Journal of microbiological methods最新文献

{"title":"High-throughput screening assay for nitrification inhibitors and the discovery of goitrin as a biological nitrification inhibitor","authors":"Fabian Beeckman ,&nbsp;Andrzej Drozdzecki ,&nbsp;Alexa De Knijf ,&nbsp;Dominique Audenaert ,&nbsp;Tom Beeckman ,&nbsp;Hans Motte","doi":"10.1016/j.mimet.2025.107201","DOIUrl":"10.1016/j.mimet.2025.107201","url":null,"abstract":"<div><div>Nitrification inhibitors are valuable in mitigating nitrogen (N) losses from agricultural fertilizers, enhancing fertilizer use efficiency, and minimizing their environmental and climatic impacts. Currently, the portfolio of approved inhibitors is limited, creating a strong demand for new alternatives. Traditional nitrification inhibition assays rely on large soil systems, batch cultures, or, at best, microbial cultures in deep-well 96-well plates, none of which are suited for high-throughput screening. Here, we present a highly robust method that enables rapid and efficient screening of thousands of molecules on the soil-borne ammonia-oxidizing bacteria <em>Nitrosomonas europaea</em> and <em>Nitrosospira multiformis</em>. Our assay utilizes 384-well plates for both screening and read-out, requiring only 50 μL of culture per sample and low amounts of compound. The assay also allows for further characterization of nitrification inhibitors and differentiation between those targeting the ammonia monooxygenase (AMO) and hydroxylamine oxidoreductase (HAO) pathways. Finally, we applied the assay to test several oxazolidine variants and discovered goitrin as a novel biological nitrification inhibitor (BNI). Overall, this assay offers promising tools for the rapid identification of novel nitrification inhibitors, contributing to sustainable agriculture.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"236 ","pages":"Article 107201"},"PeriodicalIF":1.7,"publicationDate":"2025-07-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144702661","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Site-2 protease-like protein drives organic solvent tolerance in Rhodococcus ruber","authors":"Jinfang Wu,&nbsp;Zhonghao Wu,&nbsp;Ren Peng","doi":"10.1016/j.mimet.2025.107204","DOIUrl":"10.1016/j.mimet.2025.107204","url":null,"abstract":"<div><div>Engineered overexpression of a Site-2 protease-like protein (S2plp) in <em>Rhodococcus ruber</em> SD3 significantly enhanced organic solvent tolerance. Through electro-transformation with the recombinant plasmid pNV18-<em>s2plp</em>-<em>s2plp</em>, we generated a strain exhibiting superior growth under multiple organic solvent stresses. Transcriptomic analysis identified 13 significantly upregulated genes, including those encoding aminoglycoside O-phosphotransferase, replication initiation protein, two M50 family metallopeptidases, transposase, SMC family ATPase, ATP-dependent helicase, three hypothetical proteins, and three small RNAs (sRNAs). KEGG analysis linked S2plp overexpression to enhanced DNA repair mechanisms, directly contributing to organic solvent tolerance. These results establish S2plp as a novel molecular target for boosting solvent resilience in this important bacterium, advancing its potential in biocatalysis and bioremediation.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"236 ","pages":"Article 107204"},"PeriodicalIF":1.7,"publicationDate":"2025-07-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144707790","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Advancing innovative techniques in arbuscular mycorrhizal Fungi propagation: A key to sustainable agriculture and ecosystem management","authors":"Aditi Pandit ,&nbsp;Divya Gunsola ,&nbsp;Apaarna ,&nbsp;Rahul Kumar ,&nbsp;Periyasamy Panneerselvam ,&nbsp;Debasis Mitra","doi":"10.1016/j.mimet.2025.107200","DOIUrl":"10.1016/j.mimet.2025.107200","url":null,"abstract":"<div><div>Arbuscular mycorrhizal fungi (AMF) play pivotal roles in enhancing plant nutrient acquisition and overall ecosystem sustainability. The propagation of AMF involves several methodologies tailored to address specific research or practical objectives. So, enhancing methods for propagating AMF is pivotal for fostering sustainable agriculture and maintaining ecosystem balance at the application level. However, traditional substrate-based techniques focus on the isolation, purification, and cultivation of AMF spores, thereby enabling the establishment of pure cultures. Hydroponic and aeroponic systems have emerged as valuable platforms for AMF cultivation, offering controlled environments to study spore germination, hyphal growth, and mycorrhizal colonization. In addition, this review explores the significance of root-organ culture techniques in the propagation and study of AMF. As the demand for sustainable agriculture increases, the application of AMF in agroecosystems has gained prominence. This review discusses the implementation of these techniques in conventional and organic farming systems. In conclusion, this review synthesizes the diverse methodologies employed in AMF propagation, from traditional spore-based techniques to cutting-edge approaches. The integration of these techniques not only deepens our understanding of AMF biology but also holds promise for sustainable agricultural practices and ecosystem management.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"236 ","pages":"Article 107200"},"PeriodicalIF":1.7,"publicationDate":"2025-07-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144675002","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Assessing the diagnostic utility of routine liquid enriched media in the processing of microbiological samples","authors":"Victoria Jordan ,&nbsp;Hemalatha Varadhan","doi":"10.1016/j.mimet.2025.107196","DOIUrl":"10.1016/j.mimet.2025.107196","url":null,"abstract":"<div><div>A retrospective review of the utility of routinely inoculating thioglycollate broth for select sterile fluids was performed. The broth was contributory in ≤1 % of samples, prompting removal of broth inoculation from routine processing of these sample types. Laboratories should consider reviewing the routine use of enriched broth particularly in the context of modern ancillary methods of pathogen detection.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"236 ","pages":"Article 107196"},"PeriodicalIF":1.7,"publicationDate":"2025-07-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144667766","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Biphasic bactericidal activity of nitroxoline against Acinetobacter baumannii assessed by Raman-DIP","authors":"Xiaofei Yi ,&nbsp;Xin Chen ,&nbsp;Minggui Wang ,&nbsp;Jianfeng Zhang ,&nbsp;Xiaogang Xu","doi":"10.1016/j.mimet.2025.107199","DOIUrl":"10.1016/j.mimet.2025.107199","url":null,"abstract":"<div><div>The increasing antimicrobial resistance (AMR) of <em>Acinetobacter baumannii</em> presents a challenge to clinical management and underscores its role as a critical pathogen in refractory urinary tract infections (UTIs). Nitroxoline has gained renewed interest as a potential therapeutic option. Raman deuterium stable isotope probing (Raman-DIP), which allows for the analysis of bacterial metabolic activity, has shown promise as a tool for assessing antimicrobial efficacy. The aim of this study was to evaluate the bactericidal activity of nitroxoline against <em>A. baumannii</em> and the potential of Raman-DIP to assess this activity. Thirty-four <em>A. baumannii</em> isolates were collected from patients with UTI. Minimum inhibitory concentrations (MICs), minimum bactericidal concentrations (MBCs), optimal bactericidal concentrations (OBCs), and time–killing curves were determined. Raman-DIP evaluated nitroxoline's effects on bacterial metabolism and survival, using the C−D ratio (C−D to the sum of the C − H and C − D band intensities) as a metabolic activity metric. Nitroxoline demonstrated potent antimicrobial activity against <em>A. baumannii</em>, with MIC_50/90 and MBC_50/90 values of 2/2 and 2/4 mg/L, respectively. Notably, it showed biphasic bactericidal characteristics with an OBC_50/90 value of 4/8 mg/L. Raman-DIP revealed a decrease in the C−D ratio with an increase in nitroxoline concentration, indicating reduced metabolic activity. An inverse correlation was observed between bacterial survival and the C−D ratio at concentrations above the OBC. These findings underscore the necessity to optimize dosing regimens for enhancing the efficacy of nitroxoline. Raman-DIP may serve as an effective tool for investigating the effect of nitroxoline on bacterial metabolism, thereby informing its clinical applications.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"236 ","pages":"Article 107199"},"PeriodicalIF":1.7,"publicationDate":"2025-07-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144667767","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Evaluation of digital PCR for the detection of bovine viral diarrhea virus in persistently infected cattle","authors":"Duan Sriyotee Loy ,&nbsp;Zachary Brown ,&nbsp;Enakshy Dutta ,&nbsp;John Dustin Loy","doi":"10.1016/j.mimet.2025.107198","DOIUrl":"10.1016/j.mimet.2025.107198","url":null,"abstract":"<div><div>Bovine Viral Diarrhea Virus (BVDV) is a significant pathogen of cattle, causing substantial economic losses. Identifying persistently infected (PI) animals is crucial for BVD control and prevention, with pooled ear-notch tissue testing being a standard screening method. Digital PCR (dPCR) is an emerging diagnostic tool that provides absolute quantification of animal pathogens in low abundant targets. This study evaluates the effectiveness of dPCR for BVD surveillance using field submissions of pooled ear-notch samples, comparing it to real-time quantitative reverse transcription PCR (qRT-PCR). The study compared performance using 107 pools of ear notches (4853 sample animals), including 76 BVD-positive pools (3543 sample animals) and 31 BVD-negative pools (1310 sample animals). Pools included those that contained PI animals detected by IHC (<em>n</em> = 25) and those with only virus detection that were negative by IHC (<em>n</em> = 51). Performance metrics evaluated included analytical and diagnostic sensitivity and specificity, limit of detection (LOD) and test agreement with qRT-PCR. Results showed that dPCR exhibited enhanced sensitivity and lower LOD compared to qRT-PCR, detecting as low as 0.6 viral copies/μL. dPCR achieved 100 % sensitivity (76/76) and 96.77 % specificity (30/31) compared to qRT-PCR and no detection of common bovine pathogens. The comparison between qRT-PCR and dPCR using Cohen's kappa coefficient in pooled samples was 0.98, indicating almost perfect agreement. In pooled ear-notch samples with qRT-PCR quantification cycle (Cq) values ranging from 30 to 33.99, the viral load ranged from 4.75 to 194.78 viral copies/μL. The study suggests that dPCR is a sensitive and specific method for detecting BVD in pooled ear-notches.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"236 ","pages":"Article 107198"},"PeriodicalIF":1.7,"publicationDate":"2025-07-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144655789","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Advances in molecular diagnostics of Neisseria gonorrhoeae","authors":"Choo Yee Yu ,&nbsp;Xin Rui Lim ,&nbsp;Thanh Van Phan ,&nbsp;Nurulfiza Mat Isa ,&nbsp;Chan Yean Yean ,&nbsp;Kok-Gan Chan ,&nbsp;Geik Yong Ang","doi":"10.1016/j.mimet.2025.107197","DOIUrl":"10.1016/j.mimet.2025.107197","url":null,"abstract":"<div><div><em>Neisseria gonorrhoeae</em> is a human obligate pathogen that causes the sexually transmitted infection (STI) gonorrhea. As the second most commonly reported STI of bacterial origin, gonorrhea is a growing global public health concern given that the causative pathogen has developed resistance to antibiotics that are currently used for treatment. A holistic approach is thus essential to reduce the incidence of gonorrhea and to control the spread of antimicrobial resistance <em>N. gonorrhoeae</em>. Improvement in diagnostics is one of the avenues to combat rising STI rates and this review aims to provide an overview of the strategies used for <em>N. gonorrhoeae</em> nucleic acid detection by examining nucleic acid amplification tests (NAATs) that are cleared by the United States Food and Drug Administration (FDA). The updated and comprehensive information of FDA-cleared NAATs presented in this review includes the recent clearance of NAATs for point-of-care testing and home-based specimen collection kit for gonorrhea testing that can potentially revolutionize STI services. Further progress in the molecular diagnostics for gonorrhea is anticipated along with vaccine development as the challenge remains to meet the World Health Organization goal of reducing the incidence of <em>N. gonorrhoeae</em> infection by 90 % by 2030.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"236 ","pages":"Article 107197"},"PeriodicalIF":1.7,"publicationDate":"2025-07-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144655788","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Evaluation of nucleic acid extraction methods for recovery of Cyclospora cayetanensis, Salmonella enterica, and murine norovirus from water and sludge","authors":"Amy Kahler ,&nbsp;Jessica Hofstetter ,&nbsp;Camila Rodrigues ,&nbsp;Mia Mattioli","doi":"10.1016/j.mimet.2025.107195","DOIUrl":"10.1016/j.mimet.2025.107195","url":null,"abstract":"<div><div>The coccidian parasite <em>Cyclospora cayetanensis</em> is the causative agent for foodborne outbreaks of cyclosporiasis and multiple fresh produce recalls annually. In recent years, this organism has been reported in the water near produce growing operations during outbreak investigations, prompting a call for more research on its environmental prevalence in the United States. Currently, there is a lack of performance data available on methods for conducting this research, including the performance of DNA extraction methods for molecular testing. Extraction methods for environmental samples must be efficient due to the often-limited amount of target nucleic acid and the potential for molecular inhibitors present in an environmental sample. This study assessed the performance of <em>C. cayetanensis</em> nucleic acid extraction seeded into surface water, produce wash water, and tap water by two methods designed for use with environmental samples: the PowerViral and UNEX methods. The PowerSoil extraction method (2 g) was assessed for <em>C. cayetanensis</em> extraction from seeded sewage sludge – an environmental sample type used to evaluate parasite carriage within communities. Extraction performance of the PowerViral and UNEX methods were also assessed for the detection of the foodborne bacterial pathogen <em>Salmonella</em> and a surrogate for foodborne viruses, murine norovirus (MNV) seeded into surface water, produce wash water, and tap water. The PowerViral method resulted in consistent detection (83–100 %) of <em>C. cayetanensis</em>, <em>S. enterica</em>, and MNV across all water types. Detection rates for the UNEX method ranged from 56 to 100 % prevalence for tap water and wash water, but there were no detections for any microbe from surface water. The PowerSoil method resulted in poor recovery of <em>C. cayetanensis</em> from sludge (≤1 % recovery), while both the PowerViral and UNEX methods effectively recovered <em>C. cayetanensis</em> from sludge (4–36 % recovery).</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"236 ","pages":"Article 107195"},"PeriodicalIF":1.7,"publicationDate":"2025-07-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144649631","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Evaluation of a high-throughput method for processing sponge-stick samples to detect viable, non-spore-forming biothreat agents","authors":"Vanessa L. Brisson ,&nbsp;Staci R. Kane ,&nbsp;M. Worth Calfee ,&nbsp;Stephen Cendrowski ,&nbsp;Sanjiv R. Shah","doi":"10.1016/j.mimet.2025.107194","DOIUrl":"10.1016/j.mimet.2025.107194","url":null,"abstract":"<div><div>After a bioterrorism incident, surface sampling is often used to determine the extent of contamination and exposure, guiding decontamination efforts and decisions for re-occupancy of affected sites. The sponge-stick (SS) is a preferred and commonly used device for sample collection to detect both spore-forming and non-spore-forming biothreat agents from non-porous surfaces. Here, a recently developed high-throughput method (HTM) for processing SS samples to detect viable <em>Bacillus anthracis</em> spores was adapted for detection of non-spore-forming biothreat agents, <em>Yersinia pestis</em> and <em>Francisella tularensis</em>. The scalable HTM was used to process up to 20 SS samples simultaneously, compared to the current stomacher-based method which processes one SS at a time. Comparisons of the HTM and the stomacher-based method were statistically indistinguishable for most experiments (<em>P</em> &gt; 0.05) with HTM recoveries of 37–60 % for <em>Y. pestis</em> inoculated at 10²–10³ cells/SS and held 48 h at 4 °C to mimic sample transport/storage. The HTM was integrated with Rapid Viability-Polymerase Chain Reaction (RV-PCR) analysis to detect viable <em>Y. pestis</em> in the presence of particulate contamination (Arizona Test Dust, ATD). This approach detected <em>Y. pestis</em> inoculated at 20 cells/SS and ATD did not impact detection (<em>P</em> &gt; 0.05). <em>F. tularensis</em> showed significantly lower recoveries between no-hold time and 48-h hold time (4 °C, <em>P</em> &lt; 0.05) using the HTM, which further testing showed could be due to toxicity of the neutralizing buffer used for SS pre-wetting. With modifications, this method could enhance throughput capacity while maintaining similar recovery efficiencies to current methods for other non-spore-forming bacterial pathogens.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"236 ","pages":"Article 107194"},"PeriodicalIF":1.7,"publicationDate":"2025-07-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144626574","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Effect of epinephrine on growth characteristics of Actinobacillus pleuropneumoniae field isolates","authors":"Elena Schreiber ,&nbsp;Fritjof Freise ,&nbsp;Nicole de Buhr ,&nbsp;Isabel Hennig-Pauka","doi":"10.1016/j.mimet.2025.107193","DOIUrl":"10.1016/j.mimet.2025.107193","url":null,"abstract":"<div><div>Based on empirical observations, stressors in swine populations may contribute to outbreaks due to <em>Actinobacillus pleuropneumoniae</em> (<em>App</em>) in colonized pigs. Stress hormones as catecholamines were found to impact immune mechanism but also bacterial protein expression <em>in vivo</em>, so it can be hypothesized that they are key molecules in disease pathogenesis. Culturing methods to isolate <em>App</em> from tonsillar tissue of carrier pigs for further typing are mostly not successful. In this study the effect of epinephrine in culturing media on <em>App</em> growth behavior was tested. Starting cultures of different age (24-h-old or 7-day-old cultures) of three <em>App</em> field strains per serotypes 2, 5 and 12 were compared in their growth behavior in medium supplemented with various epinephrine concentrations. The response of <em>App</em> strains to epinephrine varied between strains. Highest differences in CFU/mL and in growth curve characteristics between different epinephrine concentrations were found in serotype 5 strains. Inflection points of bacterial growth curves with 24-h-old starting cultures were delayed with 100 μM epinephrine with exception of one ST5 strain. In all ST2 and ST12 as well as one ST5 strain 100 μM epinephrine led to a growth advantage in 7-day old compared to 24-h-old starting cultures. Epinephrine did not consistently enhance growth of <em>App</em>, so that it cannot be recommended as a supplement for growth media to facilitate isolation from colonized tonsils of subclinically infected pigs.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"236 ","pages":"Article 107193"},"PeriodicalIF":1.7,"publicationDate":"2025-07-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144623648","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}