Microbiology spectrum最新文献

{"title":"Identification and drug resistance profiling of <i>Mycobacterium orygis</i> from an African elephant using whole-genome sequencing.","authors":"Hassan Ghayas, Javaria Ashraf, Noureen Saeed, Sadaf Zaka, Samreen Shafiq, Joveria Farooqi, Kauser Jabeen, Abida Rais, Aamir Ismail Rizvi, Ghulam Mustafa, Samreen Ashraf Qureshi, Yusra Askari, Naseem Salahuddin, Egon A Ozer, Rumina Hasan","doi":"10.1128/spectrum.03586-25","DOIUrl":"https://doi.org/10.1128/spectrum.03586-25","url":null,"abstract":"<p><p>Tuberculosis (TB) caused by members of the <i>Mycobacterium tuberculosis</i> complex (MTBC) has been increasingly reported in wildlife and captive animals, posing zoonotic risks. Accurate diagnosis and speciation are essential to trace the infection source and to tailor therapy accordingly. Current diagnostic tools, however, have limitations in speciating MTBC strains. In this study, we investigated a lung biopsy sample of a deceased adult female elephant (<i>Loxodonta africana</i>) for mycobacterial infection. Mycobacterial Growth Indicator Tube culture was positive, and initial immunochromatographic testing (BD MPT64) confirmed the isolate as a member of the MTBC. To further confirm species identity and assess drug resistance, whole-genome sequencing was performed. Species was identified using Genome Detective and KmerFinder. Species identification and lineage analysis consistently classified the isolate as <i>Mycobacterium orygis</i> (lineage La3)<i>,</i> a rare and often overlooked zoonotic member of the MTBC. Antimicrobial resistance profiling through TB-Profiler and PhyResSE identified resistance to isoniazid, attributed to the presence of the katG p.Ser315Thr mutation. No resistance-associated mutations were detected for rifampicin, levofloxacin, or pyrazinamide. These findings were consistent with phenotypic drug susceptibility testing. The detection of <i>M. orygis</i> in a captive elephant suggests that the infection was of zoonotic origin and underscores its potential for spreading disease, and thus the need for integrated surveillance in animal reservoirs.</p><p><strong>Importance: </strong>This study reports the first case of <i>Mycobacterium orygis</i> infection in an African elephant in captivity. Currently, the methods for differentiation of <i>Mycobacterium tuberculosis</i> complex (MTBC) subspecies have limitations. This case highlights the importance of precise species identification and drug resistance profiling of MTBC organisms and the use of whole-genome sequencing in diagnosis. Additionally, our findings underline the need for surveillance of mycobacteria and other potential pathogens in captive animals and in animal caregivers to reduce the potential for zoonotic disease transmission.</p>","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":" ","pages":"e0358625"},"PeriodicalIF":3.8,"publicationDate":"2026-05-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147840092","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":"KEGG orthology-based machine learning reveals functional determinants of antimicrobial resistance in <i>Acinetobacter baumannii</i>.","authors":"Zhihang Zheng, Bei Jiang, Abebe Mekuria Shenkutie, Jingyuan Bian, Yuyao Yan, Ruizhen Pi, Qing Xiong, Polly Hang-Mei Leung","doi":"10.1128/spectrum.02592-25","DOIUrl":"https://doi.org/10.1128/spectrum.02592-25","url":null,"abstract":"&lt;p&gt;&lt;p&gt;Antimicrobial resistance represents a critical global health threat, with &lt;i&gt;Acinetobacter baumannii&lt;/i&gt; classified as a priority pathogen due to its extensive multidrug resistance. Currently, antimicrobial susceptibility testing relies on phenotypic characterization, which provides limited mechanistic insights into resistance mechanisms and significantly delays appropriate therapy. While machine learning approaches have shown promise for rapid resistance prediction, existing computational methods rely predominantly on gene presence-absence matrices that inadequately capture functional capacity. To address this limitation, we developed a functional genomics framework using KEGG orthology annotations to predict antimicrobial resistance phenotypes directly from whole-genome sequencing data. We systematically evaluated six machine learning algorithms on 1,088 &lt;i&gt;A. baumannii&lt;/i&gt; isolates across 10 antimicrobial agents, with subsequent validation in an independent cohort of 508 isolates. KEGG orthology-based representations consistently outperformed traditional gene presence-absence encoding across all algorithms. XGBoost achieved superior performance (94.44% weighted accuracy) and was therefore selected for interpretability analysis. Identified determinants included known carbapenemases and, crucially, K18974 (&lt;i&gt;sul1&lt;/i&gt;), a ubiquitous marker of integron-mediated propagation. Additionally, K21801 (&lt;i&gt;iaaH&lt;/i&gt;) was identified as a key driver of non-conventional resistance via indole-mediated signaling. Subsequent multidrug resistance analysis identified shared determinants-overlapping KEGG orthologs across antimicrobial classes-that generate predictable cross-resistance patterns. These computationally derived molecular networks closely mirrored the observed clinical co-resistance phenotypes. Overall, this interpretable machine learning framework advances mechanistic understanding of resistance evolution and establishes a promising computational foundation. Future prospective validation is required to translate these findings into clinical utility.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Importance: &lt;/strong&gt;&lt;i&gt;Acinetobacter baumannii&lt;/i&gt; represents one of the most urgent antimicrobial resistance threats globally, and traditional diagnostic methods can take up to 48 hours to determine appropriate treatment options. This delay contributes to poor patient outcomes and drives further resistance development through inappropriate antibiotic use. This study addresses a critical gap by developing a machine learning framework that can rapidly predict antimicrobial resistance patterns directly from whole-genome sequencing data. Unlike previous approaches that rely on simple gene presence-absence data, our method uses functional genomic annotations to capture the complex biological processes underlying resistance. The framework not only achieves superior prediction accuracy but also provides interpretable insights into resistance mechanisms, identifying both known resistance gene","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":" ","pages":"e0259225"},"PeriodicalIF":3.8,"publicationDate":"2026-05-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147840080","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":"Dengue-specific T-cell memory following natural infection: a comparative analysis between people living with HIV and HIV-negative individuals.","authors":"Francesca Lombardi, Antonio Abatino, Pierluigi Francesco Salvo, Ilenia Aversa, Gianmaria Baldin, Raffaella Gallo, Enrica Tamburrini, Patrizia Laurenti, Valeria Campolattano, Simona Di Giambenedetto, Carlo Torti, Camillo Palmieri","doi":"10.1128/spectrum.02899-25","DOIUrl":"https://doi.org/10.1128/spectrum.02899-25","url":null,"abstract":"<p><p>People living with HIV (PLWH) represent a population of particular interest in the context of arboviral infections. This observational study aimed to evaluate and compare the magnitude and functionality of dengue virus (DENV)-specific T-cell memory elicited by natural DENV infection in PLWH and HIV-negative individuals. Nineteen participants infected during the 2023 autochthonous dengue outbreak in Rome (10 PLWH and 9 HIV-negative individuals) were assessed for interferon gamma (IFN-γ)-releasing cells in peripheral blood mononuclear cells (PBMCs) using an IFN-γ -ELISpot assay following stimulation with peptide pools.When examining both the overall cumulative ELISpot responses, calculated as the sum of spot-forming units (SFU) across all antigens or the binary responder status to each DENV antigen, HIV-negative individuals showed higher and broader cumulative responses than PLWH, but differences did not reach statistical significance. However, responses to prM/E/C antigens were higher in the HIV-negative group, reaching statistical significance (<i>P</i> = 0.041). Among PLWH, no association was found between T-cell reactivity and CDC stage at diagnosis. Our findings suggest that while PLWH with well-controlled infection retain dengue-specific T-cell memory, their response to structural antigens may be attenuated. Vaccination should be considered a key preventive strategy for this clinically vulnerable population.</p><p><strong>Importance: </strong>The geographic expansion of dengue virus (DENV) transmission into temperate regions highlights the need to characterize immune responses in populations with chronic conditions such as HIV. Despite immune reconstitution under antiretroviral therapy (ART), people living with HIV (PLWH) may exhibit residual immune dysregulation that could affect DENV-specific immunity and clinical outcomes. In this study, we directly compared DENV-specific T-cell memory responses in PLWH and HIV-negative individuals following natural infection during an autochthonous outbreak. While PLWH mounted detectable cellular responses, reactivity to structural antigens (prM/E/C) was significantly reduced compared with HIV-negative participants. These findings indicate that qualitative differences in antigen-specific T-cell memory may persist despite virological suppression and immune recovery, with potential implications for protection upon re-exposure. Our results provide a rationale for evaluating dengue vaccination strategies in PLWH as local transmission becomes more frequent in non-endemic settings.</p>","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":" ","pages":"e0289925"},"PeriodicalIF":3.8,"publicationDate":"2026-05-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147840098","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":"Unveiling hidden microbial diversity in Mars 2020 mission assembly cleanrooms with molecular insights into the persistence and perseverance of novel species defying metagenome sequencing.","authors":"Shobhan Karthick Muthamilselvi Sivabalan, Varsha Vijayakumar, Pratyay Sengupta, Siddhakam Palmal, Srinivasan Krishnamurthi, Nitin Kumar Singh, Nikos C Kyrpides, Karthik Raman, Kasthuri Venkateswaran","doi":"10.1128/spectrum.01273-25","DOIUrl":"https://doi.org/10.1128/spectrum.01273-25","url":null,"abstract":"&lt;p&gt;&lt;p&gt;NASA cleanrooms, which are critical for assembling space mission components, are maintained under stringent decontamination protocols to minimize biological contamination. These environments are characterized by nutrient-poor and oligotrophic conditions, leading to low microbial loads. Despite extensive cleaning, oligotrophs capable of surviving in such conditions continue to persist, often remaining undetected due to their low abundance, resistance to environmental stresses, and difficulties in biomolecule extraction. Even with shotgun metagenome sequencing technologies, these microbes may go undetected or be underrepresented due to their robust cell walls and the absence of reference genomes in publicly available databases. Over a 6-month study of Mars 2020 mission cleanrooms, 182 bacterial strains belonging to 19 families were identified using a whole-genome sequencing (WGS) approach. Among these, 14 novel Gram-positive species were discovered, including eight spore formers. Though the novel species comprised only 0.001% of the sequencing data, their successful cultivation allowed for functional characterization. Through WGS data mining, genomic traits associated with resilience in extreme conditions were revealed. These species were found to be involved in nitrogen cycling, carbohydrate metabolism, and radiation resistance, traits essential for survival in extreme environments. Furthermore, 12 biosynthetic gene clusters were identified, including those linked to ectoine and [Formula: see text]-poly-L-lysine production, suggesting potential biotechnological applications. These findings highlight the hidden microbial diversity within cleanrooms and emphasize the necessity of advanced detection strategies. A better understanding of these microbes will provide insights into extremophiles with applications in biotechnology, medical research, and life support systems for future space exploration missions.IMPORTANCEDespite strict decontamination protocols, NASA cleanrooms harbor low-biomass microbial communities adapted to nutrient-poor environments. These oligotrophic microbes often go undetected in shotgun metagenomics methods due to their low abundance, resistance to lysis, and lack of reference genomes. Standard shotgun metagenome sequencing methods fail to retrieve them, as dominant microbial DNA overshadows rare species. Over 6 months of monitoring Mars 2020 mission cleanrooms, 182 bacterial strains from 19 families were identified, including 14 novel Gram-positive species, 8 of which were spore formers. Though present at 0.001% abundance in sequencing data, we successfully cultured them, enabling functional characterization. These microbes exhibited roles in nitrogen cycling, carbohydrate metabolism, and radiation resistance, with 12 biosynthetic gene clusters linked to ectoine and [Formula: see text]-poly-L-lysine production. These findings highlight the previously underestimated microbial diversity in cleanrooms and emphasize the need","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":" ","pages":"e0127325"},"PeriodicalIF":3.8,"publicationDate":"2026-05-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147840238","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":"PRV-delgE/gI/TK immunization preserves blood-brain barrier integrity and limits CNS injury following intracerebral PRV challenge.","authors":"Tong Xu, Baoling Liu, Hanyu Li, Lei Zhao, Yangming Dai, Xin Wu, Siyuan Lai, Jian-Bo Huang, Zhi-Wen Xu, Ling Zhu","doi":"10.1128/spectrum.03930-25","DOIUrl":"https://doi.org/10.1128/spectrum.03930-25","url":null,"abstract":"<p><p>Pseudorabies virus (PRV) causes rapidly progressive and often fatal encephalitis in both natural and non-natural hosts, making the development of effective preventive strategies critically important. Among current vaccine candidates, the triple-gene-deleted PRV strain (PRV-delgE/gI/TK) has been widely studied; however, its protective effects against brain injury, particularly blood-brain barrier (BBB) damage, remain poorly characterized. To address this gap, we established an intracerebral challenge model in C57BL/6J mice to directly evaluate central nervous system (CNS) outcomes following wild-type PRV infection. Wild-type PRV challenge resulted in severe neurological deficits, uniform mortality, extensive neuronal degeneration, pronounced BBB disruption, downregulation of tight junction proteins, strong induction of MMP-2/9, and a robust inflammatory cytokine surge. In contrast, mice immunized with PRV-delgE/gI/TK exhibited only mild and transient symptoms, rapid functional recovery, markedly reduced viral replication, and complete viral clearance by 14 days. Immunization preserved BBB integrity, maintained ZO-1 and occludin expression, prevented MMP activation, and substantially blunted neuroinflammatory responses. Collectively, these results show that PRV-delgE/gI/TK immunization effectively protects against wild-type PRV-induced CNS injury in C57BL/6J mice, as evidenced by reduced viral burden, preserved BBB integrity, and attenuated neuroinflammatory responses, and provide new insight into the neuroprotective effects of attenuated PRV vaccination.IMPORTANCEPseudorabies virus (PRV) can cause severe and often fatal encephalitis, but the central nervous system (CNS)-protective effects of attenuated PRV vaccines remain insufficiently characterized, particularly with respect to blood-brain barrier (BBB) injury. Using a C57BL/6J intracerebral challenge model, this study shows that immunization with the triple-gene-deleted PRV-delgE/gI/TK strain markedly reduces viral replication, preserves BBB integrity, and attenuates neuroinflammatory responses after wild-type PRV challenge. These findings provide <i>in vivo</i> evidence that attenuated PRV immunization can protect against PRV-induced CNS injury and offer important insights for improving prevention strategies against neurotropic PRV infection.</p>","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":" ","pages":"e0393025"},"PeriodicalIF":3.8,"publicationDate":"2026-05-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147840201","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":"Prospective study of vaginal samples for the detection of <i>Gardnerella vaginalis</i> and <i>G. swidsinskii/leopoldii</i> using Gardnerella agar in CO₂ and anaerobic atmospheres.","authors":"Irena Grmek Košnik, Ana Ribnikar, Ema Ogrizek, Jelena Kovačević","doi":"10.1128/spectrum.03668-25","DOIUrl":"https://doi.org/10.1128/spectrum.03668-25","url":null,"abstract":"<p><p><i>Gardnerella vaginalis</i> is a key bacterium in the etiology of bacterial vaginosis (BV) and contributes to biofilm formation. <i>Gardnerella</i> is a facultative anaerobic bacterium, meaning it can grow both in the presence of oxygen and under anaerobic conditions. We conducted a prospective study in 2025, including 160 consecutive vaginal samples from women submitted for pathogenic bacteria. Clinical information on the referral form indicated in 87 women (54.4%), from which colpitis in 64 cases (73.6%). There were 43 pregnant women (26.9%), with an average age of 30.7 years. Each vaginal sample was cultured on Gardnerella agar and incubated in both CO₂ and anaerobic atmospheres. The culture plates were examined after 24 and 48 h of incubation, independently of other results. In cases where suspicious colonies with beta-hemolysis were observed, bacterial identification was performed using MALDI-TOF mass spectrometry (Bruker). Gram-stained smears were evaluated according to the Nugent score. The bacteria <i>Gardnerella vaginalis</i> and <i>Gardnerella leopoldii/swidsinskii</i> were detected in 46 (28.8%) samples. The sensitivity of <i>G. vaginalis</i> and <i>G. leopoldii/swidsinskii</i> cultivation on Gardnerella agar in a CO₂ atmosphere was 67.4% compared to anaerobic cultivation. Anaerobic cultivation on Gardnerella agar proved to be a superior method for the isolation of <i>G. vaginalis</i> and <i>G. leopoldii/swidsinskii</i> compared to cultivation in a CO₂ atmosphere. The diagnosis of BV in clinical practice relies on standard Amsel's criteria, gram-stained vaginal smears evaluated using the Nugent scoring system or Ison-Hay criteria, and the isolation of <i>G. vaginalis</i> and <i>G. leopoldii/swidsinskii</i>.IMPORTANCEBacterial vaginosis (BV) is the most common vaginal infection among women of reproductive age and is associated with significant adverse outcomes, including preterm birth, spontaneous abortion, and increased susceptibility to sexually transmitted infections. Accurate and timely microbiological diagnosis is therefore clinically important. Our study demonstrates that anaerobic incubation on selective Gardnerella agar improves the detection of <i>Gardnerella vaginalis</i> and <i>Gardnerella leopoldii</i>/<i>swidsinskii</i> compared with incubation in a CO₂-enriched atmosphere. Under anaerobic conditions, colonies were larger, hemolysis was more pronounced, and overall sensitivity was higher. Although BV diagnosis in clinical practice primarily relies on Amsel's criteria and Nugent scoring, optimized culture conditions provide valuable complementary microbiological evidence. These findings contribute to the optimization of laboratory procedures and may enhance the reliability of routine diagnostic workflows in women with suspected BV.</p>","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":" ","pages":"e0366825"},"PeriodicalIF":3.8,"publicationDate":"2026-05-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147840148","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":"Optimized use of the FilmArray Meningitis/Encephalitis panel for early discontinuation of antibiotic therapy.","authors":"Nathan Nicolau-Guillaumet, Marin Moutel, Chloé Plouzeau-Jayle, Gauthier Pean de Ponfilly, VIrginie Courbin, Anne-Gaëlle Ranc, Hélène Revillet, Bruno Mourvillier, Maxime Hentzien, Anaëlle Muggeo, Thomas Guillard","doi":"10.1128/spectrum.00566-26","DOIUrl":"https://doi.org/10.1128/spectrum.00566-26","url":null,"abstract":"<p><p>Suspected meningitis/encephalitis cases require prompt empirical antimicrobial therapy. Syndromic diagnostics like the FilmArray Meningitis/Encephalitis (FAME) panel offer rapid results and are increasingly used despite high costs. This study assessed FAME's integration in clinical microbiology laboratories' everyday practice and its impact on early treatment decisions. The FilmArray Meningitis/encephalitis panel Outcome Study (FAMOuS) retrospectively reviewed biological data, treatments before/after FAME, and outcomes from 783 patients across six French hospitals over a 3-year period. FAME results were 80% negative, 7% bacterial, and 13% viral, with no bacterial false negatives. Positive results in samples with <10 leukocytes were exclusively seen in immunocompromised individuals and children under 2. Positive results prompted therapeutic changes in 74% of cases, compared to 50% for negatives. FAMOuS enabled the proposal of a decision-support flowchart to optimize the rational use of FAME and support antibiotic stewardship, with the potential to increase early antibiotic discontinuation and reduce unnecessary testing.IMPORTANCEPrompt identification of infectious causes is essential in suspected meningitis and encephalitis, where timely treatment decisions can be lifesaving. The FAMOuS study evaluated the use of the FilmArray Meningitis/Encephalitis (FAME) panel across six French hospitals, analyzing nearly 800 patient episodes. The findings demonstrate that FAME offers reliable results that directly influence clinical management-guiding therapeutic adjustments in most positive cases and supporting early antibiotic discontinuation when appropriate. The study highlights specific patient contexts where FAME provides the greatest clinical value and proposes a decision-support flowchart to optimize its rational use. By integrating rapid molecular diagnostics into everyday practice, this work contributes to improved patient care and reinforces hospital antibiotic stewardship strategies.</p>","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":" ","pages":"e0056626"},"PeriodicalIF":3.8,"publicationDate":"2026-05-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147840147","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":"Microbiome in women with endometriosis and the <i>in vitro</i> effects of <i>Lactobacillus reuteri</i> on human endometrium.","authors":"Jae Hoon Lee, Gee Soo Jung, Kyungmin Kim, Hyemin Park, Yunjeong Park, Inha Lee, Min Jung Lee, Ji-Ho Lee, Young Sik Choi, SiHyun Cho","doi":"10.1128/spectrum.03689-25","DOIUrl":"https://doi.org/10.1128/spectrum.03689-25","url":null,"abstract":"<p><p>Endometriosis (EMS) is a chronic inflammatory disorder affecting ~10% of reproductive-age women, with increasing evidence implicating the microbiome in its pathogenesis through immunomodulation and estrogen metabolism. This study investigated microbiome composition in the vagina, endometrium, and peritoneal fluid (PF) of women with and without EMS and further assessed the effects of <i>Lactobacillus reuteri</i> (<i>L. reuteri</i>) on endometrial (EM) cells <i>in vitro</i>. Samples from 41 patients were analyzed using 16S rRNA gene sequencing, targeting the V3-V4 regions. Western blotting, ELISA, and LC-MS/MS were employed to evaluate protein expression and estrogen metabolism during EM-<i>L</i>. <i>reuteri</i> co-culture with or without estradiol-17-glucuronide (E2G). Microbiome analysis revealed no significant differences in alpha or beta diversity between EMS and controls across all compartments. However, LEfSe analysis identified several taxa with differential abundance, with <i>L. reuteri</i> consistently altered in both vagina and EM. Across the menstrual cycle, EM and vaginal microbiomes were stable, whereas PF microbiota showed phase-dependent variation involving 60 genera and 76 species. <i>In vitro</i>, <i>L. reuteri</i> alone did not alter endometriosis-related proteins, but in the presence of E2G, it reduced BAX/Bcl-2 ratios and increased p-NF-κB, suggesting anti-apoptotic and pro-inflammatory shifts. Progesterone receptor α/β expression decreased, while estrogen receptor levels remained unchanged. <i>L. reuteri</i> increased β-glucuronidase activity but did not enhance E2G-to-estradiol conversion. These findings highlight <i>L. reuteri</i> as a potentially important species in EMS, with <i>in vitro</i> evidence suggesting survival-promoting effects under estrogenic conditions. Further research should explore multi-species interactions and hormonal contexts to clarify microbial contributions to EMS pathogenesis.</p><p><strong>Importance: </strong>Although <i>Lactobacillus reuteri</i> appeared more abundant in the vagina and endometrium of controls, suggesting a protective role, <i>in vitro</i> findings paradoxically indicated anti-apoptotic and pro-inflammatory effects under estrogenic conditions, underscoring the need for further investigation of multi-species microbial interactions and hormonal contexts in endometriosis pathogenesis.</p>","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":" ","pages":"e0368925"},"PeriodicalIF":3.8,"publicationDate":"2026-05-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147840059","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":"Reducing culture underestimation in urine: flow cytometry-guided validation of low-dose DTT pre-treatment for biofilm dispersal.","authors":"Luigi Regenburgh De La Motte, Ivan Muradore, Fabiana Giarritiello, Erica Stefàno, Loredana Deflorio, Lorenzo Drago","doi":"10.1128/spectrum.03910-25","DOIUrl":"https://doi.org/10.1128/spectrum.03910-25","url":null,"abstract":"&lt;p&gt;&lt;p&gt;A substantial proportion of symptomatic patients with urinary tract infections (UTIs) have negative or low-count urine cultures despite clinically compatible presentations. Biofilm-associated bacterial aggregation and the presence of viable bacteria not recovered by standard culture may contribute to this diagnostic gap. Flow cytometry (FACS) can detect these viable cells, but culturability remains essential for species identification and antimicrobial susceptibility testing. We evaluated whether low-concentration dithiothreitol (DTT) can safely disperse urinary biofilm-associated aggregates and improve bacterial recovery from urine samples. Seventy-two clinical urine samples were processed in parallel (untreated vs 0.1% DTT-treated). Viability was quantified using SYTO9/PI-based flow cytometry with bead-normalized bacterial counts. Culturability was assessed on chromogenic media. Comparative analyses included paired statistics, Bland-Altman agreement, and correlation between viable and culturable fractions. DTT (0.1%) did not alter bacterial viability (&lt;i&gt;P&lt;/i&gt; = 0.47), confirming its non-toxicity at this concentration. In contrast, DTT significantly increased CFU recovery (median: 10 to 5.5 × 10³ CFU/mL; &lt;i&gt;P&lt;/i&gt; &lt; 0.0001), converting several previously negative or low-count samples into positive cultures. Agreement between viable and culturable fractions improved after DTT treatment, primarily through reduced systematic bias in Bland-Altman analysis, while correlation coefficients remained similar (r: 0.44 vs 0.45). A persistent discrepancy between FACS-derived viable counts and culture-based counts suggests the presence of viable bacterial populations not fully recovered by standard culture conditions, findings that are consistent with but do not directly demonstrate the presence of VBNC subpopulations. Low-dose DTT is a safe and effective pre-analytical treatment that markedly improves culture sensitivity without compromising bacterial viability. These findings support further investigation of DTT-assisted processing as a potential strategy to reduce culture underestimation in urinary diagnostics and to better characterize viable bacterial populations not detected by standard culture methods.IMPORTANCEStandard urine culture may fail to detect the full spectrum of bacteria present in urinary tract infections, particularly when microorganisms are embedded within mucus, biofilm-like aggregates, or enter a viable but non-culturable state. These hidden bacterial populations may contribute to persistent symptoms or recurrent infections while remaining undetected by routine culture methods. Our findings suggest that pre-treating urine samples with a low concentration of dithiothreitol may help disperse biofilm-like aggregates while preserving bacterial viability. When combined with flow cytometry, this approach may allow a more comprehensive estimation of the viable bacterial fraction and, in some cases, may increase the likelihood of bacteri","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":" ","pages":"e0391025"},"PeriodicalIF":3.8,"publicationDate":"2026-05-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147840167","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 cocktail of SARS-CoV-2 S stem helix domain and receptor binding domain human monoclonal antibodies prevents the emergence of viral escape mutants.","authors":"Yao Ma, Chengjin Ye, Michael S Piepenbrink, Sara H Mahmoud, Anastasija Cupic, Esteban Castro, Nathaniel Jackson, Mahmoud Bayoumi, Alvaro S Padron, Adolfo García-Sastre, Gregory C Ippolito, Mark R Walter, James J Kobie, Luis Martinez-Sobrido","doi":"10.1128/spectrum.00006-26","DOIUrl":"https://doi.org/10.1128/spectrum.00006-26","url":null,"abstract":"<p><p>Neutralizing antibodies (NAbs) targeting the spike (S) glycoprotein remain a crucial therapeutic strategy against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, emerging viral variants have escaped all Food and Drug Administration (FDA)-approved NAb treatments, underscoring the urgent need for effective therapeutic alternatives. Using a nanoluciferase (Nluc)-expressing attenuated recombinant SARS-CoV-2 lacking the open reading frames (ORFs) 3a and 7b (Δ3a7b-Nluc), we characterized resistance profiles of two broadly protective NAbs targeting the S receptor binding domain (RBD) in S1 (1301B7) and the stem helix domain (SH) in S2 (1249A8). Serial passaging of Δ3a7b-Nluc under selective pressure identified a 1301B7 antibody-resistant mutant (ARM-B7) harboring an RBD mutation (S371F) that conferred resistance to 1301B7 and other RBD-directed NAbs (casirivimab, SC27, and sotrovimab). In contrast, no ARM emerged under treatment with 1249A8 or an antibody cocktail of 1301B7 (RBD) + 1249A8 (SH). These findings demonstrate that escape mutations in S2 SH are not easily tolerated by the virus compared with S1 RBD, and that a NAbs cocktail therapy targeting the SARS-CoV-2 S1 RBD and S2 SH offers the most effective strategy to prevent the emergence of escape mutations. Together, our findings provide critical insights into developing next-generation resistance-evading NAb therapies against SARS-CoV-2, and potentially other coronaviruses, and demonstrate the value of using our attenuated viral platforms for the safe identification of ARM without the potential biosafety concerns of doing these experiments using wild-type (WT) forms of SARS-CoV-2.</p><p><strong>Importance: </strong>The clinical efficacy of early SARS-CoV-2 NAbs has been challenged by the emergence of escape viral variants, highlighting an urgent need to anticipate resistance. Using a luminescent attenuated SARS-CoV-2 platform, we profiled resistant mutations against two broadly protective SARS-CoV-2 NAbs. Passage of Δ3a7b-Nluc in the presence of a NAb targeting the RBD S1 domain (1301B7) readily selected for an ARM, whereas passage in the presence of an SH S2 domain NAb (1249A8) did not. Notably, a cocktail of 1301B7 and 1249A8 created a high barrier of selecting SARS-CoV-2 ARM, preventing the emergence of resistant variants. We identified an S371F mutation in the S1 RBD of ARM-B7 that confers resistance to 1301B7 and other S1 RBD-targeting NAbs. These results highlight the importance of combination therapies targeting both variable RBD S1 and conserved SH S2 domain for the efficient treatment of SARS-CoV-2 and to prevent the emergence of NAb-induced escape mutations.</p>","PeriodicalId":18670,"journal":{"name":"Microbiology spectrum","volume":" ","pages":"e0000626"},"PeriodicalIF":3.8,"publicationDate":"2026-05-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147840113","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}