Journal of microbiological methods最新文献

{"title":"Carcinoscorpius and Tachypleus lysates assay for detecting endotoxin in milk and groundwater: Toward reducing reliance on Limulus amebocyte lysate","authors":"Fauziyah ,&nbsp;Apon Zaenal Mustopa ,&nbsp;Fatimah ,&nbsp;Nabila Aprianti ,&nbsp;Rahmi Damarani ,&nbsp;Amanda Astri Pratiwi Febrianti ,&nbsp;Dina Permata Wijaya ,&nbsp;Fitri Agustriani ,&nbsp;Rozirwan","doi":"10.1016/j.mimet.2025.107119","DOIUrl":"10.1016/j.mimet.2025.107119","url":null,"abstract":"<div><div>As one of the emerging pollutants, the presence of endotoxins became an increasingly urgent issue due to their impact on human health when found in drinking water and groundwater. A simple and rapid assay for assessing bacterial biomass in these water samples was essential, particularly in situations where access to laboratory facilities was limited. The bacterial endotoxin test (BET) using the <em>Limulus</em> amebocyte lysate (LAL) test was established as an effective method. However, the application of BET employing <em>Carcinoscorpius</em> or <em>Tachypleus</em> Amebocyte Lysate (CAL/TAL), derived from <em>Carcinoscorpius rotundicauda</em> and <em>Tachypleus gigas,</em> remained rarely performed. This study aimed to explore the CAL and TAL potential for detecting bacterial endotoxins in milk and groundwater. In this assay, 94 blood samples of horseshoe crabs collected from the Banyuasin Waters of South Sumatra (79 samples of <em>C. rotundicauda</em> and 15 samples of <em>T. gigas</em>) were used. The assay was carried out using the gel-clot method. The results revealed that the CAL and TAL were able to detect small concentrations of endotoxin (up to a concentration of 0.0156 EU/mL) in raw milk, pasteurized milk, well water, and L1 HPV 52 protein based on the gel-clot test. Both CAL and TAL could be a potential substitute for the LAL assay, especially for detecting bacterial biomass in milk and groundwater. Furthermore, these findings were essential as initial scientific information for developing the CAL/TAL tests toward the development of recombinant Factor C (rFC).</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"232 ","pages":"Article 107119"},"PeriodicalIF":1.7,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143683760","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":"Engineering of a monitorable expression system to characterize β-lactamase genes in Enterobacteriaceae","authors":"Anna Schumann ,&nbsp;Ahmed Gaballa ,&nbsp;Martin Wiedmann","doi":"10.1016/j.mimet.2025.107120","DOIUrl":"10.1016/j.mimet.2025.107120","url":null,"abstract":"<div><div>Bacteria are becoming progressively more resistant to available antimicrobials. The increased ease and availability of genome sequencing has made it possible to identify putative, novel antimicrobial resistance (AMR) genes bioinformatically. However, no standardized system is available to phenotypically characterize the ability of novel AMR genes in <em>Enterobacteriaceae</em> to confer resistance and impact bacterial physiology and pathogenicity in relation to expression levels. We previously used plasmid pBAD24, which allows for arabinose-inducible expression of heterologous genes, and <em>Escherichia coli</em> Top10 to characterize mobile colistin resistance genes. Based on the pBAD24 backbone, we constructed a new plasmid (pBAD25) that carries a kanamycin resistance gene (instead of an ampicillin resistance gene). We show that our expression system allows for the characterization of five different <em>bla</em>_<em>OXA</em> genes, which differ in their ability to confer susceptibility to β-lactams, detected protein levels, and impact on bacterial growth. We characterized <em>bla</em>_{<em>OXA-48b</em>}, a close relative of <em>bla</em>_{<em>OXA-48</em>}, previously uncharacterized in <em>E. coli</em>, to be phenotypically similar to <em>bla</em>_{<em>OXA-48,</em>} and <em>bla</em>_{<em>OXA-549</em>}, a previously uncharacterized gene of the <em>bla</em>_{<em>OXA-548</em>} family, as encoding a β-lactamase that is detected intra- but not extracellularly, has moderate growth defects, and decreases susceptibility to carbapenems and ampicillin. Additionally, we found that, in <em>bla</em>_<em>OXA</em> expressing strains, (i) levels of intracellular proteins and bacterial growth negatively correlate and (ii) susceptibility to 2nd and 3rd generation cephalosporins and susceptibility to different carbapenems positively correlate. Our results demonstrate that the expression of AMR genes, specifically <em>bla</em>_<em>OXA</em> genes, through pBAD25 allows for easy characterization of putative, novel AMR genes.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"232 ","pages":"Article 107120"},"PeriodicalIF":1.7,"publicationDate":"2025-03-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143670209","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":"Innovative antigens for more accurate diagnosis of Q fever","authors":"Katarína Palkovicová ,&nbsp;Gabriela Flores-Ramírez ,&nbsp;Marco Quevedo-Diaz ,&nbsp;Frantisek Csicsay ,&nbsp;Ludovít Skultety","doi":"10.1016/j.mimet.2025.107106","DOIUrl":"10.1016/j.mimet.2025.107106","url":null,"abstract":"<div><div><em>Coxiella burnetii</em>, the causative agent of Q fever, poses a significant public health concern worldwide. Diagnosis primarily relies on serological tests. Traditional antigen production methods, typically involving embryonated hen eggs, are labor-intensive, costly, and require biosafety level 3 facilities. In this study, we tested inactivated whole-cell antigens (SAP9 and NMII/AP9) from <em>C. burnetii</em> strains grown in axenic media, offering a safer and more efficient alternative to egg-based production. These antigens were validated using an <em>in-house</em> ELISA method against human patient sera, demonstrating high sensitivity and specificity comparable to ELISA and to the Gold Standard, IFA commercial kits. Notably SAP9 and NMII/AP9 antigens showed no cross-reactivity with intracellular pathogens that cause illness with similar symptoms. This approach represents significant advancement in diagnostic antigen production for Q fever, facilitating cost-effective epidemiological studies and enhancing laboratory safety.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"232 ","pages":"Article 107106"},"PeriodicalIF":1.7,"publicationDate":"2025-03-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143634290","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Diagnostic accuracy comparison of the Fluorion MTBC QLP 2.1 real-time PCR and the COBAS TaqMan MTB assays for Mycobacterium tuberculosis detection","authors":"Salma Madihi ,&nbsp;Achraf Aainouss ,&nbsp;Mly Driss El Messaoudi ,&nbsp;Abdelouaheb Benani","doi":"10.1016/j.mimet.2025.107115","DOIUrl":"10.1016/j.mimet.2025.107115","url":null,"abstract":"<div><div>The Fluorion MTBC QLP 2.1 Real-Time PCR test is a valuable alternative for tuberculosis diagnosis, demonstrating 100 % specificity and 87.5 % sensitivity for respiratory specimens, 97.8 % specificity and 85.3 % sensitivity for non-respiratory specimens, and an overall agreement of 92.3 % with the COBAS TaqMan MTB assay.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"232 ","pages":"Article 107115"},"PeriodicalIF":1.7,"publicationDate":"2025-03-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143601463","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":"Manual and automated bacterial streak plating using permanent magnet-actuated metal balls","authors":"Tao Tao ,&nbsp;Yongchen Cai ,&nbsp;Fawaz El-Dani ,&nbsp;Oi Wah Liew ,&nbsp;Tuck Wah Ng","doi":"10.1016/j.mimet.2025.107107","DOIUrl":"10.1016/j.mimet.2025.107107","url":null,"abstract":"<div><div>The identification of microorganisms and antimicrobial susceptibility testing usually begins with culture of isolated colonies of the organism by streaking the sample onto petri dishes with appropriate nutrient agar using metal loops. An alternative method of using metal balls actuated by a permanent magnet to successfully perform the streak plate operation was described and demonstrated here. With metal balls of 2.5 mm diameter as carrier of the inoculum, a travel speed of 3 mm/s was found to be optimal for maintaining constant contact with the agar surface and to obtain isolated colonies. In applying this method using both the manual and automated approaches, five repetitions with <em>Escherichia coli</em> and yoghurt samples (likely dominated by <em>Lactobacillus</em> spp) yielded consistent streaking patterns and single colony isolation following incubation. Effective thermal sterilization (180 °C for 3 h) of the used metal balls and restoration of its magnetic properties upon cooling was demonstrated in subsequent streak plating experiments without inoculum.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"232 ","pages":"Article 107107"},"PeriodicalIF":1.7,"publicationDate":"2025-03-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143585949","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Characterization of circulating Mycoplasma hyopneumoniae variants in the Midwestern United States using Multiple-Locus Variable number tandem repeat Analysis and P146 gene sequencing","authors":"Alyssa M. Betlach ,&nbsp;Doug Marthaler ,&nbsp;Eduardo Fano ,&nbsp;Maria Pieters","doi":"10.1016/j.mimet.2025.107104","DOIUrl":"10.1016/j.mimet.2025.107104","url":null,"abstract":"<div><div>In the United States, Multiple-Locus Variable number tandem repeat Analysis (MLVA) and complete P146 gene sequence are employed to characterize <em>Mycoplasma hyopneumoniae</em> from clinical samples. However, a comparison of MLVA and P146 sequencing and interpretation of assay results has not been conducted. Therefore, the aim of this study was to characterize and compare <em>M. hyopneumoniae</em> variants detected in the Midwestern United States using MLVA and P146 gene sequencing. A total of 160 samples were analyzed for this investigation. Both molecular techniques were compared for assay sensitivity, discriminatory power, and congruence. Epidemiological relationships in clustering of <em>M. hyopneumoniae</em> variants were evaluated employing Principal Coordinate Analysis. Fair agreement (κ = 0.34) in assay outcome was calculated between the two techniques. Ability to obtain a VNTR type or a P146 sequence was dependent upon the relative bacterial load (i.e., Ct value) in the sample. Simpson's diversity index was higher for MLVA (<em>D</em> <em>=</em> 0.899) than for P146 sequencing (<em>D</em> <em>=</em> 0.844). High congruence for the number of tandem repeats detected in the poly-serine region of P146 was also obtained. Similar epidemiological inferences were generated from the two assays, as production flow explained most of the variation in the clustering of VNTR types and P146 sequences. Results from this study highlighted differences in sensitivity and discriminatory power between the two molecular techniques. Nevertheless, both techniques revealed a wide genetic diversity among <em>M. hyopneumoniae</em> variants, with similar epidemiological inferences generated. Further research utilizing whole-genome sequencing could help identify other areas within the genome that are unrepresented by MLVA and P146 sequencing, can aid characterizing <em>M. hyopneumoniae</em> variants.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"230 ","pages":"Article 107104"},"PeriodicalIF":1.7,"publicationDate":"2025-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143472565","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":"Rapid screening of primary and rationally synthesized anti-mycobacterial compounds in macrophage using double recombinant M. bovis BCG strain","authors":"Mohd Mustkim Ansari ,&nbsp;Rajendra Kumar Dhuriya ,&nbsp;Gunjan ,&nbsp;Ruchi Verma ,&nbsp;Garima Kumari ,&nbsp;Bhupendra N. Singh","doi":"10.1016/j.mimet.2025.107105","DOIUrl":"10.1016/j.mimet.2025.107105","url":null,"abstract":"<div><div>Antimycobacterial screening is done primarily at three levels; in vitro, ex vivo and in vivo. In earlier studies, we had generated a double recombinant <em>Mycobacterium bovis</em> BCG strain carrying firefly and Renilla luciferase genes as two reporters under the control of a constitutive and an inducible mycobacterial promoter. The presence of dual reporters allows simultaneous expression and analysis of two reporter enzymes within a single system. The expression profile of the firefly luciferase gene, rendered by a constitutive mycobacterial promoter, corroborates with the decline in bacterial growth in response to a wide range of antimycobacterial drugs, while the enhanced expression of Renilla luciferase mirrors the selective induction of the reporter gene expression as a result of FAS-II pathway-specific inhibition. Thus, the double recombinant strain allows the screening of both primary and rationally synthesized FAS-II pathway inhibitors in a single assay. While this was successfully used for in vitro screening, ex vivo adaptation of this screen-system posed several challenges. The constitutive hsp60pr showed appreciable expression inside macrophages, but the expression of the inducible <em>kas</em> operon promoter was found to be meager. This became a limiting factor as more number of bacilli needed per screening sample and with continued treatment the decline in CFU level worsens the detection limit of the luciferase assay. To develop a screen-system that compensate the lower level expression of a given mycobacterial promoter inside macrophages we introduced Nano luciferase reporter in recombinant mycobacteria. Nano luciferase emits several-fold brighter luminescence than firefly and Renilla luciferases and duly compensates the lower level expression of the <em>kas</em> operon promoter inside macrophages. The newly engineered double recombinant strain stays stable inside macrophages and serves as a model screen-system for general and pathway specific anti-mycobacterial ex vivo screening. The turnaround time is significantly reduced and the outcomes are similar and more consistent with those attained using conventional CFU based procedures.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"230 ","pages":"Article 107105"},"PeriodicalIF":1.7,"publicationDate":"2025-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143557186","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":"Detection of macrolide-resistant Mycoplasma pneumoniae using the Kogene Mp-DR real-time PCR assay: A clinical validation study","authors":"Bosung Park,&nbsp;Eun Jeong Won,&nbsp;Heungsup Sung,&nbsp;Mi-Na Kim","doi":"10.1016/j.mimet.2025.107102","DOIUrl":"10.1016/j.mimet.2025.107102","url":null,"abstract":"<div><div>Macrolide-resistant <em>Mycoplasma pneumoniae</em> (MRMP) requires rapid diagnosis. The Kogene Mp-DR real-time PCR assay showed 100 % sensitivity and specificity for detecting A2063G mutations in the 23S rRNA gene. Although no samples were A2064G-positive, all wild-type cases were accurately classified, highlighting the rapid diagnostic capability of this method for MRMP detection.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"230 ","pages":"Article 107102"},"PeriodicalIF":1.7,"publicationDate":"2025-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143424796","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":"Methodological aspects of investigating the resistome in pig farm environments","authors":"Valeriia Ladyhina ,&nbsp;Elisabeth Rajala ,&nbsp;Susanna Sternberg-Lewerin ,&nbsp;Leila Nasirzadeh ,&nbsp;Erik Bongcam-Rudloff ,&nbsp;Johan Dicksved","doi":"10.1016/j.mimet.2025.107103","DOIUrl":"10.1016/j.mimet.2025.107103","url":null,"abstract":"<div><div>A typical One Health issue, antimicrobial resistance (AMR) development and its spread among people, animals, and the environment attracts significant research attention. The animal sector is one of the major contributors to the development and dissemination of AMR and accounts for more than 50 % of global antibiotics usage. The use of antibiotics exerts a selective pressure for resistant bacteria in the exposed microbiome, but many questions about the epidemiology of AMR in farm environments remain unanswered. This is connected to several methodological challenges and limitations, such as inconsistent sampling methods, complexity of farm environment samples and the lack of standardized protocols for sample collection, processing and bioinformatical analysis. In this project, we combined metagenomics and bioinformatics to optimise the methodology for reproducible research on the resistome in complex samples from the indoor farm environment. The work included optimizing sample collection, transportation, and storage, as well as DNA extraction, sequencing, and bioinformatic analysis, such as metagenome assembly and antibiotic resistance gene (ARG) detection. Our studies suggest that the current most optimal and cost-effective pipeline for ARG search should be based on Illumina sequencing of sock sample material at high depth (at least 25 M 250 bp PE for AMR gene families and 43 M for gene variants). We present a computational analysis utilizing MEGAHIT assembly to balance the identification of bacteria carrying ARGs with the potential loss of diversity and abundance of resistance genes. Our findings indicate that searching against multiple ARG databases is essential for detecting the highest diversity of ARGs.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"230 ","pages":"Article 107103"},"PeriodicalIF":1.7,"publicationDate":"2025-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143425386","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Inhibition of Pseudomonas aeruginosa with silver in a new synthetic medium: Investigation on the MIC, growth rate and lag-phase at the lower limit","authors":"Jörg Böllmann,&nbsp;Ramona Riedel,&nbsp;Marion Martienssen","doi":"10.1016/j.mimet.2025.107095","DOIUrl":"10.1016/j.mimet.2025.107095","url":null,"abstract":"<div><div>The antimicrobial properties of silver are well-known and widely applied. Although it is known, that silver interacts and binds on complex organic substances like proteins, many experiments on the sterilisation efficiency and inhibiting properties are still carried out in complex culture media. Given, that silver is often applied in environments with no or few organic substrates, like cooling circuits or in the treatment of tap and process water, further insight on the minimum inhibition concentration and lethal concentration at those conditions is of interest. We have developed a defined medium for the standard bacterium <em>Pseudomonas aeruginosa</em> that is free of complex organic carbon with equal cultivation properties like commonly used nutrient solutions. With this medium we could narrow the range of the MIC between 2.5 μg to 10 μg∙L⁻¹, which very much overlaps with the bactericidic concentration depending on the initial concentration of bacteria cells. These results might help to optimise the technical application of silver. We further observed a delayed growth of bacterial cultures of up to three days compared to silver free controls, which is caused either by a partial sterilisation down to theoretical one surviving cell or by a prolonged lag phase. Based on these observations we recommend a prolonged incubation for experiments on sterilisation with silver and the use of defined media, which do not interact with the disinfecting agent.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"230 ","pages":"Article 107095"},"PeriodicalIF":1.7,"publicationDate":"2025-02-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143349695","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}