Microbiology spectrum最新文献

{"title":"Reevaluation of the gastrointestinal methanogenic archaeome in multiple sclerosis and its association with treatment.","authors":"Pei Yee Woh, Yehao Chen, Christina Kumpitsch, Rokhsareh Mohammadzadeh, Laura Schmidt, Christine Moissl-Eichinger","doi":"10.1128/spectrum.02183-24","DOIUrl":"10.1128/spectrum.02183-24","url":null,"abstract":"<p><p>The role of the gut archaeal microbiome (archaeome) in health and disease remains poorly understood. Methanogenic archaea have been linked to multiple sclerosis (MS), but prior studies were limited by small cohorts and inconsistent methodologies. To address this, we re-evaluated the association between methanogenic archaea and MS using metagenomic data from the International Multiple Sclerosis Microbiome Study. We analyzed gut microbiome profiles from 115 MS patients and 115 healthy household controls across Buenos Aires (27.8%), Edinburgh (33.9%), New York (10.4%), and San Francisco (27.8%). Metagenomic sequences were taxonomically classified using kraken2/bracken and a curated profiling database to detect archaea, specifically <i>Methanobrevibacter</i> species. Most MS patients were female (80/115), aged 25-72 years (median: 44.5), and 70% were undergoing treatment, including dimethyl fumarate (<i>n</i> = 21), fingolimod (<i>n</i> = 20), glatiramer acetate (<i>n</i> = 14), interferon (<i>n</i> = 18), natalizumab (<i>n</i> = 6), or ocrelizumab/rituximab (<i>n</i> = 1). We found no significant differences in overall archaeome profiles between MS patients and controls. However, treated MS patients exhibited higher abundances of <i>Methanobrevibacter smithii</i> and <i>M.</i> sp900766745 compared to untreated patients. Notably, <i>M.</i> sp900766745 abundance correlated with lower disease severity scores in treated patients. Our results suggest that gut methanogens are not directly associated with MS onset or progression but may reflect microbiome health during treatment. These findings highlight potential roles for <i>M. smithii</i> and <i>M.</i> sp900766745 in modulating treatment outcomes, warranting further investigation into their relevance to gut microbiome function and MS management.IMPORTANCEMultiple sclerosis (MS) is a chronic neuroinflammatory disease affecting the central nervous system, with approximately 2.8 million people diagnosed worldwide, mainly young adults aged 20-30 years. While recent studies have focused on bacterial changes in the MS microbiome, the role of gut archaea has been less explored. Previous research suggested a potential link between methanogenic archaea and MS disease status, but these findings remained inconclusive. Our study addresses this gap by investigating the gut archaeal composition in MS patients and examining how it changes in response to treatment. By focusing on methanogens, we aim to uncover novel insights into their role in MS, potentially revealing new biomarkers or therapeutic targets. This research is crucial for enhancing our understanding of the gut microbiome's impact on MS and improving patient management.</p>","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":" ","pages":"e0218324"},"PeriodicalIF":3.7,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143492962","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":"Impact of first-void urine volume on chlamydia and gonorrhea positivity rates in men who have sex with men and transgender women.","authors":"Clayton W Hall, Adam Pyke, Mikhail Davydov, Katelin Urbanoski, Tim H Guimond, Kevin Woodward","doi":"10.1128/spectrum.03072-24","DOIUrl":"10.1128/spectrum.03072-24","url":null,"abstract":"","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":" ","pages":"e0307224"},"PeriodicalIF":3.7,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143483423","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":"Pathological characterization of female reproductive organs prior to miscarriage induced by Zika virus infection in the pregnant common marmoset.","authors":"Toshifumi Imagawa, Kazuo Tanaka, Masahiko Ito, Mami Matsuda, Tadaki Suzuki, Tsuyoshi Ando, Chizuko Yaguchi, Kazuyoshi Miyamoto, Shuji Takabayashi, Ryosuke Suzuki, Tomohiko Takasaki, Hiroaki Itoh, Isao Kosugi, Tetsuro Suzuki","doi":"10.1128/spectrum.02282-24","DOIUrl":"10.1128/spectrum.02282-24","url":null,"abstract":"<p><p>While Zika virus (ZIKV) infection in pregnant women is known to increase the risk of miscarriage and stillbirth, the mechanism by which ZIKV infection leads to the inability to continue a pregnancy is not clear. In our common marmoset models of ZIKV infection in pregnant individuals, miscarriage was observed in dams infected in the first or second trimester, and preterm delivery was observed in a dam infected in the third trimester. Serum progesterone levels were significantly lower prior to miscarriage or preterm delivery in the infected marmosets. To elucidate the pathology of the placental region just before the onset of ZIKV-induced miscarriage, we newly prepared an infected marmoset in the first trimester of pregnancy and euthanized it when the serum progesterone concentration was markedly reduced. Pathological analysis revealed significant degeneration in cells at the maternal-fetal interface, presumably trophoblasts. Cleaved-caspase was widely observed in the endometrial to placental region, and TNFα at 200 pg/mL was detected in the amniotic fluid, suggesting that apoptosis may progress in the endometrium and placenta, leading to decreased trophoblast function and miscarriage. ZIKV NS1 protein was found sporadically in the cellular degeneration area and widely in the basal layer of the endometrium. Furthermore, the viral protein was frequently detected in the follicles and corpus luteum of the ovary. The developed ZIKV infection model in pregnant marmosets would be useful not only to better understand the mechanism of ZIKV-induced miscarriage but also to analyze the effects of the viral infection on female reproductive tissues.</p><p><strong>Importance: </strong>Although several viruses, including Zika virus (ZIKV), are known to increase the risk of miscarriage upon viral infection, the mechanism by which miscarriage is induced by viral infection is largely unknown. This is partly due to the difficulty of pathological analysis of maternal tissues in the period following viral infection and prior to miscarriage. In this study, we predicted the occurrence of miscarriage by monitoring serum progesterone levels and performed pathological analysis of peri-placental tissues at a time point assumed to be just before miscarriage. This is the first report of trophoblast degeneration prior to miscarriage, suggesting that the experimental method used here is useful for analyzing the pathogenesis of virus infection-related miscarriage. Further immunostaining revealed that ZIKV NS1 was distributed not only in the uterus but also in the ovaries, with particularly pronounced staining of oocytes. Whether ZIKV infection affects female reproductive function should be clarified in the future.</p>","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":" ","pages":"e0228224"},"PeriodicalIF":3.7,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143492958","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":"A novel double-antibody sandwich ELISA based on monoclonal antibodies against the viral spike protein detects porcine deltacoronavirus infection.","authors":"Yingjie Bai, Ruiming Yu, Guangqing Zhou, Liping Zhang, TianTian Wang, Ya Liu, Dongsheng Wang, Zhongwang Zhang, Yonglu Wang, Huichen Guo, Li Pan, Xinsheng Liu","doi":"10.1128/spectrum.02854-24","DOIUrl":"10.1128/spectrum.02854-24","url":null,"abstract":"<p><p>Porcine deltacoronavirus (PDCoV) is a significant emerging pathogen that causes severe enteric disease in swine, and therefore significant economic losses in the pig farming industry. Here, we developed a novel double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) based on two monoclonal antibodies directed against the PDCoV spike protein. These two monoclonal antibodies were obtained through hybridoma fusion and screening, and they can specifically react with the PDCoV spike protein. The detection limits of the DAS-ELISA for the recombinant spike protein and viral titer were approximately 0.12 ng/mL and 1.96 × 10³ copies/μL, respectively. The DAS-ELISA did not cross-react with other swine enteric coronaviruses, including porcine epidemic diarrhea virus, transmissible gastroenteritis virus, or porcine rotavirus. A total of 145 rectal swab samples were collected and tested for the presence of PDCoV with the DAS-ELISA and reverse transcription-quantitative PCR (RT-qPCR). The coincidence rate between the DAS-ELISA and RT-qPCR was 91.03%, with a kappa value of 0.814, indicating that the DAS-ELISA is a reliable method for viral antigen detection in clinical samples. DAS-ELISA had a sensitivity of 92.85% and a specificity of 89.89%. The positive predictive value and negative predictive value of this method are 85.25% and 95.24%, respectively. Furthermore, the DAS-ELISA can also be used to detect the spike protein in PDCoV vaccines, making it a valuable tool for assessing the efficacy of PDCoV vaccines.</p><p><strong>Importance: </strong>Since 2014, porcine deltacoronavirus (PDCoV) has spread widely across multiple countries and regions, causing significant economic losses to the global livestock industry. Currently, no commercially available vaccine exists for the prevention of PDCoV infection; therefore, accurate and effective diagnostic methods are crucial for its control and prevention. In this study, the PDCoV S protein expressed in Chinese Hamster Ovary (CHO) cells was used to immunize mice, and a novel double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was established based on two monoclonal antibodies. The DAS-ELISA had high sensitivity, good repeatability, strong specificity, and high consistency for detecting clinical samples and spike protein in PDCoV vaccines. Therefore, the DAS-ELISA established in this study may be a reliable and effective tool for detecting PDCoV infection and the efficacy of PDCoV vaccines.</p>","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":" ","pages":"e0285424"},"PeriodicalIF":3.7,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143516126","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":"Clinical presentation, treatment, and antimicrobial susceptibility of 155 sequential <i>Staphylococcus lugdunensis</i> infections.","authors":"Kurt D Palumbo, Natasia F Jacko, Michael Z David","doi":"10.1128/spectrum.02749-24","DOIUrl":"10.1128/spectrum.02749-24","url":null,"abstract":"<p><p><i>Staphylococcus lugdunensis</i> is known to be virulent, but there are few large-scale epidemiologic studies of this species to define types of infection, susceptibility patterns, and severity. <i>S. lugdunensis</i> isolates from any culture at four U.S. tertiary care hospitals between 1 April 2021 and 1 April 2022 were identified. For the first isolate from each subject, clinical, demographic, and outcome data were recorded. Of 291 isolates, 223 were obtained from a clinically significant infection. Of these 223 isolates, 86 (38.6%) were from monomicrobial cultures; additionally, <i>S. lugdunensis</i> was considered a true pathogen in 69/137 polymicrobial infections. Among 155 subjects with <i>S. lugdunensis</i> infections, 49.7% were female, 46.5% were black, and 41.9% were white; 51.6% of infections were community associated. The most common infection sites were skin and soft tissue (SSTI) (<i>n</i> = 98, 63.2%), urinary tract (<i>n</i> = 16, 10.3%), and sinusitis (<i>n</i> = 14, 9%). Of nine monomicrobial bloodstream infections (BSIs), two were fatal, three involved foreign bodies, and two had infective endocarditis. Greater than half of SSTIs required an invasive procedure for cure. Among 138/291 isolates from colonization or infection, tetracycline, trimethoprim-sulfamethoxazole, oxacillin, and vancomycin susceptibility rates were 94.8% (128/135), 95.9% (94/98), 84.1% (116/138), and 100% (138/138), respectively. There were similarities in types of infection comparing <i>S. lugdunensis</i> in this study and prior reports on <i>Staphylococcus aureus</i>. SSTI was the predominant <i>S. lugdunensis</i> infection type; more than 50% of SSTIs required procedural intervention. Of nine BSIs, three involved a foreign body, and there were two cases of infective endocarditis. Oxacillin resistance was identified in 16% of isolates.</p><p><strong>Importance: </strong>In recent years, <i>Staphylococcus lugdunensis</i> has been identified with increasing frequency as a human pathogen causing a wide variety of clinical syndromes, from soft tissue infections to fatal cases of bloodstream infection. Despite this, there are few large-scale epidemiologic studies examining this highly virulent organism. Our study adds to the growing literature on this emerging pathogen by analyzing a large case series of sequential <i>S. lugdunensis</i> infections at four U.S. hospitals to define its contemporary epidemiology, including the types of infections it causes, their outcomes, treatment approaches, and antimicrobial susceptibilities. These data provide valuable insights for clinicians in diagnosing and treating patients with these often debilitating infections. The findings also improve upon our understanding of the incidence of each infection syndrome and variability in antimicrobial susceptibilities of isolates to guide the design of future studies on the genomic epidemiology of this important pathogen.</p>","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":" ","pages":"e0274924"},"PeriodicalIF":3.7,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143586264","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":"Characterization of vaginal microbiomes in clinician-collected bacterial vaginosis diagnosed samples.","authors":"Hayden N Brochu, Qimin Zhang, Kuncheng Song, Ling Wang, Emily A Deare, Jonathan D Williams, Crystal R Icenhour, Lakshmanan K Iyer","doi":"10.1128/spectrum.02582-24","DOIUrl":"10.1128/spectrum.02582-24","url":null,"abstract":"<p><p>Bacterial vaginosis (BV) is a type of vaginal inflammation caused by bacterial overgrowth, upsetting the healthy microbiome of the vagina. Existing clinical testing for BV is primarily based upon physical and microscopic examination of vaginal secretions. Modern PCR-based clinical tests target panels of BV-associated microbes, such as the Labcorp NuSwab test that targets <i>Atopobium</i> (<i>Fannyhessea</i>) <i>vaginae</i>, <i>Megasphaera-1</i>, and <i>Bacterial Vaginosis Associated Bacterium (BVAB)-2</i>. Remnant clinician-collected NuSwab vaginal swabs underwent DNA extraction and 16S V3-V4 rRNA gene sequencing to profile microbes in addition to those included in the Labcorp NuSwab test. Community state types (CSTs) were determined using the most abundant taxon detected in each sample. PCR results for NuSwab panel microbial targets were compared against the corresponding microbiome profiles. Metabolic pathway abundances were characterized via metagenomic prediction from amplicon sequence variants (ASVs). 16S V3-V4 rRNA gene sequencing of 75 remnant vaginal swabs yielded 492 unique 16S V3-V4 ASVs, identifying 83 unique genera. NuSwab microbe quantification was strongly concordant with quantification by sequencing (<i>P</i> < 0.01). Samples in CST-I (18 of 18, 100%), CST-II (three of three, 100%), CST-III (15 of 17, 88%), and CST-V (one of one, 100%) were largely categorized as BV-negative via the NuSwab panel, while most CST-IV samples (28 of 36, 78%) were BV-positive or BV-indeterminate. BV-associated microbial and predicted metabolic signatures were shared across multiple CSTs. These findings highlight robust sequencing-based quantification of Labcorp NuSwab BV microbes, accurate discrimination of vaginal microbiome CSTs dominated by distinct <i>Lactobacilli</i>, and expanded the identification of BV-associated bacterial and metabolic biomarkers.IMPORTANCEBacterial vaginosis (BV) poses a significant health burden for women during reproductive years and onward. Current BV diagnostics rely on either panels of select microbes or on physical and microscopic evaluations by technicians. Here, we sequenced the microbiome profiles of samples previously diagnosed by the Labcorp NuSwab test to better understand disruptions to the vaginal microbiome during BV. We show that microbial sequencing can faithfully reproduce targeted PCR diagnostic results and can improve our knowledge of healthy and BV-associated microbial and metabolic biomarkers. This work highlights a robust, agnostic BV classification scheme with potential for future development of sequencing-based BV diagnostic tools.</p>","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":" ","pages":"e0258224"},"PeriodicalIF":3.7,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143492889","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":"Epidemiological characteristics and antimicrobial resistance of extensively drug-resistant <i>Acinetobacter baumannii</i> in ICU wards.","authors":"Jingchao Shi, Xiaoting Mao, Fengtian Sun, Jianghao Cheng, Lijia Shao, Xiaoyun Shan, Yijun Zhu","doi":"10.1128/spectrum.02619-24","DOIUrl":"10.1128/spectrum.02619-24","url":null,"abstract":"<p><p><i>Acinetobacter baumannii</i> is a significant nosocomial pathogen, particularly problematic due to its extensive drug resistance. This study investigates 56 extensively drug-resistant <i>A. baumannii</i> (XDRAB) strains collected from various ICU wards at Jinhua Central Hospital, Zhejiang Province, China. Strains were isolated from diverse clinical samples, including sputum, blood, cerebrospinal fluid, and wound secretions. Identification was confirmed via matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), and antibiotic susceptibility testing was conducted using the VITEK 2 Compact system, E-test, and Kirby-Bauer methods. All strains were susceptible to polymyxin, with four showing intermediate susceptibility to tigecycline, while resistance rates to other antibiotics were 100%. Molecular typing through pulsed-field gel electrophoresis (PFGE) classified the strains into 10 types, with the dominant type (G) primarily found in ICU3, indicating a potential clonal outbreak. Whole-genome sequencing (WGS) and multi-locus sequence typing (MLST) identified ST208 as the predominant sequence type. Resistance gene screening revealed the presence of blaOXA-23, blaTEM-1D, and aminoglycoside resistance genes in most strains. Phylogenetic analysis confirmed the clonal transmission of ST208 strains across the hospital, with a high degree of genomic similarity among the isolates. These findings highlight the importance of continuous monitoring and effective infection control measures to prevent the spread of XDRAB in healthcare settings.IMPORTANCEExtensively drug-resistant <i>Acinetobacter baumannii</i> is a critical public health threat, particularly in hospital environments where it causes a variety of infections. The global spread of extensively drug-resistant <i>A. baumannii</i> (XDRAB) and its resistance to most antibiotics make treatment options limited, increasing the risk of patient morbidity and mortality. This study provides important insights into the molecular epidemiology of XDRAB in a hospital setting, revealing the clonal transmission of the ST208 sequence type. By utilizing both pulsed-field gel electrophoresis (PFGE) and whole-genome sequencing (WGS), we identified genetic links between strains and the presence of key resistance genes. The findings underscore the urgent need for robust infection control protocols, routine surveillance, and judicious use of antibiotics to mitigate the spread of XDRAB and ensure better patient outcomes.</p>","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":" ","pages":"e0261924"},"PeriodicalIF":3.7,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143542561","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":"Outer membrane permeability of <i>Pseudomonas aeruginosa</i> through β-lactams: new evidence on the role of OprD and OpdP porins in antibiotic resistance.","authors":"Francesco Amisano, Paola Mercuri, Steven Fanara, Olivier Verlaine, Patrick Motte, Jean Marie Frère, Marc Hanikenne, Moreno Galleni","doi":"10.1128/spectrum.00495-24","DOIUrl":"10.1128/spectrum.00495-24","url":null,"abstract":"<p><p>Gram-negative bacteria are a major concern for public health, particularly due to the continuous rise of antibiotic resistance. A major factor that helps the development of resistance is the outer membrane that is essential since it acts as a strong permeability barrier to many antibiotics that are effective against other bacteria. In this study, we determine the specific permeability coefficients for various antibiotics in <i>Pseudomonas aeruginosa</i> strains, which differ from each other for their porin expressions. We showed that OprD and OpdP porins contribute both to internalize meropenem and biapenem. Using qRT-PCR, we demonstrated that their expression is dependent of the various phases of cellular growth. We were able to show how the OpdP porin is less expressed in exponential growth phases, while it tends to be produced when the bacterial culture enters into the latent phase, in an inversely proportional way compared to the OprD porin. The deletion of the OpdP porin, in the presence of meropenem at concentrations equivalent to the MIC values, contributes to the selection of carbapenem-resistant strains. Therefore, the presence of mutations/deletions of the OpdP porin should receive greater consideration from a clinical point of view as the use of meropenem at nonoptimal concentrations could lead to the appearance of resistance phenotypes.IMPORTANCECarbapenem-resistant strains of <i>Pseudomonas aeruginosa</i> are among the major threats to public health. The permeability of the outer membrane for the β-lactam antibiotics is one of the major factors that reduce the activity of the antibiotics. In this study, we measure the low permeability coefficient of the <i>P. aeruginosa</i> outer membrane to β-lactams. The methodology we develop to determine the permeability can be applied to other antibiotic families and/or pathogens.</p>","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":" ","pages":"e0049524"},"PeriodicalIF":3.7,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143542566","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":"Resistome and virulome determination in <i>Helicobacter pylori</i> using next-generation sequencing with target-enrichment technology.","authors":"Léo Gillet, Lucie Bénéjat, Quentin Jehanne, Pierre-Louis Maunet, Claudie Perreau, Astrid Ducournau, Johanna Aptel, Marine Jauvain, Philippe Lehours","doi":"10.1128/spectrum.03298-24","DOIUrl":"10.1128/spectrum.03298-24","url":null,"abstract":"<p><p>The identification of <i>Helicobacter pylori</i> infection from gastric biopsy samples requires PCR or bacterial cultures. However, it is difficult to culture <i>H. pylori</i> because it is a fragile bacterium. Next-generation sequencing (NGS) allows direct assessment of the resistome and virulome. Here we describe a new NGS method for studying the resistome and virulome of <i>H. pylori</i> directly from gastric biopsies, based on enrichment analyses and targeted sequencing of <i>H. pylori</i> DNA. In all, 19 DNA samples from human gastric biopsies that tested positive for <i>H. pylori</i> were analyzed. The Agilent SureSelectXT target-enrichment protocol was used with a custom bait library prior to sequencing using the Agilent MagnisDx NGS Library Prep System. NGS sequencing was performed on the Illumina iSeq 100 sequencer using RNA probes for virulence, resistance, and molecular typing genes. The method yielded significant results with a limit of detection of around 1.8e⁵ CFU per mL <i>H. pylori</i>. Mutations in the <i>23S rDNA</i> sequence associated with macrolide resistance and in the quinolone resistance-determining region of <i>gyrase A</i> associated with levofloxacin resistance were correctly identified. The results of MLST phylogeny analyses performed after target-enrichment were consistent with those obtained via conventional Sanger sequencing. Among the <i>cagA</i>-positive isolates, the gene was detected correctly, and the <i>vacA</i> genotype was determined. In conclusion, our enrichment method enables rapid assessment of the resistome and virulome of <i>H. pylori</i> directly from fresh gastric biopsies.IMPORTANCE<i>Helicobacter pylori</i>, a bacterium that infects at least 50% of the world population, is often treated by probabilistic antimicrobial therapies due to the lack of antimicrobial resistance data provided by clinical laboratories to clinicians. However, targeted antimicrobial therapies are increasingly recommended to achieve efficient eradication with a limited impact on the gut microbiota and with fewer adverse events for the patient. Recent advancements in next-generation sequencing strategies have opened new opportunities in the diagnosis of <i>H. pylori</i> infection. The significance of our research is the development of a novel next-generation sequencing strategy based on target-enrichment. This approach enables the identification of the resistome and the virulome of <i>H. pylori</i> directly from gastric biopsies, providing clinicians with a broad overview of therapeutic options.</p>","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":" ","pages":"e0329824"},"PeriodicalIF":3.7,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143557333","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":"Analytical assessment of metagenomic workflows for pathogen detection with NIST RM 8376 and two sample matrices.","authors":"Jason G Kralj, Stephanie L Servetas, Samuel P Forry, Monique E Hunter, Jennifer N Dootz, Scott A Jackson","doi":"10.1128/spectrum.02806-24","DOIUrl":"10.1128/spectrum.02806-24","url":null,"abstract":"<p><p>We assessed the analytical performance of metagenomic workflows using NIST Reference Material (RM) 8376 DNA from bacterial pathogens spiked into two simulated clinical samples: cerebrospinal fluid (CSF) and stool. Sequencing and taxonomic classification were used to generate signals for each sample and taxa of interest and to estimate the limit of detection (LOD), the linearity of response, and linear dynamic range. We found that the LODs for taxa spiked into CSF ranged from approximately 100 to 300 copy/mL, with a linearity of 0.96 to 0.99. For stool, the LODs ranged from 10 to 221 kcopy/mL, with a linearity of 0.99 to 1.01. Furthermore, discriminating different <i>E. coli</i> strains proved to be workflow-dependent as only one classifier:database combination of the three tested showed the ability to differentiate the two pathogenic and commensal strains. Surprisingly, when we compared the linear response of the same taxa in the two different sample types, we found those functions to be the same, despite large differences in LODs. This suggests that the \"agnostic diagnostic\" theory for metagenomics (i.e., any organism can be identified because DNA is the measurand) may apply to different target organisms and different sample types. Because we are using RMs, we were able to generate quantitative analytical performance metrics for each workflow and sample set, enabling relatively rapid workflow screening before employing clinical samples. This makes these RMs a useful tool that will generate data needed to support the translation of metagenomics into regulated use.IMPORTANCEAssessing the analytical performance of metagenomic workflows, especially when developing clinical diagnostics, is foundational for ensuring that the measurements underlying a diagnosis are supported by rigorous characterization. To facilitate the translation of metagenomics into clinical practice, workflows must be tested using control samples designed to probe the analytical limitations (e.g., limit of detection). Spike-ins allow developers to generate fit-for-purpose control samples for initial workflow assessments and inform decisions about further development. However, clinical sample types include a wide range of compositions and concentrations, each presenting different detection challenges. In this work, we demonstrate how spike-ins elucidate workflow performance in two highly dissimilar sample types (stool and CSF), and we provide evidence that detection of individual organisms is unaffected by background sample composition, making detection sample-agnostic within a workflow. These demonstrations and performance insights will facilitate the translation of the technology to the clinic.</p>","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":" ","pages":"e0280624"},"PeriodicalIF":3.7,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143586252","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}