Journal of microbiological methods最新文献_第7页

Construction and application of MS2 bacteriophage virus-like particles for SARS-CoV-2 detection using a single-plasmid system 单质粒系统检测SARS-CoV-2 MS2噬菌体病毒样颗粒的构建及应用

IF 1.9 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Journal of microbiological methods

Pub Date : 2025-11-25 DOI: 10.1016/j.mimet.2025.107344

Mengjie Liang , Yongxin Li , Jingyuan Yang , Chunyan Liu , Haojie Lin , Zhaohui Deng , Xin Zhang

Research on the compatibility of MS2 bacteriophage virus-like particles (VLPs), which are used for the detection of a range of RNA viruses, with commercially available detection kits is limited. Here, we aimed to construct a positive control for RNA virus nucleic acid detection using MS2 bacteriophage VLPs and evaluate its compatibility with commercial nucleic acid detection kits to support the standardization of molecular diagnostic quality control systems. We generated recombinant plasmids expressing MS2 bacteriophage maturation enzyme, capsid proteins, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) target genes (ORF1ab, N, and E) using a single-plasmid dual-expression system (pACYCDuet-1). The plasmids were expressed in Escherichia coli BL21, and then MS2 VLPs were purified. The VLPs were characterized, and their performance as a positive control was validated for homogeneity and stability; their detection compatibility with five commercial SARS-CoV-2 nucleic acid detection kits was also examined. We successfully generated MS2 VLPs carrying SARS-CoV-2 target genes, with a uniform particle size of 23–28 nm and target gene copy number of 1.99 × 10¹⁰ copies/μL. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis confirmed the expression of MS2 proteins, whereas functional validation revealed excellent nuclease resistance, batch–batch homogeneity, and stability at 4 °C for ≥20 days. Importantly, the positive control exhibited consistent detection performance across all five commercial kits, confirming its practicality for routine laboratory workflows. The established positive control is readily applicable for standardizing quality control in molecular diagnostics.

用于检测一系列RNA病毒的MS2噬菌体病毒样颗粒（vlp）与市售检测试剂盒的兼容性研究有限。本研究旨在利用MS2噬菌体VLPs构建RNA病毒核酸检测阳性对照，并评价其与市售核酸检测试剂盒的兼容性，为分子诊断质量控制体系的标准化提供支持。我们使用单质粒双表达系统（pACYCDuet-1）生成了表达MS2噬菌体成熟酶、衣壳蛋白和严重急性呼吸综合征冠状病毒2 （SARS-CoV-2）靶基因（ORF1ab、N和E）的重组质粒。质粒在大肠杆菌BL21中表达，然后纯化MS2 VLPs。对VLPs进行了表征，并对其作为阳性对照的性能进行了均匀性和稳定性验证；并检测其与5种市售SARS-CoV-2核酸检测试剂盒的检测兼容性。成功制备了携带SARS-CoV-2靶基因的MS2 VLPs，粒径为23-28 nm，靶基因拷贝数为1.99 × 1010 copies/μL。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳证实了MS2蛋白的表达，而功能验证显示了出色的核酸酶抗性，批批次均匀性和在4°C下≥20天的稳定性。重要的是，阳性对照在所有五种商用试剂盒中表现出一致的检测性能，证实了其在常规实验室工作流程中的实用性。所建立的阳性对照易于应用于分子诊断的标准化质量控制。

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引用次数: 0

Single-cell RNA seq reveals heterogeneous survival strategies of Vibrio parahaemolyticus against ampicillin 单细胞RNA测序揭示副溶血性弧菌对氨苄西林的异质生存策略

IF 1.9 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Journal of microbiological methods

Pub Date : 2025-11-24 DOI: 10.1016/j.mimet.2025.107343

Zhangxi Gong , Yu Zhou , Yongbing Ba , Yao Yang , Yingjie Pan , Yong Zhao , Haiquan Liu

Bulk RNA-Seq only captures the average gene expression level of bacterial populations and has limitations in analysing functional differences between bacterial subpopulations. Therefore, the study innovatively applied droplet-based single-cell RNA sequencing (scRNA-seq) to resolve ampicillin resistance heterogeneity in Vibrio parahaemolyticus (V. parahaemolyticus). Findings indicate that high levels of drug resistance (MIC = 1000 μg/mL) demonstrate a significant impact on the phenotypes of interest, particularly when subinhibitory concentrations are considered. To further elucidate the phenotypic heterogeneity results, functionally specialized cellular subpopulations was identified by scRNA-seq, such as an oxidative stress defense subpopulation mediated by gorA upregulation, virulence modulation achieved through exsD-mediated repression of the type III secretion system, drug resistance conferred by MATE family efflux pumps, protein homeostasis maintained by Hsp20, metabolic reprogramming driven by NirD/YgiW/YdeI family proteins, and membrane damage repair facilitated by pspA upregulation. The phenotypic heterogeneity analysis results support some of the newly discovered functional annotations of resistant subpopulations, demonstrating the biological basis of bacterial heterogeneity under antibiotic pressure. The discovery of these functional drug-resistant subpopulations provides deeper insight into V. parahaemolyticus resistance and paves the way for developing novel antimicrobial strategies targeting key signaling proteins for population-level coordination.

Bulk RNA-Seq仅捕获细菌群体的平均基因表达水平，在分析细菌亚群体之间的功能差异方面存在局限性。因此，本研究创新性地应用基于液滴的单细胞RNA测序技术（scRNA-seq）来分析副溶血性弧菌（V. parahaemolyticus）氨苄西林耐药异质性。研究结果表明，高水平的耐药（MIC = 1000 μg/mL）对感兴趣的表型有显著影响，特别是当考虑亚抑制浓度时。为了进一步阐明表型异质性结果，通过scRNA-seq鉴定了功能特化的细胞亚群，例如由gorA上调介导的氧化应激防御亚群，通过exsd介导的III型分泌系统抑制实现的毒力调节，MATE家族外排泵赋予的耐药性，由Hsp20维持的蛋白稳态，由nrd /YgiW/YdeI家族蛋白驱动的代谢重编程。以及pspA上调促进的膜损伤修复。表型异质性分析结果支持一些新发现的耐药亚群的功能注释，证明了抗生素压力下细菌异质性的生物学基础。这些功能性耐药亚群的发现提供了对副溶血性弧菌耐药性的更深入了解，并为开发针对关键信号蛋白的新型抗菌策略铺平了道路，以实现群体水平的协调。

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引用次数: 0

Bioconversion of naringin to naringenin by a characterized Naringinase from aspergillus Niger HO32: Toward the valorization of citrus flavonoids 来自黑曲霉HO32的柚皮酶将柚皮苷生物转化为柚皮素：柑橘类黄酮的增殖研究。

IF 1.9 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Journal of microbiological methods

Pub Date : 2025-11-22 DOI: 10.1016/j.mimet.2025.107342

Nour Eddine Bentouhami , Sarra Yousfi , Meryem Idrissi Yahyaoui , Sara Moumnassi , Fatima Brahmi , Reda Bellaouchi , Houssam Abouloifa , Nabil Ghabbour , Ennouamane Saalaoui , Bassem Jaouadi , Abdelkarim Abousalham , Adem Gharsallaoui , Nour Eddine Chihib , Loubna Firdaous , Abdeslam Asehraou

In this study, we investigated the production and characterization of a naringinase by Aspergillus niger HO32, after which the enzyme was precipitated by fractionation using 80% ammonium sulfate and dialyzed at 4 °C overnight. The obtained results showed that its optimum pH and temperature are 5.5 and 55 °C, respectively, and it was stable at a pH range between 3 and 7 and at temperatures of 40, 50, and 60 °C. Naringinase is also capable of hydrolyzing the glycosides naringin, hesperidin, and others. HPLC-MS analysis, demonstrating the bioconversion of naringin to naringenin with a simultaneous increase of naringenin content. The K_m, V_max, and k_cat parameters using naringin substrate were 2 mM, 1428.6 U/mg, and 340.14 min⁻¹, respectively. The results obtained suggest that our naringinase has revealed an immense potential for application in the food sciences and industrial biotechnology fields, mainly for the bio-debittering of orange juices, and for the biotransformation of various foods.

在这项研究中，我们研究了黑曲霉HO32的柚皮苷酶的生产和表征，之后用80 %硫酸铵分离酶，并在4 °C下透析过夜。结果表明，其最适pH值为5.5，最适温度为55 °C，最适pH值为3 ~ 7，最适温度为40、50、60 °C。柚皮苷酶还能水解柚皮苷、橙皮苷和其他苷类。高效液相色谱-质谱分析表明，柚皮苷的生物转化为柚皮素，同时柚皮素含量增加。柚皮苷底物的Km、Vmax和kcat参数分别为2 mM、1428.6 U/mg和340.14 min-1。研究结果表明，我们的柚皮酶在食品科学和工业生物技术领域具有巨大的应用潜力，主要用于橙汁的生物脱臭和各种食品的生物转化。

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引用次数: 0

Evaluation of IgG anti-L. tropica antibody response as a biomarker for cutaneous leishmaniasis using ELISA in endemic regions of Pakistan IgG抗l抗体的评价。热带抗体反应作为皮肤利什曼病在巴基斯坦流行地区使用ELISA的生物标志物

IF 1.9 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Journal of microbiological methods

Pub Date : 2025-11-22 DOI: 10.1016/j.mimet.2025.107340

Bashair Sheikh Pervez , Arshad Islam , Azhar Minhas , Yusuf Ozbel , Seray Toz , Shahid Waseem , Obaid Hayat , Shumaila Naz

Cutaneous leishmaniasis (CL) has diverse clinical manifestations that overlap with other skin conditions. Conventional diagnostic methods, such as microscopy, often lack sensitivity and make diagnosis a challenge due to varying sensitivity and specificity, highlighting the need for sensitive and cost-effective immunodiagnostic alternatives. The present study aimed to evaluate anti-leishmanial immunoglobulin G (IgG anti-L. tropica) antibody responses as a biomarker of CL. The lesional aspirate and serum samples from CL-infected individuals (n = 216) and healthy controls (n = 157) were collected from CL endemic regions of Punjab, Khyber Pakhtunkhwa (KPK) and Balochistan in Pakistan. Leishmania tropica (L. tropica) was identified as a dominant species via real-time ITS-1 PCR. The soluble Leishmania antigen was prepared from cultured L. tropica and utilized to detect levels of Leishmania-specific IgG anti-L. tropica antibodies in sera employing enzyme-linked immunosorbent assay (ELISA). The ELISA demonstrated significantly higher IgG anti-L. tropica levels in CL patients (P < 0.0001) as compared to healthy controls. A cut-off value of 0.22 yielded a sensitivity of 79.35 % and specificity of 84.40 %, with an Area Under Curve (AUC) of 0.79. IgG anti-L. tropica levels. A significant difference in IgG anti-L. tropica levels of antibody was observed among CL patients with different lesion sizes and numbers. The developed ELISA highlighted the importance of employing reliable diagnostic techniques for CL. The serological approach may complement conventional diagnostics and contribute to better disease management in endemic settings.

皮肤利什曼病（CL）具有多种临床表现，与其他皮肤病重叠。传统的诊断方法，如显微镜，往往缺乏灵敏度，并且由于不同的灵敏度和特异性，使诊断成为一项挑战，突出需要敏感和具有成本效益的免疫诊断替代方案。本研究旨在评价抗利什曼免疫球蛋白G （IgG）抗l。抗体反应作为CL的生物标志物。从巴基斯坦旁遮普省、开伯尔-普赫图赫瓦省（KPK）和俾路支省的CL流行地区收集CL感染者（ = 216）和健康对照（ = 157）的病变抽吸液和血清样本。实时ITS-1 PCR鉴定热带利什曼原虫（L. tropica）为优势种。从培养的热带乳杆菌中制备可溶性利什曼原虫抗原，用于检测利什曼原虫特异性IgG抗- l水平。采用酶联免疫吸附试验（ELISA）检测血清中的热带虫抗体。ELISA结果显示IgG抗l明显升高。CL患者的热带气旋水平(P

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引用次数: 0

Rapid microbiological method for analysis of clindamycin-based product potency and its equivalence to UV and HPLC methods 克林霉素产品效价的快速微生物学分析方法及其与紫外和高效液相色谱法的等效性。

IF 1.9 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Journal of microbiological methods

Pub Date : 2025-11-21 DOI: 10.1016/j.mimet.2025.107338

Isadora Alves Lustosa, Ana Carolina Kogawa

Clindamycin is an antimicrobial sold in capsules, injectable solution, or gel for topical use. Despite its wide use, current official compendia and literature lack microbiological methods to assess its potency, relying mainly on physicochemical assays such as high-performance liquid chromatography (HPLC). These techniques, however, do not evaluate the biological activity of the drug. In this context, the present study proposes, for the first time, aimed to develop and validate a rapid microbiological method to determine the potency of clindamycin in capsule form and to compare its results with those obtained from UV and HPLC methods. The method employed Escherichia coli ATCC 8739, BHI broth as the growth medium, and a hydroalcoholic diluent (ethanol: water, 1:1 v/v) for solution preparation. Concentrations tested were 8, 16, and 32 μg mL⁻¹, with incubation at 35 ± 2 °C for 4 h under agitation (70 rpm), followed by absorbance measurement at 530 nm. The method exhibited linearity (r > 0.99), selectivity, precision (RSD 3.16 %, 3.57 %, 3.14 %), accuracy (average recovery of 99.47 %), and robustness under most tested conditions, as percentage of inoculum, brand of BHI broth, and volume of BHI broth used in the tube. Statistical comparison confirmed the equivalence of the proposed method with UV and HPLC (F_cal 0.68 < F_tab 5.14). This validated microbiological method offers a fast, reliable, and biologically relevant alternative for routine potency determination of clindamycin capsules, addressing a major analytical gap in the quality control of antimicrobial products.

克林霉素是一种抗菌剂，以胶囊、注射溶液或凝胶形式出售，用于局部使用。尽管其用途广泛，但目前的官方药典和文献缺乏微生物学方法来评估其效力，主要依赖于高效液相色谱（HPLC）等理化分析。然而，这些技术并不能评价药物的生物活性。在此背景下，本研究首次提出建立并验证一种快速测定克林霉素胶囊效价的微生物学方法，并将其结果与紫外法和高效液相色谱法进行比较。该方法以大肠杆菌ATCC 8739、BHI肉汤为生长培养基，以乙醇：水1:1 v/v的水醇稀释液配制溶液。测试浓度为8、16和32 μg mL-1，在35 ± 2 °C下孵育4 h，搅拌（70 rpm）， 530 nm处测定吸光度。在接种量、菌种、培养液体积等条件下，该方法均具有良好的线性（r > 0.99）、选择性、精密度（RSD 3.16 %、3.57 %、3.14 %）、准确度（平均回收率99.47 %）和稳健性。统计比较证实了该方法与UV和HPLC的等效性（Fcal 0.68 tab 5.14）。该方法为克林霉素胶囊的常规效价测定提供了一种快速、可靠、具有生物学相关性的替代方法，解决了抗菌产品质量控制的主要分析空白。

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引用次数: 0

CRISPR technology for diagnosis and treatment of human brucellosis CRISPR技术用于人类布鲁氏菌病的诊断和治疗

IF 1.9 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Journal of microbiological methods

Pub Date : 2025-11-20 DOI: 10.1016/j.mimet.2025.107339

Cia-Hin Lau , Xiaojun Li , Qing-Le Liang , Rui Guo , Xiangyang Ren , Zhenna Xu , Yanqiu Zhu , Ying Lai , Guoqing Liu , Yumei Huang , Weidong Wu , Haibao Zhu , Jinhui Chen , Xianpeng Zhang

The global burden of Brucella infection in livestock and human health is substantial, particularly in developing countries or rural areas. Currently, brucellosis diagnosis primarily relies on PCR, microbiological culture, and serological tests. However, these approaches have several drawbacks such as long experimental duration, lengthy procedure, low positive detection rates, high variability in results, interspecies cross-reactivity, and require expensive equipment and professional operators. Herein, we review how recent emerging CRISPR/Dx technology can address some of these shortcomings to realize field-deployable detection of Brucella in domestic animals and point-of-care testing (POCT) for human brucellosis. CRISPR technology has been successfully used to treat brucellosis by deleting or inactivating the genes associated with the Brucella replication or survival. Therefore, we also discuss how CRISPR technology can be potentially used to treat brucellosis, as antibiotic therapy may lose efficacy when encountering multidrug-resistant Brucella strains and the treatment is long-lasting in infected individuals to prevent relapse. Lastly, we critically discuss the advances, pitfalls, and future perspectives of CRISPR technology for the diagnosis and treatment of brucellosis in humans and livestock. Ultimately, the continued refinement of CRISPR technology will pave the road for field-deployable pathogen diagnostics and home self-tests of brucellosis to mitigate global Brucella infections.

布鲁氏菌感染对牲畜和人类健康造成的全球负担是巨大的，特别是在发展中国家或农村地区。目前，布鲁氏菌病的诊断主要依靠聚合酶链反应、微生物培养和血清学检测。然而，这些方法有一些缺点，如实验时间长、程序长、阳性检出率低、结果变异性大、种间交叉反应性强、需要昂贵的设备和专业的操作人员。在此，我们回顾了最近出现的CRISPR/Dx技术如何解决这些缺点，以实现家畜布鲁氏菌的现场部署检测和人类布鲁氏菌病的点护理检测（POCT）。CRISPR技术已被成功地用于治疗布鲁氏菌病，通过删除或使与布鲁氏菌复制或存活相关的基因失活。因此，我们还讨论了CRISPR技术如何潜在地用于治疗布鲁氏菌病，因为抗生素治疗可能在遇到耐多药布鲁氏菌菌株时失去疗效，并且在感染个体中治疗是持久的，以防止复发。最后，我们批判性地讨论了CRISPR技术在人类和牲畜布鲁氏菌病诊断和治疗方面的进展、缺陷和未来前景。最终，CRISPR技术的不断完善将为布鲁氏菌病的现场部署病原体诊断和家庭自我检测铺平道路，以减轻全球布鲁氏菌感染。

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引用次数: 0

Development of gene manipulation methods for Enterococcus cecorum 盲肠球菌基因操作方法的研究进展。

IF 1.9 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Journal of microbiological methods

Pub Date : 2025-11-14 DOI: 10.1016/j.mimet.2025.107331

Daisuke Takamatsu , Mariko Okamoto , Takashi Mada , Ayako Watanabe-Yanai , Koume Matsubara , Masatoshi Okura

Enterococcus cecorum is an emerging pathogen that causes economic losses in the poultry industry; however, our understanding of its pathogenesis remains limited. Because the lack of gene manipulation methods has hampered E. cecorum studies, we developed genetic manipulation methods for this organism using plasmids used in other gram-positive cocci.

盲肠球菌是一种新兴病原体，给家禽业造成经济损失；然而，我们对其发病机制的了解仍然有限。由于缺乏基因操作方法阻碍了盲肠杆菌的研究，我们利用其他革兰氏阳性球菌的质粒开发了这种生物的遗传操作方法。

引用次数: 0

Identification of Legionella anisa, Legionella longbeachae and Legionella pneumophila using MALDI-TOF MS: A method validation study with environmental isolates MALDI-TOF质谱法鉴定兔军团菌、长滩军团菌和嗜肺军团菌：环境分离菌株的方法验证研究

IF 1.9 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Journal of microbiological methods

Pub Date : 2025-11-13 DOI: 10.1016/j.mimet.2025.107330

Ellinoora Savonen , Silja Mentula , Jenni Ikonen , Ilkka T. Miettinen , Pekka M. Rossi , Marjo Niittynen

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) allows species-level microbial identification quickly and cost-effectively. The aim of this study was to validate MALDI-TOF MS for the identification of Legionella anisa, Legionella longbeachae and Legionella pneumophila and to examine whether incubation time or sample preparation method affect identification results.

Environmental Legionella strains and ATCC reference strains were used in the study. The strains underwent 16S rRNA sequencing and latex agglutination and fluorescence tests to which MALDI-TOF MS results were compared. The strains, incubated for 24 and 48 h, were analysed with MALDI-TOF MS using direct transfer and extraction methods. Following performance characteristics were calculated from the results: sensitivity, specificity, relative accuracy, predictive values, repeatability and reproducibility.

Species-level identification was achieved for 63 out of the 69 environmental and reference strains regardless of incubation time or preparation method. MALDI-TOF MS proved to be very specific (100 %) and sensitive (94.6–100 %) to the Legionella species analysed. With the extraction method, incubation time had a statistically significant, but species-dependent effect on the interpretative criteria, i.e. score values, in all species studied. No significant difference in any of the species' score values was found between the direct transfer and extraction methods with 24 h incubation. MALDI-TOF MS score values showed high repeatability (std 0.01–0.23) and reproducibility (std 0.02–0.18) for all Legionella species analysed. Based on the results, MALDI-TOF MS suits well for the analysis of environmental Legionella strains already after 24 h of incubation and using direct transfer method.

基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）可以快速、经济地进行物种级微生物鉴定。本研究的目的是验证MALDI-TOF MS对兔军团菌、长滩军团菌和嗜肺军团菌的鉴定效果，并考察培养时间和制样方法对鉴定结果的影响。采用环境军团菌和ATCC对照菌株进行研究。对菌株进行16S rRNA测序、胶乳凝集和荧光检测，并与MALDI-TOF MS结果进行比较。培养24和48 h后，采用MALDI-TOF MS直接转移法和萃取法对菌株进行分析。根据结果计算以下性能特征：灵敏度、特异性、相对准确度、预测值、可重复性和再现性。无论孵育时间或制备方法如何，69株环境和参考菌株中有63株获得了种水平的鉴定。结果表明，MALDI-TOF MS对军团菌的特异度（100% %）和敏感性（94.6 ~ 100% %）均较高。在提取方法中，孵育时间对所有研究物种的解释标准（即得分值）具有统计学意义，但具有物种依赖性。孵育24 h后，直接转移和提取两种方法对各物种的评分值均无显著差异。所有军团菌的MALDI-TOF MS评分值具有较高的重复性（标准为0.01 ~ 0.23）和重复性（标准为0.02 ~ 0.18）。结果表明，MALDI-TOF质谱法适用于已培养24 h并采用直接转移法的环境军团菌菌株分析。

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引用次数: 0

Utility of a commercially available transwell system for assessment of MSC-mediated mitigation of biofilms 利用市售的transwell系统评估msc介导的生物膜缓解作用。

IF 1.9 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Journal of microbiological methods

Pub Date : 2025-11-12 DOI: 10.1016/j.mimet.2025.107325

Sarah M. Khatibzadeh , Linda A. Dahlgren , Clayton C. Caswell , William A. Ducker , Stephen R. Werre , Sophie H. Bogers

Mesenchymal stromal cells (MSC) reduce and prevent biofilm infections in a paracrine fashion by secretion of antimicrobial proteins. However, studies to-date have used conditioned cell culture medium alone rather than allowing real-time MSC-biofilm interaction. A co-culture method to evaluate MSC efficacy in reducing established biofilms is needed to facilitate MSC-bacterial interactions while enabling biofilm separation for imaging and quantification after co-culture. We describe the development of a novel in vitro method to co-culture established S. aureus and E. coli biofilms with equine bone marrow-derived MSC ± amikacin sulfate using a commercially available transwell plate system. The system was used to co-culture MSC from 6 horses with mature biofilms and perform analysis of biomass by crystal violet assay, bioburden by colony forming unit analysis and biofilm size by quantitative photographic analysis using Image J. Our method allowed downstream quantification of biofilm biomass, live bacterial counts, and photographic analysis of biofilm size and may be used for high-throughput evaluation of MSC as a treatment for biofilm infections.

间充质间质细胞（MSC）通过分泌抗菌蛋白以旁分泌方式减少和预防生物膜感染。然而，迄今为止的研究都是单独使用条件细胞培养基，而不是允许msc -生物膜实时相互作用。需要一种共培养方法来评估MSC减少已建立的生物膜的功效，以促进MSC与细菌的相互作用，同时使生物膜分离，以便在共培养后进行成像和定量。我们描述了一种新的体外方法的发展，该方法使用市售的transwell板系统与马骨髓来源的MSC ± 硫酸阿米卡星共同培养已建立的金黄色葡萄球菌和大肠杆菌生物膜。该系统用于6匹马的MSC与成熟生物膜共培养，并通过结晶紫法分析生物量，菌落形成单位分析生物负荷，通过Image j定量摄影分析生物膜大小。我们的方法允许下游定量生物膜生物量，活细菌计数和生物膜大小的摄影分析，并可用于高通量评估MSC作为生物膜感染治疗方法。

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引用次数: 0

Unmasking metabolic endotoxemia: Proteinase K–enhanced detection of free and protein-associated endotoxin in human serum 揭示代谢性内毒素血症：人血清中游离内毒素和蛋白相关内毒素的蛋白酶k增强检测。

IF 1.9 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Journal of microbiological methods

Pub Date : 2025-11-12 DOI: 10.1016/j.mimet.2025.107329

Alejandra Vargas-Caraveo , Keren E. Tovar-Presas , Dalia S. Flores-Molina , Diego J. Hernandez-Castro , Katya A. Carrasco-Urrutia , Ana L. Arellano-Ortiz

Metabolic endotoxemia is a low-grade endotoxin exposure that leaks from the gut into the bloodstream, contributing to low-grade chronic inflammation. The most used method to quantify endotoxins is the Limulus Amebocyte Lysate (LAL) assay; however, its accuracy is limited due to the strong affinity of endotoxins for plasma proteins like albumin and lipoproteins, which can mask them and interfere with detection by factor C.

The aim of this study was to evaluate the use of proteinase K pretreatment to unmask protein-bound endotoxins in human serum, allowing quantification of total, free, and bound fractions using the LAL assay. Participants were classified as normal-weight or obese based on anthropometric and biochemical criteria. Blood samples were obtained under fasting conditions and 180 min after a balanced meal. Each sample was split into two aliquots: one untreated and the other digested with proteinase K. Protein degradation was confirmed by SDS-PAGE, and a formula was applied to estimate the percentage of protein-bound endotoxin.

Results showed an approximately fivefold increase in detectable endotoxin levels after proteolysis under fasting conditions in both groups, and a smaller, though significant, postprandial increase. Obese participants showed a lower postprandial percentage of protein-bound endotoxin than normal-weight individuals, despite similar endotoxin after proteolysis levels in both groups.

These findings highlight the need for proteolysis in accurately measuring endotoxin concentrations. Furthermore, the proportion of protein-bound endotoxin may serve as a marker of physiological detoxification capacity. The study suggests that inflammation risk is more closely tied to endotoxin bioavailability than total circulating levels.

代谢性内毒素血症是一种低级别内毒素暴露，从肠道渗漏到血液中，导致低级别慢性炎症。定量内毒素最常用的方法是鲎试剂（LAL）测定法；然而，由于内毒素与血浆蛋白（如白蛋白和脂蛋白）有很强的亲和力，其准确性受到限制，这可能掩盖它们并干扰因子c的检测。本研究的目的是评估蛋白酶K预处理在人血清中揭开蛋白结合内毒素的面具的使用，允许使用LAL测定法定量总、游离和结合部分。根据人体测量和生化标准，参与者被分为正常体重或肥胖。在禁食条件下和均衡膳食后180 分钟采集血液样本。每个样品被分成两等份：一份未经处理，另一份用蛋白酶k消化。通过SDS-PAGE确认蛋白质降解，并应用公式估计蛋白质结合内毒素的百分比。结果显示，在两组空腹条件下，蛋白水解后可检测到的内毒素水平增加了大约5倍，餐后的增加幅度较小，但很明显。肥胖参与者的餐后蛋白质结合内毒素百分比低于体重正常的人，尽管两组在蛋白质水解后的内毒素水平相似。这些发现强调了蛋白质水解在准确测量内毒素浓度方面的必要性。此外，蛋白质结合内毒素的比例可以作为生理解毒能力的标志。研究表明，炎症风险与内毒素生物利用度的关系比与总循环水平的关系更密切。

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