Journal of microbiological methods最新文献

{"title":"Campylobacter fetus subspecies specific PCR assays inferred from comparative genomic analysis for accurate subspecies identification","authors":"Linda van der Graaf - van Bloois ,&nbsp;Aldert L. Zomer ,&nbsp;Birgitta Duim ,&nbsp;Jaap A. Wagenaar","doi":"10.1016/j.mimet.2024.107049","DOIUrl":"10.1016/j.mimet.2024.107049","url":null,"abstract":"<div><div>Bovine Genital Campylobacteriosis (BGC) is caused by <em>Campylobacter fetus</em> subsp. <em>venerealis</em> and is a notifiable disease to the WOAH (World Organisation for Animal Health). For an effective BGC control program, the reliable differentiation of <em>Campylobacter fetus</em> subsp. <em>venerealis</em> (Cfv) from the closely related <em>Campylobacter fetus</em> subsp. <em>fetus</em> (Cff) is required<em>.</em> However, the available molecular <em>C. fetus</em> subspecies identification assays lack sensitivity and specificity to differentiate <em>C. fetus</em> isolates based on their phenotypic or genotypic differences. Furthermore, the current biochemical subspecies identification is not fully congruent with the genomic differentiation of <em>C. fetus</em> strains.</div><div>In this study, the genome sequences of 41<em>C. fetus</em> strains with well identified subspecies, were analyzed with the large-scale BLAST score ratio (LS-BSR) pipeline to identify Cff and Cfv specific sequences. With this analysis, the <em>asd</em> gene encoding an aspartate-semialdehyde dehydrogenase was identified, which contained a 6-bp Cff-specific sequence, and this 6-bp sequence was absent in the <em>asd</em> gene of Cfv strains. This sequence was used for the development of PCR assays to differentiate Cff and Cfv strains. The <em>C. fetus</em> subspecies identification of the developed <em>asd</em> PCR assays was in full congruence with the genomic classification of strains and are recommended for molecular identification of <em>C. fetus</em> subspecies in BGC control programs.</div><div>The <em>asd</em> PCR can be assessed on sequenced genomes using a web interface containing the Cfvcatch tool, which includes placement of the tested genome in a phylogenetic tree with reference <em>C. fetus</em> genomes to distinguish the two subspecies and to detect antimicrobial resistance genes.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"226 ","pages":"Article 107049"},"PeriodicalIF":1.7,"publicationDate":"2024-09-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142348448","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":"Optimized protocol for collecting root canal biofilms for in vitro studies","authors":"Rafael da Silva Goulart ,&nbsp;Mariana Oliveira-Silva ,&nbsp;Milton Faria-Junior ,&nbsp;Yara Teresinha Correa Silva-Sousa ,&nbsp;Carlos Eduardo Saraiva Miranda ,&nbsp;André Pitondo-Silva","doi":"10.1016/j.mimet.2024.107048","DOIUrl":"10.1016/j.mimet.2024.107048","url":null,"abstract":"<div><div>Endodontic retreatment is often necessitated by several factors, including the persistence of microorganisms in the root canal system (RCS). Their complex organization in biofilms increases their pathogenic potential, necessitating new disinfection strategies. This study aimed to standardize a new <em>in vitro</em> protocol for collecting biofilm from the RCS. Thirty-four bovine incisors were used in the study, divided into two experimental groups with two collection steps each: (a) biofilm collection protocol and (b) absorbent paper points protocol. Twelve specimens from each group were selected for counting colony-forming units (CFUs), while eight specimens were prepared for scanning electron microscopy (SEM). Two additional specimens served as sterilization controls to ensure that experiments were free of contamination. The coronal region was removed and standardized at 15 mm. After preparation with ProTaper up to F5, the apical foramen was sealed with composite resin, and the roots were stabilized with acrylic resin in 1.5-mL Eppendorf tubes. The specimens were sterilized and inoculated with <em>Enterococcus faecalis</em> NTCT 775 every 24 h for 21 days. After this period, each group underwent biofilm collection protocols, and CFU and scanning electron microscopy (SEM) data were analyzed. The Shapiro–Wilk test was performed to assess the normality of log-transformed data, and the results indicated a normal distribution for all groups, allowing parametric testing. The Levene test was used to evaluate the equality of variances. The proposed biofilm collection method yielded significantly higher CFU counts compared with the absorbent paper points method, particularly when analyzed on a log₁₀ scale. An independent samples <em>t</em>-test confirmed a statistically significant difference between the two methods (<em>p</em> &lt; 0.0001). The proposed protocol achieved an efficiency rate of 95.85 % ± 1.15 %, whereas the absorbent paper points protocol yielded a lower efficiency of 5.46 % ± 1.37 %. Therefore, the biofilm collection protocol proposed in this study proved to be more effective for biofilm removal from the RCS.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"226 ","pages":"Article 107048"},"PeriodicalIF":1.7,"publicationDate":"2024-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142348449","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 comparative study of a rapid phenotypic antimicrobial susceptibility testing system directly from positive blood cultures to the disk diffusion and VITEK 2 methods","authors":"Merav Strauss,&nbsp;Shereen Affan Suleiman,&nbsp;Najwa Lauz,&nbsp;Bela Reznik-Gitlitz,&nbsp;Dana Sagas,&nbsp;Raul Colodner","doi":"10.1016/j.mimet.2024.107046","DOIUrl":"10.1016/j.mimet.2024.107046","url":null,"abstract":"<div><h3>Background</h3><div>Sepsis is a life-threatening condition that impacts 49 million people annually and causes 11 million deaths worldwide. Surviving bloodstream infections (BSIs) depends on the rapid administration of effective antimicrobial treatment, underscoring a need for rapid antimicrobial susceptibility testing (AST).</div></div><div><h3>Aim</h3><div>To evaluate the performance of Quantamatrix's dRAST v2.5 system (Seoul, South Korea) for AST directly from positive blood cultures as compared to the Disk-Diffusion (DD) and VITEK 2 methods.</div></div><div><h3>Methods</h3><div>The study included 191 positive blood cultures from clinical samples and spiked blood culture bottles. Following Gram staining and species-level identification, AST was performed by VITEK 2 and standard DD methods using CLSI (2021) interpretation.</div></div><div><h3>Results</h3><div>dRAST demonstrated very good AST performance for a Gram-negative isolate, and good performance for Gram-positive isolates, meeting CLSI criteria for the acceptance of a new method. Antimicrobials that were not considered verified compared to VITEK 2 and DD were cefazolin, ceftazidime, meropenem, and trimethoprim/sulfamethoxazole for Gram-negatives and clindamycin, erythromycin, penicillin, and oxacillin for Gram-positives. dRAST ESBL detection results were strongly correlated with the ESBL phenotypes obtained with other methods. Additional resistance mechanisms were in concordance with traditional tests.</div></div><div><h3>Conclusions</h3><div>dRAST demonstrated good AST performance, meeting CLSI criteria for most relevant antibiotics. dRAST was associated with a significant reduction in time-to-results, labor, and the subjectivity of result analyses, making it a valuable addition to efforts supporting the treatment of patients with bacteremia.</div><div>AST (antimicrobial susceptibility test), blood culture, dRAST, rapid methods, sepsis, turnaround time (TAT).</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"226 ","pages":"Article 107046"},"PeriodicalIF":1.7,"publicationDate":"2024-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142289361","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":"“Curing of common plasmids in gram-negative bacteria using a Cas9-based conjugative vector”","authors":"Kelly K. Yen ,&nbsp;Austin J. Terlecky ,&nbsp;Mingju Hao ,&nbsp;Vanessa Cienfuegos ,&nbsp;Albert Rojtman ,&nbsp;Liang Chen ,&nbsp;Barry N. Kreiswirth","doi":"10.1016/j.mimet.2024.107047","DOIUrl":"10.1016/j.mimet.2024.107047","url":null,"abstract":"<div><div>We report the creation of 17 <em>Escherichia coli</em> strains harboring the conjugative plasmid pLCasCureT with a CRISPR-Cas9 system to surgically “cure” the most common plasmids among <em>Enterobacterales</em> species. This approach can create isogenic pairs of strains to study host-plasmid interactions, correlate plasmid genotype and phenotype, and create plasmid-free cloning strains.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"226 ","pages":"Article 107047"},"PeriodicalIF":1.7,"publicationDate":"2024-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142289363","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 simple culture medium for phenotypic characterization and long-term storage of medically relevant fusarioid fungi","authors":"Ruan Campos Monteiro ,&nbsp;Maria Cecília Zorat Yu ,&nbsp;Somayeh Dolatabadi ,&nbsp;Rossana de Aguiar Cordeiro ,&nbsp;Edlâny Pinho Romão Milanez ,&nbsp;Sarah Santos Gonçalves ,&nbsp;Zoilo Pires de Camargo ,&nbsp;Ana Luisa Höfling-Lima ,&nbsp;Anderson Messias Rodrigues","doi":"10.1016/j.mimet.2024.107042","DOIUrl":"10.1016/j.mimet.2024.107042","url":null,"abstract":"<div><div>Fusarioid fungi, particularly <em>Neocosmospora solani</em> and <em>Fusarium oxysporum</em>, are emerging as significant human pathogens, causing infections ranging from localized mycoses to life-threatening systemic diseases. Accurate identification and preservation of these fungi in clinical laboratories remain challenging because of their diverse morphologies and specific growth requirements. This study evaluated a novel milk-honey and malt agar (MHM) against conventional media for cultivating and preserving 60 clinical fusarioid isolates, including <em>Neocosmospora</em> spp. (<em>n</em> = 47), <em>Bisifusarium</em> spp. (<em>n</em> = 5), and <em>Fusarium</em> spp. (<em>n</em> = 8). Compared with Sabouraud dextrose 2 % agar (SDA) and malt extract agar (ME2), MHM significantly increased conidia production (<em>p</em> &lt; 0.0001, mean = 3.4 × 10³, standard deviation (SD) = ±1.3 × 10³), with results similar to those of carnation leaf agar (CLA). MHM facilitated superior preservation of fusarioid viability for up to one year at room temperature on slant cultures and over two years on swabs in Amies gel with charcoal, outperforming current methods such as Castellani (water) or cryopreservation. Morphological characterization of fusarioid fungi grown on MHM revealed distinct growth patterns and conidial structures for <em>Neocosmospora</em>, <em>Bisifusarium</em>, and <em>Fusarium</em> species, aiding in identifying these genera. The superior performance of MHM in stimulating conidiation, maintaining viability, and preserving morphology underscore its potential as a reference medium for medically relevant fusarioid fungi, with broad implications for clinical mycology laboratories and resource-limited settings.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"226 ","pages":"Article 107042"},"PeriodicalIF":1.7,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142289362","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":"Immunofluorescence detection of Ecytonucleospora hepatopenaei (EHP) in Penaeus vannamei","authors":"Sungman Cho ,&nbsp;Deborah A. Schaefer ,&nbsp;Hung N. Mai ,&nbsp;Michael W. Riggs ,&nbsp;Arun K. Dhar","doi":"10.1016/j.mimet.2024.107039","DOIUrl":"10.1016/j.mimet.2024.107039","url":null,"abstract":"<div><p>Hepatopancreatic microsporidiosis (HPM), caused by the microsporidium <em>Ecytonucleospora hepatopenaei</em> (EHP) leads to retarded growth and enhanced susceptibility to other diseases in shrimp resulting in a major loss for the shrimp industry worldwide. It is little understood how EHP infects its host and hijacks its cellular machinery to replicate and exert clinical manifestations in infected shrimp. Since the initial record of HPM, histopathology and polymerase chain reaction (PCR)-based assays were developed for the detection of EHP to prevent spread of the disease. Availability of an antibody-based detection method would complement these existing diagnostic tools and be useful in studying EHP pathogenesis. We describe here an immunofluorescence assay (IFA) for detecting EHP using monoclonal antibodies (mAbs) that were originally developed against <em>Cryptosporidium parvum</em>, a coccidian parasite that infects calves (<em>Bos taurus</em>), other agriculturally important animals, and humans. Forty-one mAbs were screened and two mAbs, 3E2 and 3A12, were found to detect EHP successfully. The utility of these mAbs in detecting EHP was further assessed by testing 36 experimentally challenged EHP-infected shrimp (<em>Penaeus vannamei)</em>. EHP-detection data from infected shrimp were compared by Hematoxylin and Eosin (H&amp;E) histology, real-time PCR, and immunofluorescence. The data show IFA using mAbs 3E2 and 3A12 could successfully detect EHP and that the sensitivity of detection is comparable to H&amp;E histology and quantitative PCR. Availability of mAbs that can detect EHP is expected to be immensely beneficial in HPM diagnosis. Since the pathobiology of <em>C. parvum</em> has been so widely studied, these cross-reactive mAbs may also aid in gaining some insight into EHP pathogenesis and disease.</p></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"226 ","pages":"Article 107039"},"PeriodicalIF":1.7,"publicationDate":"2024-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142172625","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":"Optical detection and enumeration of Escherichia coli and Salmonella enterica using a low-magnification light microscope","authors":"Yuzhen Zhang,&nbsp;Zili Gao,&nbsp;Lili He","doi":"10.1016/j.mimet.2024.107041","DOIUrl":"10.1016/j.mimet.2024.107041","url":null,"abstract":"<div><p>A rapid and cost-effective method for detecting bacterial cells from surfaces is critical to food safety, clinical hygiene, and pharmacy quality. Herein, we established an optical detection method based on a gold chip coating with 3-mercaptophenylboronic acid (3-MPBA) to capture bacterial cells, which allows for the detection and quantification of bacterial cells with a standard light microscope under low-magnification (10×) objective lens. Then, integrate the developed optical detection method with swab sampling to detect bacterial cells loading on stainless-steel surfaces. Using <em>Salmonella enterica</em> (SE1045) and <em>Escherichia coli</em> (<em>E. coli</em> OP50) as model bacterial cells, we achieved a capture efficiency of up to 76.0 ± 2.0 % for SE1045 cells and 81.1 ± 3.3 % for <em>E. coli</em> OP50 cells at 10³ CFU/mL upon the optimized conditions, which slightly decreased with the increasing bacterial concentrations. Our assay showed good linear relationships between the concentrations of bacterial cells with the cell counting in images in the range of 10³ -10⁷ CFU/mL for SE1045, and 10³ -10⁸ CFU/mL for <em>E. coli</em> OP50 cells. The limit of detection (LOD) was 10³ CFU/mL for both SE1045 and <em>E. coli</em> OP50 cells. A further increase in sensitivity in detecting <em>E. coli</em> OP50 cells was achieved through a heat treatment, enabling the LOD to be reduced as low as 10² CFU/mL. Furthermore, a preliminary application succeeded in assessing bacterial contamination on stainless-steel surfaces following integration with the approximately 40 % recovery rate, suggesting prospects for evaluating the bacteria from surfaces. The entire process was completed within around 2 h, costing merely a few dollars per sample. Considering the low cost of standard light microscopes, our method holds significant potential for practical industrial applications in bacterial contamination control on surfaces, especially in low-resource settings.</p></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"226 ","pages":"Article 107041"},"PeriodicalIF":1.7,"publicationDate":"2024-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142232664","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Evaluation of cobas® Liat® Cdiff, STANDARD™ M10 C. difficile and Xpert® C. difficile BT assays for rapid detection of toxigenic Clostridioides difficile","authors":"Juulia Suominen ,&nbsp;Suvi Korhonen ,&nbsp;Heini Kutvonen ,&nbsp;Hanna Jarva ,&nbsp;Anu Pätäri-Sampo ,&nbsp;Raisa Loginov","doi":"10.1016/j.mimet.2024.107043","DOIUrl":"10.1016/j.mimet.2024.107043","url":null,"abstract":"<div><p>We evaluated the analytical performance of three commercial molecular assays for rapid detection of <em>Clostridioides difficile</em> toxin B in stool samples. The results were compared with results from the BD MAX™ Cdiff assay. We analyzed forty negative and thirty-two positive stool samples with three rapid assays: Roche cobas® Liat® Cdiff, SD Biosensor STANDARD™ M10 <em>C. difficile</em> and Cepheid Xpert® <em>C. difficile</em> BT. The assays demonstrated a sensitivity of 96.9 %, 96.9 % and 100.0 % and a specificity of 100 %, 97.5 % and 97.5 %, respectively. There is limited data available on the analytical performance of the newly introduced STANDARD™ M10 <em>C. difficile</em> assay. In this study, all three rapid assays demonstrated similarly high analytical performance and can be used for detection of toxigenic <em>C. difficile</em>.</p></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"226 ","pages":"Article 107043"},"PeriodicalIF":1.7,"publicationDate":"2024-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S0167701224001556/pdfft?md5=00756b5b8da14dc99fad7302cefecc61&pid=1-s2.0-S0167701224001556-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142240765","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":"Use of a synthetic oligonucleotide to detect false positives caused by cross-contamination in nested PCR","authors":"Alexandre S. Maekawa ,&nbsp;Luciene S. Santos ,&nbsp;Paulo E.N.F. Velho ,&nbsp;Marina R. Drummond","doi":"10.1016/j.mimet.2024.107040","DOIUrl":"10.1016/j.mimet.2024.107040","url":null,"abstract":"<div><p>Nested PCR is a useful tool for identifying low-abundance target sequences of pathogens and avoiding false negatives. However, it carries an increased risk of cross-contamination, especially with its positive control. Here, we propose using customized synthetic oligonucleotides to detect false positives due to cross-contamination.</p></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"226 ","pages":"Article 107040"},"PeriodicalIF":1.7,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142172626","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":"Prediction strategy for screening functional Haloarchaea strains with qPCR assays","authors":"Xinyu Hu ,&nbsp;Wenxiang Sun ,&nbsp;Meng Zhang ,&nbsp;Wenjun Guo ,&nbsp;Shujing Yang ,&nbsp;Lin Zhu ,&nbsp;Xiang Xiao ,&nbsp;Xiangru Xu ,&nbsp;Wei Wei","doi":"10.1016/j.mimet.2024.107029","DOIUrl":"10.1016/j.mimet.2024.107029","url":null,"abstract":"<div><p>As an extremophile resource, functional Haloarchaea strains are extremely time-consuming to screen. Here, taking the screening of low-salt-tolerant strains as an example, based on the qPCR assays that shortened time by 4–7 times and achieved 100 % accuracy, a universal strategy for rapid and accurate screening of functional Haloarchaea strains was established.</p></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"226 ","pages":"Article 107029"},"PeriodicalIF":1.7,"publicationDate":"2024-09-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142154390","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}