Journal of microbiological methods最新文献_第7页

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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引用次数: 0

Determining the best method for assessing microbiological contamination on flexible endoscopes without a working channel: a pilot study 确定评估无工作通道柔性内窥镜微生物污染的最佳方法：一项试点研究。

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.2025.107328

Yana Halmans , David J. Wellenstein , Michael Romijn , Suzan A.J. Cremers , Joost Hopman , Robert P. Takes , Guido B. van den Broek

Background: Flexible endoscopes (FEs) without a working channel can become extensively contaminated. Adequate reprocessing is critical and the quality of these processes can be verified by microbiological sampling. This study aimed to determine the ability to detect colony-forming units (CFUs) after sampling used FEs without a working channel for four different sampling techniques. Methods: FEs without a working channel were collected after clinical use. Manual pre-cleaning with tap water was performed. A sample was collected from the distal 8-10 cm and tip of the FE by one of four sampling techniques: rolling over a Trypticase Soy Agar with tween and lecithin plate (group 1), rolling over a Plate Count Agar + additives (group 2), swab technique (group 3) or broth technique (group 4). After incubation, a CFU count was performed to assess microbiological contamination. Results: 160 FEs without a working channel were evenly distributed among the four sampling groups. The CFUs on the FEs were classified into four groups: 0 CFUs, 1–50 CFUs, 50–500 CFUs and > 500 CFUs. All four sampling techniques showed a variable number of CFUs, ranging from 0 to uncountable CFUs (i.e., >500) (p ≤0.001). No statistically significant difference was found in how often >0 CFUs were found between the sampling techniques (p = 0.21). Conclusions: All four sampling techniques were able to detect CFUs on used FEs without a working channel, indicating all four sampling techniques can be used for assessing microbiological contamination.

背景：没有工作通道的柔性内窥镜（FEs）可能会被广泛污染。充分的再处理是至关重要的，这些过程的质量可以通过微生物取样来验证。本研究旨在确定四种不同采样技术使用无工作通道的FEs采样后检测菌落形成单位（cfu）的能力。方法：临床使用后收集无工作通道的FEs。用自来水进行手动预清洗。通过以下四种取样技术中的一种，从FE的远端8-10 cm和尖端处采集样本：在带有间体和卵磷脂的胰酶大豆琼脂板上滚动（1组），在平板计数琼脂+添加剂上滚动（2组），拭子技术（3组）或肉汤技术（4组）。孵育后，进行CFU计数以评估微生物污染。结果：160个无工作通道的FEs均匀分布在4个采样组中。FEs上的cfu分为4组：0 cfu、1-50 cfu、50-500 cfu和 > 500 cfu。所有四种采样技术都显示了不同数量的cfu，范围从0到不可数的cfu（即bbb500）（p ≤0.001）。两种采样技术之间发现>0 cfu的频率无统计学差异（p = 0.21）。结论：所有四种采样技术都能够在没有工作通道的情况下检测到使用过的FEs中的CFUs，这表明所有四种采样技术都可用于评估微生物污染。

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引用次数: 0

A rapid MALDI Biotyper based approach for screening anovaginal colonization by Streptococcus agalactiae in pregnant women 基于MALDI生物分型快速筛选孕妇无乳链球菌阴道定植的方法。

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.2025.107327

Suellen Bernardo de Queiroz , Carlos Gabriel Andrade Barbosa , Marcelo Henrique da Silva Pereira , Aryana Jacom Isbarrola dos Santos , Moises Dantas Cartaxo de Abreu Pereira , Eloiza Helena Campana , Eduardo Sergio Soares Sousa , Vinicius Pietta Perez

Introduction

Colonization of pregnant women with Streptococcus agalactiae is the main risk factor for vertical transmission to neonates and the occurrence of early onset neonatal disease. Screening for maternal carriage is used to guide the administration of intrapartum antibiotics and can significantly reduce disease occurrence. Thus, we propose and evaluate a new protocol for detecting maternal colonization by S. agalactiae using the MALDI-TOF platform directly from enrichment broth, referred to as the MALDI-Biotyper Rapid Group B Identification Assay (MBT-RBA).

Methods

The ability of the MBT-RBA protocol to detect S. agalactiae was evaluated in mixed cultures. Additionally, the performance of MBT-RBA in comparison with the standard culture method was evaluated in 137 anovaginal swabs obtained from pregnant women, and positive MBT-RBA samples were confirmed by amplification of the cfb gene using qPCR.

Results

The MBT-RBA detected S. agalactiae growth in mixed cultures with high confidence and accuracy. The prevalence of maternal colonization was 17.5 %, and MBT-RBA showed a sensitivity of 75 %, specificity of 100 %, positive predictive value of 100 %, and negative predictive value of 94.9 %.

Conclusions

The proposed protocol can detect anovaginal colonization with high confidence after only 6 h of incubation. This is a feasible and rapid test, complementary to culture or molecular assays, for screening pregnant women with unknown colonization status.

前言：孕妇的无乳链球菌定植是垂直传播给新生儿和发生早发型新生儿疾病的主要危险因素。产妇妊娠筛查用于指导产时抗生素的使用，可显著减少疾病的发生。因此，我们提出并评估了一种利用MALDI-TOF平台直接从富集肉汤中检测S. agalactiae母体定植的新方案，即maldi -生物型快速B群鉴定试验（MBT-RBA）。方法：对MBT-RBA方案在混合培养中检测无乳链球菌的能力进行评价。此外，在137份孕妇阴道拭子中比较MBT-RBA与标准培养法的性能，并通过qPCR扩增cfb基因确认MBT-RBA阳性样本。结果：MBT-RBA在混合培养中检测无乳链球菌的生长，具有较高的置信度和准确性。母体定殖率为17.5 %，MBT-RBA敏感性为75 %，特异性为100 %，阳性预测值为100 %，阴性预测值为94.9 %。结论：所提出的方案可以在孵育6 h后高置信度地检测无阴道定植。这是一种可行和快速的测试，补充培养或分子分析，用于筛选未知定植状态的孕妇。

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引用次数: 0

Improving bacterial smear detection with Wright-Giemsa staining: A multicenter study in clinical microbiology laboratories 改进细菌涂片检测赖特-吉姆萨染色：临床微生物实验室的多中心研究。

IF 1.9 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Journal of microbiological methods

Pub Date : 2025-11-11 DOI: 10.1016/j.mimet.2025.107326

Pengyu Ji , Shuqiao Yuan , Tingting Zeng , Bo Wu , Jing Ma , Zuoliang Zhang , Liqiong Yao , Na Li

Improvement in bacterial detection in smear is needed because pathogens in smears and positive blood cultures (PBCs) can be missed by microscopy if Gram staining is used. This study assessed the efficacy of Wright-Giemsa staining in remedying bacterial detection failures. We conducted a prospective multicenter study involving 5667 sputum specimens and 5502 PBCs collected from five distinct centers. When initial Gram staining did not identify bacteria, Wright-Giemsa staining was performed. The study also analyzed cases of missed detections and the undetected microorganism types. Among the sputum specimens, 3.0 % (170 cases) that were negative for Gram staining were successfully identified using Wright-Giemsa staining, revealing 321 previously undetected microbial forms. Of the PBCs, 5.1 % (283 cases) exhibited no microorganisms upon initial Gram staining, and subsequent Wright-Giemsa staining identified bacteria in 119 PBCs (42.0 % of the 283 cases). Culture identification further confirmed the bacterial presence in 77 PBCs (64.7 % of the 119 cases). The predominant contaminants were coagulase-negative staphylococci, Micrococcus spp., Corynebacterium spp., and alpha-hemolytic streptococci, which accounted for 64.9 % of the total isolates. For both specimens, the missed detection rate in primary hospitals was significantly higher than in regional central hospitals (p < 0.001). Incorporating Wright-Giemsa staining can substantially decrease the missed detection rate in bacterial smears, potentially harmonizing inter-laboratory discrepancies. For Gram-staining-negative sputum specimens or PBCs, Wright-Giemsa staining may enhances detection in microbiology laboratories, particularly in primary hospitals with limited specimens or diagnostic experience.

需要改进涂片中的细菌检测，因为如果使用革兰氏染色，涂片和阳性血培养（pbc）中的病原体可能会在显微镜下被遗漏。本研究评估了赖特-吉姆萨染色在纠正细菌检测失败方面的功效。我们进行了一项前瞻性多中心研究，包括来自五个不同中心的5667份痰标本和5502份PBCs。当最初的革兰氏染色未发现细菌时，进行赖特-吉姆萨染色。对漏检病例和未检出的微生物类型进行了分析。在痰标本中，3.0 %（170例）的革兰氏染色阴性通过Wright-Giemsa染色成功鉴定，揭示了321种以前未检测到的微生物形式。在最初的革兰氏染色中，5.1% %（283例）未发现微生物，随后的Wright-Giemsa染色在119例pbc中发现了细菌（283例中的42.0% %）。培养鉴定进一步证实77例pbc中存在细菌（119例中64.7% %）。主要污染物为凝固酶阴性葡萄球菌、微球菌、棒状杆菌和溶血性链球菌，占总分离物的64.9% %。两种标本在基层医院的漏检率均显著高于区域中心医院（p . 539）

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引用次数: 0

Rapid estimation of bacterial abundance in mouse feces by flow cytometry with SYBR green I and counting beads 用SYBR绿I和计数珠流式细胞术快速估计小鼠粪便中细菌丰度。

IF 1.9 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Journal of microbiological methods

Pub Date : 2025-11-10 DOI: 10.1016/j.mimet.2025.107323

Virginia A. Piqueras, Silvia G. Correa

Aims

To present a standardized and reproducible flow cytometry (FCM) protocol for the rapid estimation of bacterial abundance in mouse fecal samples using SYBR Green I and counting beads.

Methods and results

The protocol uses nucleic acid staining and reference beads to estimate SYBR Green I-positive events compatible with bacterial cells. Optimization included titration of dye and beads, gating strategy, and acquisition settings. The method was applied to fecal samples from male and female C57BL/6 and Foxp3-EGFP mice under specific-pathogen-free conditions. Measurements performed seven days apart showed high repeatability. Significant differences in bacterial abundance were observed between mouse strains, though not between sexes.

Conclusions

This FCM-based method enables rapid and consistent estimation of bacterial-like particles in fecal suspensions, though it does not distinguish live/dead cells or taxonomic identity.

Significance

The protocol provides a rapid, accessible approach for comparative or screening studies of gut microbiota dynamics, especially where detailed compositional analysis is not required or feasible.

目的：提出一种标准化和可重复的流式细胞术（FCM）方案，用于使用SYBR Green I和计数珠快速估计小鼠粪便样品中的细菌丰度。方法和结果：该方案采用核酸染色和参考珠来估计与细菌细胞相容的SYBR Green i阳性事件。优化包括染料和珠滴滴定，浇注策略和采集设置。该方法应用于C57BL/6和Foxp3-EGFP雄性和雌性小鼠在特定无病原体条件下的粪便样本。间隔7天进行的测量显示高重复性。在小鼠品系之间观察到细菌数量的显著差异，但在性别之间没有。结论：这种基于fcm的方法能够快速和一致地估计粪便悬浮液中的细菌样颗粒，尽管它不能区分活细胞/死细胞或分类身份。意义：该方案为肠道微生物群动力学的比较或筛选研究提供了一种快速，易于获取的方法，特别是在不需要或不可行的情况下进行详细的成分分析。

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引用次数: 0