Pub Date : 2025-11-03DOI: 10.1016/j.mimet.2025.107312
Huihui Zhi , Shijie Liu , Yueyu Bai , Miaoyun Li , Dong Liang , Lijun Zhao , Jong-Hoon Lee , Zihou Liu , Qian Ding , Manhong Zhan , Lingxia Sun , Yangyang Ma , Yaodi Zhu
To achieve rapid identification and detection of lactic acid bacteria (LAB) from different genera and species, six types of lactic acid bacteria were selected as the research objects, including Lactobacillus plantarum L1, Lactobacillus gasseri Y2, Lactobacillus sakei C3, Pediococcus pentosaceus W6, Streptococcus lactis S18 and Pediococcus lactis R9. Silver nanoparticle (AgNPs) were prepared as the SERS substrate material. The SERS spectra of the six LAB strains were analyzed, and a classification and identification model was constructed.The results showed that AgNPs, as the SERS substrate material, exhibited good signal enhancement performance and excellent reproducibility (RSD < 6 %), which could clearly distinguish the differences in SERS spectral peaks among the six LAB strains. Through Principal Component Analysis (PCA) and Linear Discriminant Analysis (LDA), Various types of lactic acid bacteria can be fully visualized for classification and identification, with a model accuracy rate of 100 %. This study proposes a potential technical solution for quick qualitative identification of lactic acid bacteria.
{"title":"Rapid qualitative identification and application of six common lactic acid bacteria based on surface enhanced Raman spectroscopy","authors":"Huihui Zhi , Shijie Liu , Yueyu Bai , Miaoyun Li , Dong Liang , Lijun Zhao , Jong-Hoon Lee , Zihou Liu , Qian Ding , Manhong Zhan , Lingxia Sun , Yangyang Ma , Yaodi Zhu","doi":"10.1016/j.mimet.2025.107312","DOIUrl":"10.1016/j.mimet.2025.107312","url":null,"abstract":"<div><div>To achieve rapid identification and detection of lactic acid bacteria (LAB) from different genera and species, six types of lactic acid bacteria were selected as the research objects, including <em>Lactobacillus plantarum</em> L1, <em>Lactobacillus gasseri</em> Y2, <em>Lactobacillus sakei</em> C3, <em>Pediococcus pentosaceus</em> W6, <em>Streptococcus lactis</em> S18 and <em>Pediococcus lactis</em> R9. Silver nanoparticle (AgNPs) were prepared as the SERS substrate material. The SERS spectra of the six LAB strains were analyzed, and a classification and identification model was constructed.The results showed that AgNPs, as the SERS substrate material, exhibited good signal enhancement performance and excellent reproducibility (<em>RSD</em> < 6 %), which could clearly distinguish the differences in SERS spectral peaks among the six LAB strains. Through Principal Component Analysis (PCA) and Linear Discriminant Analysis (LDA), Various types of lactic acid bacteria can be fully visualized for classification and identification, with a model accuracy rate of 100 %. This study proposes a potential technical solution for quick qualitative identification of lactic acid bacteria.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"239 ","pages":"Article 107312"},"PeriodicalIF":1.9,"publicationDate":"2025-11-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145452150","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}
Pub Date : 2025-11-03DOI: 10.1016/j.mimet.2025.107320
Justin Mark Carpani , John R. Barta , Rebecca A. Guy
Metabarcoding is widely used for detecting microorganisms in fecal samples, but its effectiveness is often limited by the co-amplification of abundant non-target DNA. In this study, a novel metabarcoding assay was developed to amplify a near-complete 18S rRNA gene fragment suitable for long-read nanopore sequencing, enhancing taxonomic resolution. The primers were optimized to maximize detection of parasitic taxa while minimizing off-target amplification of bacterial and archaeal sequences, thereby improving assay specificity. In this study, the 18S metabarcoding assay worked well on clinical fecal samples containing clinically relevant levels of parasites. However, analysis of ungulate fecal samples revealed that fungal and plant sequences vastly outnumbered other eukaryotic taxa in many samples, obscuring the detection of low-abundance protozoan and helminth parasites. To address this, Suppression/Competition PCR was developed, a novel method that selectively reduces amplification of unwanted DNA. This approach reduced fungal and plant reads by over 99 %, enabling sequences from other taxa to comprise an average of over 98 % of total reads as opposed to an initial 36 %. Utilizing this newly-developed metabarcoding assay in either the standard or Suppression/Competition configuration on fecal DNA extracts from a range of host species, parasites of interest such as Cryptosporidium sp., Cyclospora cayetanensis, Blastocystis sp., Entamoeba sp., Eimeria sp., Ancylostoma sp., and Toxocara sp. were detected, demonstrating its broad applicability.
{"title":"Suppression/competition PCR: A novel method to minimize unwanted amplicons in metabarcoding, with applications to parasite detection in fecal samples","authors":"Justin Mark Carpani , John R. Barta , Rebecca A. Guy","doi":"10.1016/j.mimet.2025.107320","DOIUrl":"10.1016/j.mimet.2025.107320","url":null,"abstract":"<div><div>Metabarcoding is widely used for detecting microorganisms in fecal samples, but its effectiveness is often limited by the co-amplification of abundant non-target DNA. In this study, a novel metabarcoding assay was developed to amplify a near-complete 18S rRNA gene fragment suitable for long-read nanopore sequencing, enhancing taxonomic resolution. The primers were optimized to maximize detection of parasitic taxa while minimizing off-target amplification of bacterial and archaeal sequences, thereby improving assay specificity. In this study, the 18S metabarcoding assay worked well on clinical fecal samples containing clinically relevant levels of parasites. However, analysis of ungulate fecal samples revealed that fungal and plant sequences vastly outnumbered other eukaryotic taxa in many samples, obscuring the detection of low-abundance protozoan and helminth parasites. To address this, Suppression/Competition PCR was developed, a novel method that selectively reduces amplification of unwanted DNA. This approach reduced fungal and plant reads by over 99 %, enabling sequences from other taxa to comprise an average of over 98 % of total reads as opposed to an initial 36 %. Utilizing this newly-developed metabarcoding assay in either the standard or Suppression/Competition configuration on fecal DNA extracts from a range of host species, parasites of interest such as <em>Cryptosporidium</em> sp., <em>Cyclospora cayetanensis</em>, <em>Blastocystis</em> sp., <em>Entamoeba</em> sp., <em>Eimeria</em> sp., <em>Ancylostoma</em> sp., and <em>Toxocara</em> sp. were detected, demonstrating its broad applicability.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"239 ","pages":"Article 107320"},"PeriodicalIF":1.9,"publicationDate":"2025-11-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145452194","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}
Pub Date : 2025-11-01DOI: 10.1016/j.mimet.2025.107314
Boyu Liu , Yifan Li , Zaixing Yang , Jingqi Wu , Yuxuan Jiang , Lili Zhao , Junwei Ge
Canine adenovirus type 2 (CAdV-2) is an important pathogen causing infectious tracheobronchitis (ITB) and viral enteritis in puppies, often exacerbating clinical symptoms through co-infection with other viruses. However, existing diagnostic methods for CAdV-2 exhibit notable limitations. Specifically, they are time-consuming, require additional nucleic acid purification steps, depend on expensive detection equipment, and necessitate operation by professional personnel. Collectively, these limitations prevent the achievement of rapid and accurate CAdV-2 detection in resource-limited settings. In this study, we established a novel CAdV-2 detection method by integrating CRISPR/Cas13a collateral cleavage activity with HUDSON rapid nucleic acid extraction, recombinase-aided amplification (RAA), and a lateral flow strip. This isothermal assay allows for visual, naked-eye result interpretation and achieves a sensitivity of 102 copies/μL as read by lateral flow strips (corresponding to approximately 750 copies per reaction). It showed excellent specificity with no cross-reactivity observed against five other major canine viruses. When tested on 20 clinical samples, the assay demonstrated a 95 % concordance rate with the conventional simplex PCR results. The entire detection process is simple to perform, requires only basic equipment, and delivers results within 90 min. The developed CRISPR/Cas13a-based detection method exhibits significant application potential for CAdV-2 detection. This study develops a CRISPR/Cas13a-based point-of-care diagnostic tool for CAdV-2, delivering rapid, sensitive, and visual detection that significantly facilitates field-based pathogen surveillance and control efforts, while advancing the application of CRISPR diagnostics in veterinary infectious diseases.
{"title":"A rapid and visual detection for canine Adenovirus-2 using CRISPR-Cas13a-based SHERLOCK technology","authors":"Boyu Liu , Yifan Li , Zaixing Yang , Jingqi Wu , Yuxuan Jiang , Lili Zhao , Junwei Ge","doi":"10.1016/j.mimet.2025.107314","DOIUrl":"10.1016/j.mimet.2025.107314","url":null,"abstract":"<div><div>Canine adenovirus type 2 (CAdV-2) is an important pathogen causing infectious tracheobronchitis (ITB) and viral enteritis in puppies, often exacerbating clinical symptoms through co-infection with other viruses. However, existing diagnostic methods for CAdV-2 exhibit notable limitations. Specifically, they are time-consuming, require additional nucleic acid purification steps, depend on expensive detection equipment, and necessitate operation by professional personnel. Collectively, these limitations prevent the achievement of rapid and accurate CAdV-2 detection in resource-limited settings. In this study, we established a novel CAdV-2 detection method by integrating CRISPR/Cas13a collateral cleavage activity with HUDSON rapid nucleic acid extraction, recombinase-aided amplification (RAA), and a lateral flow strip. This isothermal assay allows for visual, naked-eye result interpretation and achieves a sensitivity of 10<sup>2</sup> copies/μL as read by lateral flow strips (corresponding to approximately 750 copies per reaction). It showed excellent specificity with no cross-reactivity observed against five other major canine viruses. When tested on 20 clinical samples, the assay demonstrated a 95 % concordance rate with the conventional simplex PCR results. The entire detection process is simple to perform, requires only basic equipment, and delivers results within 90 min. The developed CRISPR/Cas13a-based detection method exhibits significant application potential for CAdV-2 detection. This study develops a CRISPR/Cas13a-based point-of-care diagnostic tool for CAdV-2, delivering rapid, sensitive, and visual detection that significantly facilitates field-based pathogen surveillance and control efforts, while advancing the application of CRISPR diagnostics in veterinary infectious diseases.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"239 ","pages":"Article 107314"},"PeriodicalIF":1.9,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145431631","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}
Pub Date : 2025-10-31DOI: 10.1016/j.mimet.2025.107317
Anurag Kumar Bari , Basil Britto Xavier , Tim Severs , Bhanu Sinha , John W.A. Rossen , on behalf of the DRAIGON Consortium
Background
Pseudomonas aeruginosa and Enterobacter cloacae, both members of the ESKAPE group, are multidrug-resistant pathogens that pose significant challenges in clinical care. The high GC-content (∼67 %) of the P. aeruginosa genome complicates DNA extraction and long-read sequencing, with downstream effects on genome assembly and analyses. We compared three commercial extraction kits for isolating high-quality DNA suitable for Oxford Nanopore sequencing from clinical isolates of P. aeruginosa and E. cloacae.
Methods
Genomic DNA was extracted from P. aeruginosa (n = 63; 3 × 21) and E. cloacae (n = 96; 3 × 32) clinical isolates using the MagAttract HMW DNA Kit (Qiagen), the DNeasy UltraClean Microbial Kit (Qiagen), and the MagMAX™ Microbiome Ultra Nucleic Acid Isolation Kit (ThermoFisher). DNA quantity/quality was assessed by spectrophotometry (NanoDrop, Thermo Fisher Scientific), fluorometry (Qubit, ThermoFisher), and capillary electrophoresis (TapeStation 2200, Agilent). Libraries were prepared and sequenced using Oxford Nanopore Technologies platforms. Assemblies were generated with Unicycler (v.0.5.1) and Flye (v.2.9.6); quality was assessed with QUAST (v.5.3). Genome completeness was evaluated by CheckM (v.1.1.6). Antimicrobial resistance determinants were identified with AMRFinderPlus (v.4.1.19).
Results
DNeasy yielded up to 4.7× higher DNA and ∼ 50 % higher sequencing output than MagAttract, while MagAttract produced higher DNA integrity and more contiguous assemblies. The choice of assembly had a greater impact on the detection of AMR determinants than the extraction method alone. Across workflows, Flye outperformed Unicycler, increasing detection by 2–14 percentage points. The best-performing combination (DNeasy + Flye) achieved 95.2 % AMR determinants, compared to 67.8 % for MagMAX + Unicycler, with the most difference (37.5 %) in efflux pump genes.
Conclusions
This systematic comparison highlights trade-offs between DNA yield, integrity, and downstream assembly performance, demonstrating that assembler choice critically impacts the detection of AMR determinants. These findings provide practical guidance for optimizing long-read-based sequencing workflows to support AMR surveillance and genomic epidemiology.
{"title":"Comparison of three commercial DNA extraction kits and assemblers for AMR determinant detection in Pseudomonas aeruginosa and Enterobacter cloacae using long-read sequencing","authors":"Anurag Kumar Bari , Basil Britto Xavier , Tim Severs , Bhanu Sinha , John W.A. Rossen , on behalf of the DRAIGON Consortium","doi":"10.1016/j.mimet.2025.107317","DOIUrl":"10.1016/j.mimet.2025.107317","url":null,"abstract":"<div><h3>Background</h3><div><em>Pseudomonas aeruginosa</em> and <em>Enterobacter cloacae</em>, both members of the ESKAPE group, are multidrug-resistant pathogens that pose significant challenges in clinical care. The high GC-content (∼67 %) of the <em>P. aeruginosa</em> genome complicates DNA extraction and long-read sequencing, with downstream effects on genome assembly and analyses. We compared three commercial extraction kits for isolating high-quality DNA suitable for Oxford Nanopore sequencing from clinical isolates of <em>P. aeruginosa</em> and <em>E. cloacae</em>.</div></div><div><h3>Methods</h3><div>Genomic DNA was extracted from <em>P. aeruginosa</em> (<em>n</em> = 63; 3 × 21) and <em>E. cloacae</em> (<em>n</em> = 96; 3 × 32) clinical isolates using the MagAttract HMW DNA Kit (Qiagen), the DNeasy UltraClean Microbial Kit (Qiagen), and the MagMAX™ Microbiome Ultra Nucleic Acid Isolation Kit (ThermoFisher). DNA quantity/quality was assessed by spectrophotometry (NanoDrop, Thermo Fisher Scientific), fluorometry (Qubit, ThermoFisher), and capillary electrophoresis (TapeStation 2200, Agilent). Libraries were prepared and sequenced using Oxford Nanopore Technologies platforms. Assemblies were generated with Unicycler (v.0.5.1) and Flye (v.2.9.6); quality was assessed with QUAST (v.5.3). Genome completeness was evaluated by CheckM (v.1.1.6). Antimicrobial resistance determinants were identified with AMRFinderPlus (v.4.1.19).</div></div><div><h3>Results</h3><div>DNeasy yielded up to 4.7× higher DNA and ∼ 50 % higher sequencing output than MagAttract, while MagAttract produced higher DNA integrity and more contiguous assemblies. The choice of assembly had a greater impact on the detection of AMR determinants than the extraction method alone. Across workflows, Flye outperformed Unicycler, increasing detection by 2–14 percentage points. The best-performing combination (DNeasy + Flye) achieved 95.2 % AMR determinants, compared to 67.8 % for MagMAX + Unicycler, with the most difference (37.5 %) in efflux pump genes.</div></div><div><h3>Conclusions</h3><div>This systematic comparison highlights trade-offs between DNA yield, integrity, and downstream assembly performance, demonstrating that assembler choice critically impacts the detection of AMR determinants. These findings provide practical guidance for optimizing long-read-based sequencing workflows to support AMR surveillance and genomic epidemiology.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"239 ","pages":"Article 107317"},"PeriodicalIF":1.9,"publicationDate":"2025-10-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145431783","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}
Pub Date : 2025-10-30DOI: 10.1016/j.mimet.2025.107315
Abhinaba Chakraborty, Bomba Dam
A rapid and efficient sample preparation method for visualising surface-associated microbial biofilms using Field Emission-Scanning Electron Microscopy (FE-SEM) was developed by optimising the fixative concentration and dehydration process. First, different concentrations of the fixative, glutaraldehyde (5–50 %), were tested on Escherichia coli biofilms formed on smooth glass surface for a fixation period of 30 min, followed by a 10-min dehydration each in increasing grades (10–90 %, with 10 % increment) of alcohol. The highest (50 %) glutaraldehyde concentration resulted in the sharpest biofilm micrographs. Further, the incubation period in each alcohol grade was reduced to 2 min to lower the sample preparation time. Improved imaging using the developed protocol was quantified using a newly developed metric, ‘Cellular Integrity Index’ (CII), which evaluates the morphological integrity of biofilm-cells. The protocol preserved the cellular integrity of individual biofilm-associated cells of Aeromonas hydrophila, A. salmonicida, Pseudomonas fluorescens, and Bacillus mycoides. The optimised method generated distinctive high-resolution micrographs of E. coli biofilms pre-formed on different medically-, industrially-, and environmentally-relevant surfaces, such as polypropylene plastic, catheter, and paper, all with high CII values (95–97 %) with least deformation. Further, the method was used for visualising naturally-formed biofilms on poultry ceca, plant roots, and rock surfaces with impeccable clarity, even effectively resolving different microorganisms, like fungus, algae and bacteria Thus, the developed method will be a valuable asset for any research dealing with the visualisation of naturally-formed or laboratory-developed biofilms on any sort-of surface, be it of a single individual or mixed species, thus enriching environmental, industrial, and medical research.
{"title":"A rapid and efficient method for visualisation of microbial biofilms on natural, and industrially- and medically-relevant surfaces, using field emission- scanning electron microscopy","authors":"Abhinaba Chakraborty, Bomba Dam","doi":"10.1016/j.mimet.2025.107315","DOIUrl":"10.1016/j.mimet.2025.107315","url":null,"abstract":"<div><div>A rapid and efficient sample preparation method for visualising surface-associated microbial biofilms using Field Emission-Scanning Electron Microscopy (FE-SEM) was developed by optimising the fixative concentration and dehydration process. First, different concentrations of the fixative, glutaraldehyde (5–50 %), were tested on <em>Escherichia coli</em> biofilms formed on smooth glass surface for a fixation period of 30 min, followed by a 10-min dehydration each in increasing grades (10–90 %, with 10 % increment) of alcohol. The highest (50 %) glutaraldehyde concentration resulted in the sharpest biofilm micrographs. Further, the incubation period in each alcohol grade was reduced to 2 min to lower the sample preparation time. Improved imaging using the developed protocol was quantified using a newly developed metric, ‘Cellular Integrity Index’ (CII), which evaluates the morphological integrity of biofilm-cells. The protocol preserved the cellular integrity of individual biofilm-associated cells of <em>Aeromonas hydrophila</em>, <em>A. salmonicida</em>, <em>Pseudomonas fluorescens</em>, and <em>Bacillus mycoides</em>. The optimised method generated distinctive high-resolution micrographs of <em>E. coli</em> biofilms pre-formed on different medically-, industrially-, and environmentally-relevant surfaces, such as polypropylene plastic, catheter, and paper, all with high CII values (95–97 %) with least deformation. Further, the method was used for visualising naturally-formed biofilms on poultry ceca, plant roots, and rock surfaces with impeccable clarity, even effectively resolving different microorganisms, like fungus, algae and bacteria Thus, the developed method will be a valuable asset for any research dealing with the visualisation of naturally-formed or laboratory-developed biofilms on any sort-of surface, be it of a single individual or mixed species, thus enriching environmental, industrial, and medical research.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"239 ","pages":"Article 107315"},"PeriodicalIF":1.9,"publicationDate":"2025-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145426513","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}
Pub Date : 2025-10-30DOI: 10.1016/j.mimet.2025.107318
Agata Famà , Germana Lentini , Alessia Berbiglia , Riccardo Galasso , Giuseppe Valerio De Gaetano , Francesco Coppolino , Concetta Beninati
Studying bacterial gene expression directly in infected tissues is crucial for understanding host–pathogen interactions, however recovery of intact bacterial RNA in vivo remains technically challenging. Contamination with host RNA and the instability of bacterial transcripts often limit downstream applications. Here, we developed an optimized protocol for the selective isolation of Streptococcus agalactiae RNA from infected mouse peritoneal lavage fluids. The method combines differential centrifugation, selective osmotic lysis of eukaryotic cells, and mechanical disruption of bacteria using a high-frequency bead-beating system with 106 μm glass beads. This workflow maximized RNA yield and purity, while minimizing host RNA contamination. Optimization experiments established bead size, homogenization time, and bacterial input as key parameters, enabling the recovery of detectable bacterial RNA from as few as 4 × 105 CFU/mL. In vivo, RNA preparations displayed acceptable purity (A260/280 > 1.8), with moderate integrity (RINe 5.5), yet were suitable for downstream applications. As a proof of concept, quantitative RT-PCR revealed an 11-fold upregulation of the adhesin gene pbsP in vivo compared to in vitro conditions. This protocol provides a reproducible approach for obtaining bacterial RNA from host tissues, facilitating targeted gene expression studies during infection. Although not yet optimal for full transcriptomic profiling, the method provides a practical tool for investigating virulence factor expression in vivo and can be adapted to other bacterial pathogens.
{"title":"A workflow for selective isolation of bacterial RNA from Streptococcus agalactiae during in vivo infection","authors":"Agata Famà , Germana Lentini , Alessia Berbiglia , Riccardo Galasso , Giuseppe Valerio De Gaetano , Francesco Coppolino , Concetta Beninati","doi":"10.1016/j.mimet.2025.107318","DOIUrl":"10.1016/j.mimet.2025.107318","url":null,"abstract":"<div><div>Studying bacterial gene expression directly in infected tissues is crucial for understanding host–pathogen interactions, however recovery of intact bacterial RNA <em>in vivo</em> remains technically challenging. Contamination with host RNA and the instability of bacterial transcripts often limit downstream applications. Here, we developed an optimized protocol for the selective isolation of <em>Streptococcus agalactiae</em> RNA from infected mouse peritoneal lavage fluids. The method combines differential centrifugation, selective osmotic lysis of eukaryotic cells, and mechanical disruption of bacteria using a high-frequency bead-beating system with 106 μm glass beads. This workflow maximized RNA yield and purity, while minimizing host RNA contamination. Optimization experiments established bead size, homogenization time, and bacterial input as key parameters, enabling the recovery of detectable bacterial RNA from as few as 4 × 10<sup>5</sup> CFU/mL. <em>In vivo</em>, RNA preparations displayed acceptable purity (A260/280 > 1.8), with moderate integrity (RINe 5.5), yet were suitable for downstream applications. As a proof of concept, quantitative RT-PCR revealed an 11-fold upregulation of the adhesin gene <em>pbsP in vivo</em> compared to <em>in vitro</em> conditions. This protocol provides a reproducible approach for obtaining bacterial RNA from host tissues, facilitating targeted gene expression studies during infection. Although not yet optimal for full transcriptomic profiling, the method provides a practical tool for investigating virulence factor expression <em>in vivo</em> and can be adapted to other bacterial pathogens.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"239 ","pages":"Article 107318"},"PeriodicalIF":1.9,"publicationDate":"2025-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145426497","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}
Pub Date : 2025-10-30DOI: 10.1016/j.mimet.2025.107310
Yan Liu, Qianfang Hu, Shuliang Guo
Context
The etiological confirmation of pulmonary tuberculosis typically relies on sputum or bronchoalveolar lavage fluid (BALF). Oral sampling offers a non-invasive and non-sputum alternative, but evidence varies widely due to inconsistent methods. This study employed the Cepheid Gene Xpert® MTB/RIF Ultra (Xpert-Ultra) to compare the diagnostic performance of tongue swabs, pharyngeal swabs, and posterior oropharyngeal saliva (POS) collected from the same patient to identify the optimal option.
Methods
The diagnosis was based on a composite microbiological reference standard. Tongue swabs, pharyngeal swabs, and POS were sequentially collected from each participant. Sputum and BALF underwent acid-fast bacilli smear microscopy, Cepheid Gene Xpert® MTB/RIF (Xpert), and culture, while oral samples were tested with Xpert-Ultra.
Results
All three oral sample types demonstrated 100 % specificity. Compared to tongue swabs and pharyngeal swabs, POS showed the highest sensitivity, both overall and in subgroup analyses, along with the highest bacterial load and the lowest cycle threshold values.
Conclusion
Compared with tongue swabs and pharyngeal swabs, POS exhibits the best diagnostic efficacy, and is more suitable for no-sputum or paucibacillary tuberculosis patients, and shows potential as an alternative to BALF.
{"title":"Comparison of diagnostic efficacy of three different oral samples in pulmonary tuberculosis using cepheid gene Xpert® MTB/RIF ultra","authors":"Yan Liu, Qianfang Hu, Shuliang Guo","doi":"10.1016/j.mimet.2025.107310","DOIUrl":"10.1016/j.mimet.2025.107310","url":null,"abstract":"<div><h3>Context</h3><div>The etiological confirmation of pulmonary tuberculosis typically relies on sputum or bronchoalveolar lavage fluid (BALF). Oral sampling offers a non-invasive and non-sputum alternative, but evidence varies widely due to inconsistent methods. This study employed the Cepheid Gene Xpert® MTB/RIF Ultra (Xpert-Ultra) to compare the diagnostic performance of tongue swabs, pharyngeal swabs, and posterior oropharyngeal saliva (POS) collected from the same patient to identify the optimal option.</div></div><div><h3>Methods</h3><div>The diagnosis was based on a composite microbiological reference standard. Tongue swabs, pharyngeal swabs, and POS were sequentially collected from each participant. Sputum and BALF underwent acid-fast bacilli smear microscopy, Cepheid Gene Xpert® MTB/RIF (Xpert), and culture, while oral samples were tested with Xpert-Ultra.</div></div><div><h3>Results</h3><div>All three oral sample types demonstrated 100 % specificity. Compared to tongue swabs and pharyngeal swabs, POS showed the highest sensitivity, both overall and in subgroup analyses, along with the highest bacterial load and the lowest cycle threshold values.</div></div><div><h3>Conclusion</h3><div>Compared with tongue swabs and pharyngeal swabs, POS exhibits the best diagnostic efficacy, and is more suitable for no-sputum or paucibacillary tuberculosis patients, and shows potential as an alternative to BALF.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"239 ","pages":"Article 107310"},"PeriodicalIF":1.9,"publicationDate":"2025-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145426599","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}
Pub Date : 2025-10-30DOI: 10.1016/j.mimet.2025.107319
Bedin Frederic , Benoit Vincent , Rubens Agnes , Lacoux Xavier , Foucault Frederic , Imbaud Pierre
Proximity Extension Assay (PEA) is a simple, minimally invasive assay developed for proteomic applications and biomarker detection in biological samples. The method is the combination of a homogeneous-phase immunoassay with quantitative PCR-based detection. With the aim of providing a fail-safe check for the successive steps of a new PEA-derived protocol, from sample addition to quantitative PCR, an internal control (IC) was designed. The IC consisted of a synthetic peptide made of the concatenation of three Amino Dioxa-octanoic Acid spacers inserted between two epitope-tags. The efficiency of the peptide design was checked in plasma spiked with the IC. It has been shown that the IC can be co-detected with at least one biomarker of diagnostic interest in human plasma specimens (here procalcitonin or PCT), without PCR cross-interference, and with no evidence of major interference with plasma proteins. The dynamic range of the IC peptide detection encompasses 3 orders of magnitude in concentration. At a given concentration, when tested in parallel with PCT on several human samples, Ct values for the IC remained stable while the Ct values for PCT decreased with increasing PCT concentrations. The work shown here represents a preliminary study aimed at establishing proof-of-concept for the use of a peptide containing two epitope tags as a suitable internal control in proximity assays.
{"title":"Development of a bi-epitope peptide as fail-safe internal control for a modified proximity extension assay and implementation in the detection of procalcitonin in human plasma","authors":"Bedin Frederic , Benoit Vincent , Rubens Agnes , Lacoux Xavier , Foucault Frederic , Imbaud Pierre","doi":"10.1016/j.mimet.2025.107319","DOIUrl":"10.1016/j.mimet.2025.107319","url":null,"abstract":"<div><div>Proximity Extension Assay (PEA) is a simple, minimally invasive assay developed for proteomic applications and biomarker detection in biological samples. The method is the combination of a homogeneous-phase immunoassay with quantitative PCR-based detection. With the aim of providing a fail-safe check for the successive steps of a new PEA-derived protocol, from sample addition to quantitative PCR, an internal control (IC) was designed. The IC consisted of a synthetic peptide made of the concatenation of three Amino Dioxa-octanoic Acid spacers inserted between two epitope-tags. The efficiency of the peptide design was checked in plasma spiked with the IC. It has been shown that the IC can be co-detected with at least one biomarker of diagnostic interest in human plasma specimens (here procalcitonin or PCT), without PCR cross-interference, and with no evidence of major interference with plasma proteins. The dynamic range of the IC peptide detection encompasses 3 orders of magnitude in concentration. At a given concentration, when tested in parallel with PCT on several human samples, Ct values for the IC remained stable while the Ct values for PCT decreased with increasing PCT concentrations. The work shown here represents a preliminary study aimed at establishing proof-of-concept for the use of a peptide containing two epitope tags as a suitable internal control in proximity assays.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"239 ","pages":"Article 107319"},"PeriodicalIF":1.9,"publicationDate":"2025-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145426638","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}
The Dye Reduction-based Electron-transfer Activity Monitoring (DREAM) assay offers a rapid, scalable, and culture-independent method for assessing bacterial respiratory activity. In this study, we have developed and applied a kinetic framework to extract six quantitative metrics from DREAM assay absorbance-time profiles: two derived from raw absorbance data – Dye-reduction Quotient (DQ) and Maximal Slope Interval (MSI) – and four from inverse-sigmoidal curve fitting – Baseline Absorbance (BA), Reduction Amplitude (RA), Time of Maximum Kinetic Shift (TMKS), and Redox Completion Index (RCI). These metrics complement traditional bacterial count methods by offering functional insights into metabolic activity, though they must be interpreted in a comparative rather than absolute context. We have applied this framework to two experimental conditions with mixed bacterial cultures, viz., varying bacterial concentrations and different substrate environments, to demonstrate how these metrics collectively capture the magnitude, timing, and dynamics of electron transfer processes in practical situations. This comprehensive kinetic quantification enhances the interpretive power of the DREAM assay and broadens its applicability for evaluating microbial performance in the bioprocessing and biotechnology industry and in environmental samples.
{"title":"Kinetic analysis of dye reduction profiles provide deeper insights into the electron transfer activity of bacterial cultures","authors":"S.K. Shakthi Thangavel , Sahashransu S. Mahapatra , Prajal Chettri , Shailesh Srivastava , A.S. Vishwanathan","doi":"10.1016/j.mimet.2025.107313","DOIUrl":"10.1016/j.mimet.2025.107313","url":null,"abstract":"<div><div>The Dye Reduction-based Electron-transfer Activity Monitoring (DREAM) assay offers a rapid, scalable, and culture-independent method for assessing bacterial respiratory activity. In this study, we have developed and applied a kinetic framework to extract six quantitative metrics from DREAM assay absorbance-time profiles: two derived from raw absorbance data – Dye-reduction Quotient (DQ) and Maximal Slope Interval (MSI) – and four from inverse-sigmoidal curve fitting – Baseline Absorbance (BA), Reduction Amplitude (RA), Time of Maximum Kinetic Shift (TMKS), and Redox Completion Index (RCI). These metrics complement traditional bacterial count methods by offering functional insights into metabolic activity, though they must be interpreted in a comparative rather than absolute context. We have applied this framework to two experimental conditions with mixed bacterial cultures, viz., varying bacterial concentrations and different substrate environments, to demonstrate how these metrics collectively capture the magnitude, timing, and dynamics of electron transfer processes in practical situations. This comprehensive kinetic quantification enhances the interpretive power of the DREAM assay and broadens its applicability for evaluating microbial performance in the bioprocessing and biotechnology industry and in environmental samples.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"239 ","pages":"Article 107313"},"PeriodicalIF":1.9,"publicationDate":"2025-10-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145409225","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}
Pub Date : 2025-10-29DOI: 10.1016/j.mimet.2025.107316
Vo Thi Hoang Lan , Chau Quoc Cuong , Luc Mai Thanh , Nguyen Thi My Trinh , Nguyen Hieu Nghia
We developed a fusion phytase combining acid- and alkaline-active phytases, displayed on Saccharomyces cerevisiae cells. Recombinant replicative vectors were constructed to harbor phytase expression cassettes, enabling rapid screening of fusion enzyme constructs. This system provides a preliminary approach for optimizing phytase display prior to genomic integration.
{"title":"Streamlined construction of episomal vectors for rapid assessment of fusion phytase display in Saccharomyces cerevisiae","authors":"Vo Thi Hoang Lan , Chau Quoc Cuong , Luc Mai Thanh , Nguyen Thi My Trinh , Nguyen Hieu Nghia","doi":"10.1016/j.mimet.2025.107316","DOIUrl":"10.1016/j.mimet.2025.107316","url":null,"abstract":"<div><div>We developed a fusion phytase combining acid- and alkaline-active phytases, displayed on <em>Saccharomyces cerevisiae</em> cells. Recombinant replicative vectors were constructed to harbor phytase expression cassettes, enabling rapid screening of fusion enzyme constructs. This system provides a preliminary approach for optimizing phytase display prior to genomic integration.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"239 ","pages":"Article 107316"},"PeriodicalIF":1.9,"publicationDate":"2025-10-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145418328","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}
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