization","authors":"Dante Castagnini ,&nbsp;Karina Palma ,&nbsp;Jorge Jara-Wilde ,&nbsp;Nicolás Navarro ,&nbsp;María José González ,&nbsp;Jorge Toledo ,&nbsp;Nicole Canales-Huerta ,&nbsp;Paola Scavone ,&nbsp;Steffen Härtel","doi":"10.1016/j.mimet.2024.106927","DOIUrl":"https://doi.org/10.1016/j.mimet.2024.106927","url":null,"abstract":"<div><p>Bacterial biofilms form when bacteria attach to surfaces and generate an extracellular matrix that embeds and stabilizes a growing community. Detailed visualization and quantitative analysis of biofilm architecture by optical microscopy are limited by the law of diffraction. Expansion Microscopy (ExM) is a novel Super-Resolution technique where specimens are physically enlarged by a factor of ∼4, prior to observation by conventional fluorescence microscopy. ExM requires homogenization of rigid constituents of biological components by enzymatic digestion. We developed an ExM approach capable of expanding 48-h old <em>Proteus mirabilis</em> biofilms 4.3-fold (termed PmbExM), close to the theoretic maximum expansion factor without gross shape distortions. Our protocol, based on lytic and glycoside-hydrolase enzymatic treatments, degrades rigid components in bacteria and extracellular matrix. Our results prove PmbExM to be a versatile and easy-to-use Super-Resolution approach for enabling studies of <em>P. mirabilis</em> biofilm architecture, assembly, and even intracellular features, such as DNA organization.</p></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":null,"pages":null},"PeriodicalIF":2.2,"publicationDate":"2024-03-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140332535","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":"Single primer site-specific nested PCR for accurate and rapid genome-walking","authors":"Xinyue Guo ,&nbsp;Yisong Zhu ,&nbsp;Zhenkang Pan ,&nbsp;Hao Pan ,&nbsp;Haixing Li","doi":"10.1016/j.mimet.2024.106926","DOIUrl":"10.1016/j.mimet.2024.106926","url":null,"abstract":"<div><p>Genome-walking is a molecular tool used to unveil uncharacterized DNA regions flanking a known DNA, which has been widely used in bioscience and related areas. This study developed a reliable and efficient PCR-based genome-walking approach, named as single primer site-specific nested PCR (SPN-PCR). A SPN-PCR set sequentially consists of three single-primer nested PCR amplifications. The primary relaxed thermal cycle promotes outmost nested site-specific primer (NSSP) to partially combine with numerous places on DNA template, synthesizing many single-stranded DNAs (ssDNA). Among them, the target ssDNA is exponentially amplified in the subsequent stringent cycles, as its 3′ part possesses the outmost NSSP complement; but a non-target ssDNA cannot be amplified, because it does not possess such a complement. Stringent secondary and tertiary PCRs also exclusively enrich this target DNA. Finally, the target DNA product becomes predominant. The feasibility of SPN-PCR was validated by genome-walking several selected genes from two divergent species.</p></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":null,"pages":null},"PeriodicalIF":2.2,"publicationDate":"2024-03-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140329895","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":"Diagnosis of abdominal tuberculosis: Detection of mycobacterial CFP-10 and HspX proteins by gold nanoparticle-PCR amplified immunoassay","authors":"Bhawna Dahiya ,&nbsp;Preeti Mor ,&nbsp;Anam Rais ,&nbsp;Tulika Prasad ,&nbsp;Abhishek Sheoran ,&nbsp;Reetu Sheoran ,&nbsp;Suman Sharma ,&nbsp;Mahesh K. Seth ,&nbsp;Sunil K. Srivastava ,&nbsp;Promod K. Mehta","doi":"10.1016/j.mimet.2024.106925","DOIUrl":"10.1016/j.mimet.2024.106925","url":null,"abstract":"<div><p>Attempts were made to improve the efficacy of PCR amplified immunoassay (I-PCR) for diagnosing abdominal TB cases by utilizing the gold nanoparticle (AuNP)-based I-PCR, where AuNPs were functionalized with detection antibodies/oligonucleotides that exhibited 84.3% sensitivity and 95.1% specificity. This assay would improve the ongoing algorithms used in abdominal TB diagnosis.</p></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":null,"pages":null},"PeriodicalIF":2.2,"publicationDate":"2024-03-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140326688","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 knock-out in Mycobacterium abscessus using Streptococcus thermophilus CRISPR/Cas","authors":"Suriya Akter ,&nbsp;Elisabeth Kamal ,&nbsp;Carsten Schwarz ,&nbsp;Astrid Lewin","doi":"10.1016/j.mimet.2024.106924","DOIUrl":"10.1016/j.mimet.2024.106924","url":null,"abstract":"<div><p>The CRISPRi system using dCas9_Sth1 from <em>Streptococcus thermophilus</em> developed for <em>Mycobacterium tuberculosis</em> and <em>M. smegmatis</em> was modified to allow gene knock-out in <em>M. abscessus</em>. Efficacy of the knock-out system was evaluated by applying deletions and insertions to the <em>mps1</em> gene. A comparative genomic analysis of mutants and wild type validated the target specificity.</p></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":null,"pages":null},"PeriodicalIF":2.2,"publicationDate":"2024-03-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S0167701224000368/pdfft?md5=19f8de3f3f2a514d976ca77fd80869a2&pid=1-s2.0-S0167701224000368-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140318506","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":"Braided silk sutures coated with photoreduced silver nanoparticles for eradicating Staphylococcus aureus and Streptococcus mutans infections","authors":"Shilpa Mathew ,&nbsp;K. Vijaya Kumar ,&nbsp;Ashwini Prabhu ,&nbsp;Rajesh P. Shastry ,&nbsp;K.S. Rajesh","doi":"10.1016/j.mimet.2024.106923","DOIUrl":"10.1016/j.mimet.2024.106923","url":null,"abstract":"<div><h3>Background</h3><p>Infections resulting from surgical procedures and wound closures continue to pose significant challenges in healthcare settings. To address this issue, the investigators have developed antibacterial non-resorbable braided silk sutures using <em>in situ</em> deposited silver nanoparticles (AgNPs) and investigated their efficacy in eradicating <em>Staphylococcus aureus</em> and <em>Streptococcus mutans</em> infections.</p></div><div><h3>Methods</h3><p>The braided silk sutures were modified through a simple and efficient <em>in situ</em> photoreduction method, resulting in the uniform distribution of AgNPs along the suture surface. The synthesized AgNPs were characterized using scanning electron microscopy (SEM), dynamic light scattering analysis (DLS) and Fourier Transform Infrared Spectroscopy analysis (FTIR) confirming their successful integration onto the silk sutures. The antibacterial activity of the nanoparticle coated sutures were compared and evaluated with non-coated braided silk sutures through <em>in vitro</em> assays against both <em>S. aureus</em> and <em>S. mutans</em>.</p></div><div><h3>Results</h3><p>The surface and cross-sectional analysis of the treated sutures revealed a uniform and homogeneous distribution of silver particles achieved through the photoreduction of silver solution. This observation confirms the successful coating of silver nanoparticles (AgNPs) on the sutures. The antimicrobial studies conducted, demonstrated significant reductions in bacterial colonies when exposed to the silver nanoparticle-coated sutures. Notably, the width of the inhibition zone surrounding the coated sutures remained consistently wide and stable for duration up to 7 days. This sustained and robust inhibitory effect against gram-positive bacteria, specifically <em>S. aureus</em> and <em>S. mutans</em>, serves as strong evidence of the antibacterial efficacy of the coated sutures.</p></div><div><h3>Conclusion</h3><p>The coating of silk sutures with AgNPs provided a significant and effective antibacterial capacity to the surgical sutures, with this activity being sustained for a period of 7 days. This suggests that AgNPs-<em>in situ</em> photoreduction deposited sutures have the potential to effectively manage <em>S. aureus</em> and <em>S. mutans</em> infections.</p></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":null,"pages":null},"PeriodicalIF":2.2,"publicationDate":"2024-03-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140193959","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 closed-tube, calcein-based loop-mediated isothermal amplification assay to detect Salmonella spp. in raw meat samples","authors":"Khristine B. Balaga ,&nbsp;Rance Derrick N. Pavon ,&nbsp;Alyzza Marie B. Calayag,&nbsp;Christine Aubrey C. Justo,&nbsp;Davin Edric V. Adao,&nbsp;Windell L. Rivera","doi":"10.1016/j.mimet.2024.106922","DOIUrl":"10.1016/j.mimet.2024.106922","url":null,"abstract":"<div><p>Foodborne pathogens compromise food safety and public health, and <em>Salmonella</em> spp. are among the major pathogenic bacteria that cause outbreaks worldwide. Proper surveillance through timely and cost-effective detection methods across the food animal production chain is crucial to prevent <em>Salmonella</em> outbreaks and agricultural losses. Traditional culture methods are labor- and resource-intensive, with lengthy turnaround times. Meanwhile, conventional molecular tools, such as PCR and qPCR, are expensive and require technical skills and equipment. Loop-mediated isothermal amplification (LAMP) is a simple, rapid, inexpensive, highly sensitive, and specific molecular assay that does not require expensive equipment. Hence, this study developed and optimized a closed-tube, calcein-based LAMP assay to detect <em>Salmonella</em> using the <em>invA</em> gene and performed evaluation and validation against conventional PCR. The LAMP assay showed high specificity and sensitivity. It showed 10-fold higher sensitivity than conventional PCR, at &lt;1 ng/μL DNA concentrations. Meanwhile, for CFU/mL, LAMP assay showed 1000-fold higher sensitivity than conventional PCR at 4.8 × 10³ cells/mL than 4.8 × 10⁷ cells/mL, respectively. For parallel testing of 341 raw meat samples, after conventional culture enrichment (until Rappaport-Vassiliadis broth), the optimized LAMP assay showed 100% detection on all samples while conventional PCR showed 100%, 99.04%, and 96.64% for raw chicken, beef, and pork samples, respectively. Meanwhile, a shortened enrichment protocol involving 3-h incubation in buffered peptone water only, showed lower accuracy in tandem with the optimized LAMP assay ranging from 55 to 75% positivity rates among samples. These suggest that the optimized LAMP assay possesses higher sensitivity over conventional PCR for <em>invA</em> gene detection when coupled with conventional enrichment culture methods. Hence, this assay has potential as a powerful complementary or alternative <em>Salmonella</em> detection method to increase surveillance capacity and protect consumer food safety and public health worldwide.</p></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":null,"pages":null},"PeriodicalIF":2.2,"publicationDate":"2024-03-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140184655","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":"Comparing DNA isolation and sequencing strategies for 16S rRNA gene amplicon analysis in biofilm containing environments","authors":"Ilgaz Cakin ,&nbsp;Barbara Morrissey ,&nbsp;Matthew Gordon ,&nbsp;Paul P.J. Gaffney ,&nbsp;Lucio Marcello ,&nbsp;Kenneth Macgregor ,&nbsp;Mark A. Taggart","doi":"10.1016/j.mimet.2024.106921","DOIUrl":"10.1016/j.mimet.2024.106921","url":null,"abstract":"<div><p>Bacteria are primarily responsible for biological water treatment processes in constructed wetland systems. Gravel in constructed wetlands serves as an essential substrate onto which complex bacterial biofilms may successfully grow and evolve. To fully understand the bacterial community in these systems it is crucial to properly isolate biofilms and process DNA from such substrates. This study looked at how best to isolate bacterial biofilms from gravel substrates in terms of bacterial richness. It considered factors including the duration of agitation during extraction, extraction temperature, and enzyme usage. Further, the 16S taxonomy data subsequently produced from Illumina MiSeq reads (using the SILVA 132 ribosomal RNA (rRNA) database on the DADA2 pipeline) were compared with the 16S data produced from Oxford Nanopore Technologies (ONT) MinION reads (using the NCBI 16S database on the EPI2ME pipeline). Finally, performance was tested by comparing the taxonomy data generated from the Illumina MiSeq and ONT MinION reads using the same (SILVA 132) database. We found no significant differences in the effective number of species observed when using different bacterial biofilm detachment techniques. However, enzyme treatment enhanced the total concentration of DNA. In terms of wetland community profiles, relative abundance differences within each sample type were clearer at the genus level. For genus-level taxonomic classification, MinION sequencing with the EPI2ME pipeline (NCBI database) produced bacterial abundance information that was poorly correlated with that from the Illumina MiSeq and DADA2 pipelines (SILVA132 database). When using the same database for each sequencing technology (SILVA132), the correlation between relative abundances at genus-level improved from negligible to moderate. This study provides detailed information of value to researchers working on constructed wetlands regarding efficient biofilm detachment techniques for DNA isolation and 16 s metabarcoding platforms for sequencing and data analysis.</p></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":null,"pages":null},"PeriodicalIF":2.2,"publicationDate":"2024-03-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140143699","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}