Microbiology spectrum最新文献

{"title":"Evaluation of a microfluidic-based point-of-care prototype with customized chip for detection of bacterial clusters.","authors":"Janina Treffon, Nicole Isserstedt-John, Richard Klemm, Claudia Gärtner, Alexander Mellmann","doi":"10.1128/spectrum.00862-24","DOIUrl":"10.1128/spectrum.00862-24","url":null,"abstract":"<p><p>Bacterial infection clusters cause high mortality rates and healthcare costs due to excessive therapy and hygiene measures. The aim of this study was to develop an automated real-time PCR-based point-of-care (POC) system with a customized microfluidic chip that facilitates fast detection of bacterial cluster isolates by targeting cluster-specific single-nucleotide polymorphisms (SNPs). For cluster detection of <i>Acinetobacter baumannii</i>, <i>Staphylococcus aureus</i>, and <i>Escherichia coli</i>, nine TaqMan real-time PCR assays targeting cluster-specific SNPs were designed. Additionally, for DNA input control, a universal PCR amplifying the 16S rDNA was constructed. All reactions were implemented into a microfluidic chip that was analyzed by a POC prototype enabling automated sample processing, fluid handling, and signal detection. Performance of the prototype was evaluated using 45 chips loaded with defined bacterial solutions, including swab eluates. For seven PCRs, implementation into the microfluidic chip was successful, leading to correct identification of all SNPs specific for <i>A. baumannii</i> and <i>E. coli</i> cluster isolates and delineation of all non-cluster strains within 70 min. The remaining three reactions failed in the chip, which resulted in misidentification of the <i>S. aureus</i> cluster. Sensitivity, specificity, and accuracy of the prototype were 43%, 88%, and 55%, respectively. The detection limit was PCR dependent and ranged between 10³ and 10⁵ colony-forming units/mL. Once optimized, the microfluidic POC system for cluster detection could be applied as time-saving and easy-to-use method to complement whole-genome sequencing efforts during screening for bacterial clusters.</p><p><strong>Importance: </strong>Especially in medical facilities, where morbid people are nursed in close distance to each other, pathogenic bacteria can accumulate and spread. To contain such infection clusters, usually time- and labor-intensive large-scale screening assays are conducted, where patients and patient-side surfaces are sampled, and PCR or whole-genome sequencing analyses are conducted to confirm or deny cluster affiliation of cultivated bacteria. Hence, fast solutions with easy application are required to complement the current state-of-the-art technology for cluster surveillance. Here, we developed a fully automated microfluidic point-of-care prototype that identified bacterial cluster isolates within 70 min from bacterial solutions, including swab eluates. The system requires only low hands-on time and can be applied apart from laboratory infrastructures near the patient, which considerably reduces the time from sampling to result. This ensures fast implementation of hygiene measures and quick containment of the infection cluster, which would enhance patients' safety and outcome.</p>","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-11-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142583253","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"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 <i>Helicobacter pylori</i> infection: serology vs. urea breath test.","authors":"Miguel Imperial, Kennard Tan, Chris Fjell, Yin Chang, Mel Krajden, Michael T Kelly, Muhammad Morshed","doi":"10.1128/spectrum.01084-24","DOIUrl":"10.1128/spectrum.01084-24","url":null,"abstract":"<p><p>The objective of the study was to ascertain an optimal <i>Helicobacter pylori</i> diagnostic strategy using population-level laboratory data comparing the performance of serology against urea breath test (UBT). <i>H. pylori</i> diagnostic test results for serology and UBT from two laboratories over a 12-year period (2006-20017) were extracted, linked, and analyzed. A subset of this population underwent both methods of testing within days of each other, enabling a direct comparison of the two methods. The average prevalence of <i>H. pylor</i>i positivity was 21.3% by serology and 17.5% by UBT. There were 2,612 individuals who had serology performed first, followed by UBT within 14 days. For this subset, the sensitivity of serology compared with UBT was 96.5% with a specificity of 79.2%. The negative predictive value for serology was 98.4%. Contrary to various recent clinical guidelines, the data show that serology still has utility as a sensitive enough test to be used as an initial <i>H. pylori</i> screening test in a lower prevalence population. Negative serology can be used with confidence to rule out active infection, whereas a positive serology could be followed up with a UBT or a similar performing test such as stool antigen to differentiate active from past infection. For population-based diagnostic recommendations, such a strategy may be ideal since serology generally costs less than UBT and may be combined with a blood draw being done for other diagnostic tests. Continuing to offer serology increases options for patients and may provide economic benefits for single-payer health care systems or health maintenance organizations.</p><p><strong>Importance: </strong>This study compares the performance of serology with urea breath test in the diagnosis of <i>Helicobacter pylori</i> in a population-level data set and mimics a head-to-head direct comparison as the study population had both tests performed within 2 weeks of each other. This provides new information supporting the use of serology in a diagnostic algorithm. There are several instances where serology could be preferable to patients to rule out disease, despite some guidelines suggesting serology should not be used.</p>","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11540150/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142350117","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Altitudinal variation in rhizosphere microbial communities of the endangered plant <i>Lilium tsingtauense</i> and the environmental factors driving this variation.","authors":"Boda Liu, Jinming Yang, Wanpei Lu, Hai Wang, Xuebin Song, Shaobo Yu, Qingchao Liu, Yingkun Sun, Xinqiang Jiang","doi":"10.1128/spectrum.00966-24","DOIUrl":"10.1128/spectrum.00966-24","url":null,"abstract":"<p><p>The rhizosphere soil properties and microbial communities of <i>Lilium tsingtauense</i>, an endangered wild plant, have not been examined in previous studies. Here, we characterized spatial variation in soil properties and microbial communities in the rhizosphere of <i>L. tsingtauense</i>. We measured the abundance of <i>L. tsingtauense</i> at different altitudes and collected rhizosphere and bulk soils at three representative altitudes. The results showed that <i>L. tsingtauense</i> was more abundant, and the rhizosphere soil was richer in nitrogen, phosphorus, potassium, water content, and organic matter and more acidic at high altitudes than at lower altitudes. The diversity and richness of rhizosphere bacteria and fungi increased with altitude and were higher in rhizosphere soil than in bulk soil. In addition, ectomycorrhizal fungi, endophytic fungi, and nitrogen-fixing bacteria were more abundant, and plant-pathogenic fungi were less abundant at high altitudes. Co-occurrence network analysis identified four key phyla (Bacteroidota, Proteobacteria, Ascomycota, and Basidiomycota) in the microbial communities. We identified a series of microbial taxa (Acidobacteriales, Xanthobacteraceae, and Chaetomiaceae) and rhizosphere soil metabolites (phosphatidylcholine and phosphatidylserine) that are crucial for the survival of <i>L. tsingtauense</i>. Correlation analysis and random forest analysis showed that some environmental factors were closely related to the rhizosphere soil microbial community and played an important role in predicting the distribution and growth status of <i>L. tsingtauense</i>. In sum, the results of this study revealed altitudinal variation in the rhizosphere microbial communities of <i>L. tsingtauense</i> and the factors driving this variation. Our findings also have implications for habitat restoration and the conservation of this species.</p><p><strong>Importance: </strong>Our study highlighted the importance of the rhizosphere microbial community of the endangered plant <i>L. tsingtauense</i>. We found that soil pH plays an important role in the survival of <i>L. tsingtauense</i>. Our results demonstrated that a series of microbial taxa (Acidobacteriales, Xanthobacteraceae, Aspergillaceae, and Chaetomiaceae) and soil metabolites (phosphatidylcholine and phosphatidylserine) could be essential indicators for <i>L. tsingtauense</i> habitat. We also found that some environmental factors play an important role in shaping rhizosphere microbial community structure. Collectively, these results provided new insights into the altitudinal distribution of <i>L. tsingtauense</i> and highlight the importance of microbial communities in their growth.</p>","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11536999/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142391822","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"VSV infection and LPS treatment alter serum bile acid profiles, bile acid biosynthesis, and bile acid receptors in mice.","authors":"Yamei Li, Yan Luo, Chao Wang, Lei Xu, Xinhua Dai, Yunfei An, Lin He, Dongmei Zeng, Yangjuan Bai, Hua Zhang","doi":"10.1128/spectrum.00836-24","DOIUrl":"10.1128/spectrum.00836-24","url":null,"abstract":"<p><p>Pathogen infections remain a significant public health problem worldwide. Accumulating evidence regarding the crosstalk between bile acid (BA) metabolism and immune response reveals that BA metabolism regulates host immunity and microbial pathogenesis, making it an attractive target for disease prevention and infection control. However, the effect of infection on circulating BA profiles, the biosynthesis-related enzymes, and their receptors remains to be depicted. Here, we investigated the effect of viral (vesicular stomatitis virus, VSV) and bacterial (lipopolysaccharide, LPS) infections on BA metabolism and signaling. Infection models were successfully established by intraperitoneally injecting VSV and LPS, respectively. VSV and LPS injection significantly changed the circulating BA profiles, with highly increased levels of taurine-conjugated BAs and significant decreases in unconjugated BAs. Consistent with the decreased levels of circulating cholic acid (CA) and chenodeoxycholic acid (CDCA), the expression of BA biosynthesis-related rate-limiting enzymes (<i>Cyp7a1</i>, <i>Cyp27a1</i>, <i>Cyp8b1,</i> and <i>Hsd3b7</i>) were significantly reduced. Furthermore, hepatic and pulmonary BA receptors (BARs) expression varied in different infection models. LPS treatment had an extensive impact on tested hepatic and pulmonary BARs, resulting in the upregulation of TGR5, S1PR2, and VDR, while VSV infection only promoted VDR expression. Our study provides insights into the involvement of BA metabolism in the pathophysiology of infection, which may provide potential clues for targeting BA metabolism and BAR signaling to boost innate immunity and control infection.</p><p><strong>Importance: </strong>This study focuses on the crosstalk between bile acid (BA) metabolism and immune response in VSV infection and LPS treatment models and depicts the effect of infection on circulating BA profiles, the biosynthesis-related enzymes, and their receptors. These findings provide insights into the effect of infection on BA metabolism and signaling, adding a more comprehensive understanding to the relationship between infection, BA metabolism and immune responses.</p>","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11537081/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142291324","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Antimicrobial susceptibility to last-resort antibiotics in carbapenemase-producing bacteria from Ukrainian patients.","authors":"Nelianne J Verkaik, Cornelia C H Wielders, Hans den Boer, Diana Langerak, Marius Vogel, Sandra Witteveen, Angela de Haan, Jeroen Bos, Mireille van Westreenen, Daan W Notermans, Antoni P A Hendrickx","doi":"10.1128/spectrum.01142-24","DOIUrl":"10.1128/spectrum.01142-24","url":null,"abstract":"<p><p>Since March 2022, an increase was observed in multidrug-resistant microorganisms (MDRO), associated with the hospital transfer of Ukrainian patients. The goal was to collect phenotypic susceptibility data and assess clinical implications. Carbapenemase-producing Enterobacterales (CPE, <i>n</i> = 96), <i>Pseudomonas aeruginosa</i> (CPPA, <i>n</i> = 20), and carbapenem-resistant <i>Acinetobacter baumannii-calcoaceticus</i> (CRAB, <i>n</i> = 6) from Ukrainian patients were obtained from March to December 2022 from the Dutch MDRO surveillance. Antimicrobial susceptibility testing was performed using broth microdilution (BMD) when available, fosfomycin agar dilution, disk diffusion (DD) for cefiderocol, and diverse gradient strips. All isolates were sequenced with Illumina next-generation sequencing. For meropenem, aminoglycosides, ceftazidime-avibactam, ceftolozane-tazobactam, and imipenem-relebactam, susceptibility rates were low (0%-30%), due to the high number of <i>bla</i>_NDM-positive isolates (79/122; 65%). For cefiderocol, results depended on reading with or without microcolonies, applying EUCAST or CLSI breakpoints, and whether DD or BMD was used; e.g., for <i>Klebsiella pneumoniae</i>, 30%-97% were susceptible. For colistin, 103/111 (93%) non-intrinsically resistant CPE/CPPA/CRAB isolates were susceptible. For most CPE, a low minimal inhibitory concentration (MIC) of <0.5 mg/L was measured for tigecycline and ceftazidime-avibactam-aztreonam. For CPPA, cefiderocol tested susceptible in 65%-100% of isolates. For CRAB, ampicillin-sulbactam MICs were ≥128 mg/L; for sulbactam-durlobactam, 1-2 mg/L. Admission in a Ukrainian hospital in the last year was a risk factor for MDRO, and majority were screening isolates (79%). There is extensive phenotypic resistance to last-resort antibiotics in MDRO from Ukrainian patients. Interpretation of cefiderocol susceptibility results depends on several variables. When treating patients recently admitted in Ukraine, suspected for Gram-negative bacterial infection, this should be taken into consideration.</p><p><strong>Importance: </strong>Since March 2022, multidrug-resistant microorganisms associated with Ukrainian patients have been detected in national surveillance systems of several European countries. We studied the phenotypic antimicrobial susceptibility to last-resort antibiotics of multidrug-resistant microorganisms from Ukrainian patients in the Netherlands and assessed clinical implications. Our research revealed that there was extensive phenotypic resistance to last-resort antibiotics. Healthcare professionals should be aware of multidrug-resistant microorganisms when treating patients recently admitted in Ukraine, suspected for Gram-negative bacterial infection.</p>","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11537089/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142308140","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Characterizing the rhythmic oscillations of gut bacterial and fungal communities and their rhythmic interactions in male cynomolgus monkeys.","authors":"Yunpeng Yang, Meiling Yu, Yong Lu, Changshan Gao, Ruxue Sun, Wanying Zhang, Yanhong Nie, Xinyan Bian, Zongping Liu, Qiang Sun","doi":"10.1128/spectrum.00722-24","DOIUrl":"10.1128/spectrum.00722-24","url":null,"abstract":"<p><p>The circadian oscillation of gut microbiota plays vital roles in the normal physiology and health of the host. Although the diurnal oscillation of intestinal bacteria has been extensively studied, little relevant work has been done on intestinal fungi. Besides, the rhythmic correlations between bacterial and fungal microbes are also scarcely reported. Here, we investigated the diurnal oscillations of bacterial and fungal communities in male cynomolgus monkeys by performing 16S rRNA and ITS amplicon sequencing. As for bacterial genera, we found that the relative abundance of <i>Prevotella</i>, norank_f_Eubacterium_coprostanoligenes_group, and <i>Peptococcus</i> underwent significant changes at ZT12 (19:00) and exhibited obvious rhythmic oscillations. Consequently, most of the bacterial functions varied at ZT12 and were positively correlated with the bacterial genera norank_f_Eubacterium_coprostanoligenes_group and <i>Prevotella</i>. Among the fungal genera, the relative abundance of <i>Aspergillus</i> and <i>Talaromyces</i> decreased at ZT18 (1:00) and showed slight rhythmic oscillations. As for the fungal function, the undefined saprotroph showed slight rhythmic oscillation and was positively correlated with the fungal genus <i>Aspergillus</i>. Notably, we characterized the correlations between intestinal bacteria and fungi every 6 h over the course of a day and found that the bacterial and fungal microbes interacted closely, with the most bacteria-fungi interactions occurring at ZT12. Our study contributed to a more comprehensive understanding of the diurnal oscillation patterns of bacterial and fungal microbes in male cynomolgus monkeys and uncovered their correlations during a diurnal cycle.</p><p><strong>Importance: </strong>The rhythmic oscillation of gut microbiota can impact the physiology activity and disease susceptibility of the host. Until now, most of the studies are focused on bacterial microbes, ignoring other components of gut microbes, such as fungal microbes (mycobiota). Besides, only few studies have addressed the rhythmic correlations between gut bacteria and fungi. Here, we analyzed the rhythmic oscillations of bacterial and fungal communities in male cynomolgus monkeys by performing 16S rRNA and ITS amplicon sequencing. Apart from identifying the rhythmically oscillated bacterial and fungal microbes, we conducted the correlation analysis between these two microbial communities and found that the intestinal bacteria and fungi exhibited close interactions rhythmically, with the most interactions occurring at ZT12. Thus, our study not only investigated the rhythmic oscillations of gut bacterial and fungal communities in male cynomolgus monkeys but also uncovered their rhythmic interactions.</p>","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11537094/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142350116","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Excellent anti-mildew effect of essential oil impregnation on sliced veneer plybamboo and its anti-mildew mechanism.","authors":"Qichao Bao, Yingying Shan, Fei Yang, Jiawei Zhu, Chungui Du, Yuting Wang, Yuran Shao, Chunlin Liu, Shiqin Chen, Ying Ran, Wenxiu Yin","doi":"10.1128/spectrum.01652-24","DOIUrl":"10.1128/spectrum.01652-24","url":null,"abstract":"<p><p>Sliced bamboo veneer used as a high-end decoration material is a highly innovative material for the deep processing of bamboo. However, bamboo is rich in starch and small molecular soluble sugars, making it susceptible to mildew infection and limiting the wide application of sliced veneer plybamboo. Spice essential oils are considered green and safe antimildew agents, which are cheap and accessible. The natural phenolic substances in plant essential oil have a good inhibitory effect on bamboo mildew. In this study, three types of spice essential oils (clove essential oil, oregano essential oil, and fennel essential oil) were employed, and their antifungal activity against bamboo mildews was assessed using the Oxford cup method, scanning electron microscopy, transmission electron microscopy, and a micro pH meter. The results demonstrated that the diameters of the Inhibition Zone against four common bamboo mildews (AN<i>, Aspergillus niger;</i> TV<i>, Trichoderma viride;</i> PC, <i>Penicillium citrinum</i>; MM, Mixed Mildews) caused by clove essential oil were 25.68 mm, 23.22 mm, 30.68 mm, and 25.43 mm, respectively. As an explanation, clove essential oil can inhibit or eliminate mildew by damaging and disrupting the cell membrane of the bamboo mildew, leading to significant shrinkage, distortion, surface roughness, formation of holes, or partial structural cracks in the mildew's mycelium. Additionally, it may interfere with and disrupt the pH balance of the intracellular and extracellular fluids within the cell. Furthermore, we also report that sliced veneer plybamboo impregnated with clove essential oil on each layer showed fine inhibition rates of 50%, 75%, 100%, and 25% against AN, TV, PC, and MM, respectively. This research underscores a sustainable approach to mildew prevention, crucial for advancing bamboo's utilization in high-value furniture decor applications.</p><p><strong>Importance: </strong>Mildew growth in sliced veneer plybamboo poses a significant challenge, particularly in its use for high-end furniture and decor. Traditionally, chemical treatments have been the primary solution though they often raise environmental concerns. Essential oils, with their well-documented antimicrobial properties, have emerged as an important natural and eco-friendly alternative for preventing mildew. These oils inhibit mildew growth effectively while offering a sustainable, non-toxic solution that reduces harm to both the environment and human health. By leveraging essential oils, it becomes possible to extend the lifespan of bamboo products, making them more durable and suitable for broader applications in furniture and decor, all while addressing the ecological limitations of conventional mildew prevention methods.</p>","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11537055/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142470066","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Rapid detection of SARS-CoV-2 variants by molecular-clamping technology-based RT-qPCR.","authors":"Shuo Shen, Andrew Y Fu, Maidar Jamba, Jonathan Li, Zhen Cui, Larry Pastor, Daniel Cataldi, Qing Sun, Joseph A Pathakamuri, Daniel Kuebler, Michael Rohall, Madison Krohn, Daniel Kissinger, Jocelyn Neves, Isaac Archibeque, Aiguo Zhang, Chuanyi M Lu, Michael Y Sha","doi":"10.1128/spectrum.04248-23","DOIUrl":"10.1128/spectrum.04248-23","url":null,"abstract":"<p><p>Given the challenges that SARS-CoV-2 variants have caused in terms of rapid spread and reduced vaccine efficacy, a rapid and cost-effective assay that can detect new and emerging variants is greatly needed worldwide. We have successfully applied the xenonucleic acid-based molecular-clamping technology to develop a multiplex reverse-transcription quantitative real-time PCR assay for SARS-CoV-2 multivariant detection. The assay was used to test 649 nasopharyngeal swab samples that were collected for clinical diagnosis or surveillance. The assay was able to correctly identify all 36 Delta variant samples as it accurately detected the D614G, T478K, and L452R mutations. In addition, the assay was able to correctly identify all 34 Omicron samples by detecting the K417N, T478K, N501Y, and D614G mutations. This technique reliably detects a variety of variants and has an analytical sensitivity of 100 copies/mL. In conclusion, this novel assay can serve as a rapid and cost-effective tool to facilitate large-scale detection of SARS-CoV-2 variants.</p><p><strong>Importance: </strong>We have developed a multiplex reverse-transcription quantitative real-time PCR (RT-qPCR) testing platform for the rapid detection of SARS-CoV-2 variants using the xenonucleic acid (XNA)-based molecular-clamping technology. The XNA-based RT-qPCR assay can achieve high sensitivity with a limit of detection of about 100 copies/mL for variant detection which is much better than the next-generation sequencing (NGS) assay. Its turnaround time is about 4 hours with lower cost and a lot of Clinical Laboratory Improvement Amendments (CLIA) labs own the instrument and meet skillset requirements. This assay provides a rapid, reliable, and cost-effective testing platform for rapid detection and monitoring of known and emerging SARS-CoV-2 variants. This testing platform can be adopted by laboratories that perform routine SARS-CoV-2 PCR testing, providing a rapid and cost-effective method in lieu of NGS-based assays, for detecting, differentiating, and monitoring SARS-CoV-2 variants. This assay is easily scalable to any new variant(s) should it emerge.</p>","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11537085/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142470100","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Template plasmids optimized for deletion of multiple genes in yeast <i>Saccharomyces cerevisiae</i>.","authors":"Yi-He Feng, Jing-Zhen Song, Jing Zhu, Zhiping Xie","doi":"10.1128/spectrum.01320-24","DOIUrl":"10.1128/spectrum.01320-24","url":null,"abstract":"<p><p>For <i>Saccharomyces cerevisiae</i>, gene knockout is routinely performed by transformation with a linear DNA cassette consisting of a selection marker gene flanked by upstream and downstream sequences homologous to a target gene. Over the years, several plasmid sets containing a variety of selection marker genes have been developed. Targeting fidelity under this strategy was high when performing the first gene knockout in a strain. However, we found that targeting fidelity decreased substantially when performing subsequent gene knockouts. The majority of the transformants were \"incorrect,\" in which the new selection marker gene replaced a pre-existing selection marker gene instead of its intended target. This was caused by the presence of shared regions in the knockout DNA cassettes. To minimize shared regions among knockout cassettes, we developed a set of template plasmids, in which each selection marker open reading frame is flanked by a unique promoter/terminator combination. Our SJZ series templates cover eight selection markers, namely, <i>URA3</i> (<i>C. a</i>.), <i>TRP1</i> (<i>K.l</i>.), <i>his5</i> (<i>S.p</i>.), <i>LEU2</i> (<i>K.l</i>.), <i>nat</i>, <i>hph</i>, <i>kan</i>, and <i>amdS</i>. When using our templates, targeting fidelity in subsequent gene knockouts was restored to as high as that of the first knockout, with essentially all the transformants being correct. Our templates can therefore bring efficiency improvements in future research projects involving multi-gene knockouts.</p><p><strong>Importance: </strong>When knocking out multiple genes in yeast, recombination among selection markers produces a large portion of false-positive transformants. We developed a new set of templates designed to minimize shared regions among selection markers. The use of this new template set resulted in essentially all transformants being correct knockouts.</p>","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11537097/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142470104","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"First report on the identification and characterization of mammalian orthoreovirus from sheep in China.","authors":"Dengshuai Zhao, Ping Li, Yuanhang Zhang, Dixi Yu, Tianyu Wang, Keshan Zhang","doi":"10.1128/spectrum.00847-24","DOIUrl":"10.1128/spectrum.00847-24","url":null,"abstract":"","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11537088/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142470067","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}