Pub Date : 2024-05-21DOI: 10.1016/j.mimet.2024.106958
Shinobu Oda , Sonomi Karasawa , Kurea Satoh
A novel method for the quantification of antifungal activity of fungicides and painted surfaces, mycelial invasion distance (MID) method, was developed and applied to the quantification of activities of parabens and an antifungal paint. In this method, the MID of aerial mycelia on a test paper or a panel placed on a nutrient agar plate was measured with a stereoscopic microscope and a micro-ruler. The antifungal activities of the parabens and painted surfaces were expressed as the MID. The higher the hydrophobicity of parabens, the longer the MID, that is the lower the antifungal activity, were observed. Conversely, relatively polar parabens, such as methyl and ethyl parabens, exhibited stronger antifungal activity, that is shorter MID. The most hydrophobic paraben, benzyl paraben, showed the weakest antifungal activity. Furthermore, it was confirmed that the MID method was effective for the evaluation of the painted surfaces.
{"title":"A novel procedure for the quantification of antifungal activity against filamentous fungi, mycelial invasion distance (MID) method","authors":"Shinobu Oda , Sonomi Karasawa , Kurea Satoh","doi":"10.1016/j.mimet.2024.106958","DOIUrl":"10.1016/j.mimet.2024.106958","url":null,"abstract":"<div><p>A novel method for the quantification of antifungal activity of fungicides and painted surfaces, mycelial invasion distance (MID) method, was developed and applied to the quantification of activities of parabens and an antifungal paint. In this method, the MID of aerial mycelia on a test paper or a panel placed on a nutrient agar plate was measured with a stereoscopic microscope and a micro-ruler. The antifungal activities of the parabens and painted surfaces were expressed as the MID. The higher the hydrophobicity of parabens, the longer the MID, that is the lower the antifungal activity, were observed. Conversely, relatively polar parabens, such as methyl and ethyl parabens, exhibited stronger antifungal activity, that is shorter MID. The most hydrophobic paraben, benzyl paraben, showed the weakest antifungal activity. Furthermore, it was confirmed that the MID method was effective for the evaluation of the painted surfaces.</p></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"222 ","pages":"Article 106958"},"PeriodicalIF":2.2,"publicationDate":"2024-05-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141081484","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 : 2024-05-20DOI: 10.1016/j.mimet.2024.106957
Christen Rune Stensvold , Alba Martí-Marco , Samantha Moratal , Marianne Lebbad , David Carmena
As data accumulate in GenBank, the difficulties of delineating species of Cryptosporidium based on nuclear small subunit ribosomal RNA (ssu rRNA) gene information alone becomes increasingly evident. Here, we summarize currently available evidence suggesting that several ssu rDNA sequences primarily referred to as Cryptosporidium suis (some of them from non-suid hosts) should be considered Cryptosporidium occultus.
{"title":"Cryptosporidium occultus in disguise","authors":"Christen Rune Stensvold , Alba Martí-Marco , Samantha Moratal , Marianne Lebbad , David Carmena","doi":"10.1016/j.mimet.2024.106957","DOIUrl":"10.1016/j.mimet.2024.106957","url":null,"abstract":"<div><p>As data accumulate in GenBank, the difficulties of delineating species of <em>Cryptosporidium</em> based on nuclear small subunit ribosomal RNA (<em>ssu</em> rRNA) gene information alone becomes increasingly evident. Here, we summarize currently available evidence suggesting that several <em>ssu</em> rDNA sequences primarily referred to as <em>Cryptosporidium suis</em> (some of them from non-suid hosts) should be considered <em>Cryptosporidium occultus.</em></p></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"222 ","pages":"Article 106957"},"PeriodicalIF":2.2,"publicationDate":"2024-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S0167701224000691/pdfft?md5=9d99ec5a1358bca3541d3b6a6a19d42b&pid=1-s2.0-S0167701224000691-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141081490","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}
Pub Date : 2024-05-15DOI: 10.1016/j.mimet.2024.106954
Azam Yaghoobi , Ramin Abiri , Amirhoushang Alvandi , Iraj Manouchehri , Elham Arkan , Ali R. Jalalvand
Bacterial meningitis is an acute infection which requires rapid diagnosis and treatment due to the high mortality and serious consequences of the disease. The purpose of this study was to design a homemade multiplex PCR and a novel fluorescence biosensor on chip (FBC) to detect three important agents of meningitis including Streptococcus pneumoniae (S. pneumoniae), Neisseria meningitidis (N. meningitidis), and Haemophilus influenzae (H. influenzae). The homemade multiplex PCR can diagnose three bacterial species simultaneously. Fabrication of FBC was carried out based on the deposition of lead nanoparticles on a quartz slide using the thermal evaporation method. Then, the SH-Cap Probe/Target ssDNA /FAM-Rep probe was loaded on lead film. The evaluation of the fluorescence reaction when the probes bind to the target ssDNA was assessed by a Cytation 5 Cell Imaging Multimode Reader Bio-Tek. The limit of detections (LOD) in homemade PCR and FBC to identify S. pneumoniae were 119 × 102 CFU/mL (0.27 ng/μL) and 380 CFU/mL (9 pg/μL), respectively. The LODs of homemade PCR and FBC for detection of N. meningitidis were 4.49 CFU/mL (1.1 pg/μL) and 13 × 103 CFU/mL (30 pg/μL), respectively. Our results confirmed the LODs of homemade PCR and FBC in detection of H. influenzae were 15.1 CFU/mL (30 fg/μL) and 41 × 102 CFU/mL (90 pg/ μL), respectively. Both techniques had appropriate sensitivity and specificity in detection of S. pneumoniae, N. meningitidis and H. influenzae.
{"title":"A novel biosensing strategy for identification of three important bacteria causing meningitis","authors":"Azam Yaghoobi , Ramin Abiri , Amirhoushang Alvandi , Iraj Manouchehri , Elham Arkan , Ali R. Jalalvand","doi":"10.1016/j.mimet.2024.106954","DOIUrl":"https://doi.org/10.1016/j.mimet.2024.106954","url":null,"abstract":"<div><p>Bacterial meningitis is an acute infection which requires rapid diagnosis and treatment due to the high mortality and serious consequences of the disease. The purpose of this study was to design a homemade multiplex PCR and a novel fluorescence biosensor on chip (FBC) to detect three important agents of meningitis including <em>Streptococcus pneumoniae (S. pneumoniae)</em>, <em>Neisseria meningitidis (N. meningitidis),</em> and <em>Haemophilus influenzae (H. influenzae)</em>. The homemade multiplex PCR can diagnose three bacterial species simultaneously. Fabrication of FBC was carried out based on the deposition of lead nanoparticles on a quartz slide using the thermal evaporation method. Then, the SH-Cap Probe/Target ssDNA /FAM-Rep probe was loaded on lead film. The evaluation of the fluorescence reaction when the probes bind to the target ssDNA was assessed by a Cytation 5 Cell Imaging Multimode Reader Bio-Tek. The limit of detections (LOD) in homemade PCR and FBC to identify <em>S. pneumoniae</em> were 119 × 10<sup>2</sup> CFU/mL (0.27 ng/μL) and 380 CFU/mL (9 pg/μL), respectively. The LODs of homemade PCR and FBC for detection of <em>N. meningitidis</em> were 4.49 CFU/mL (1.1 pg/μL) and 13 × 10<sup>3</sup> CFU/mL (30 pg/μL), respectively. Our results confirmed the LODs of homemade PCR and FBC in detection of <em>H. influenzae</em> were 15.1 CFU/mL (30 fg/μL) and 41 × 10<sup>2</sup> CFU/mL (90 pg/ μL), respectively. Both techniques had appropriate sensitivity and specificity in detection of <em>S. pneumoniae</em>, <em>N. meningitidis</em> and <em>H. influenzae</em>.</p></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"222 ","pages":"Article 106954"},"PeriodicalIF":2.2,"publicationDate":"2024-05-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140951251","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 : 2024-05-15DOI: 10.1016/j.mimet.2024.106956
Valeria Poscente , Luciana Di Gregorio , Manuela Costanzo , Roberta Bernini , Annamaria Bevivino
Flow cytometry (FCM) provides unique information on bacterial viability and physiology, allowing a real-time early warning antimicrobial and antibiofilm monitoring system for preventing the spread risk of foodborne disease. The present work used a combined culture-based and FCM approach to assess the in vitro efficacy of essential oils (EOs) from condiment plants commonly used in Mediterranean Europe (i.e., thyme EO, oregano EO, basil EO, and lemon EO) against planktonic and sessile cells of food-pathogenic Listeria monocytogenes 56 LY, and contaminant and alterative species Escherichia coli ATCC 25922 and Pseudomonas fluorescens ATCC 13525. Evaluation of the bacterial response to the increasing concentrations of natural compounds posed FCM as a crucial technique for the quantification of the live/dead, and viable but non-culturable (VBNC) cells when antimicrobial agents exert no real bactericidal action. Furthermore, the FCM results displayed higher numbers of viable bacteria expressed as Active Fluorescent Units (AFUs) with a greater level of repeatability compared with outcomes of the plate-count method. Overall, accurate counting of viable microbial cells is a critically important parameter in food microbiology, and flow cytometry provides an innovative approach with high-throughput potential for applications in the food industry as “flow microbiology”.
{"title":"Flow cytometry: Unravelling the real antimicrobial and antibiofilm efficacy of natural bioactive compounds","authors":"Valeria Poscente , Luciana Di Gregorio , Manuela Costanzo , Roberta Bernini , Annamaria Bevivino","doi":"10.1016/j.mimet.2024.106956","DOIUrl":"https://doi.org/10.1016/j.mimet.2024.106956","url":null,"abstract":"<div><p>Flow cytometry (FCM) provides unique information on bacterial viability and physiology, allowing a real-time early warning antimicrobial and antibiofilm monitoring system for preventing the spread risk of foodborne disease. The present work used a combined culture-based and FCM approach to assess the in vitro efficacy of essential oils (EOs) from condiment plants commonly used in Mediterranean Europe (i.e., thyme EO, oregano EO, basil EO, and lemon EO) against planktonic and sessile cells of food-pathogenic <em>Listeria monocytogenes</em> 56 LY, and contaminant and alterative species <em>Escherichia coli</em> ATCC 25922 and <em>Pseudomonas fluorescens</em> ATCC 13525. Evaluation of the bacterial response to the increasing concentrations of natural compounds posed FCM as a crucial technique for the quantification of the live/dead, and viable but non-culturable (VBNC) cells when antimicrobial agents exert no real bactericidal action. Furthermore, the FCM results displayed higher numbers of viable bacteria expressed as Active Fluorescent Units (AFUs) with a greater level of repeatability compared with outcomes of the plate-count method. Overall, accurate counting of viable microbial cells is a critically important parameter in food microbiology, and flow cytometry provides an innovative approach with high-throughput potential for applications in the food industry as “flow microbiology”.</p></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"222 ","pages":"Article 106956"},"PeriodicalIF":2.2,"publicationDate":"2024-05-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140951250","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}
We aim to objectify the evaluation criteria of agglutination rate estimation in the Microscopic Agglutination Test (MAT). This study proposes a deep learning method that extracts free leptospires from dark-field microscopic images and calculates the agglutination rate. The experiments show the effect of objectification with real pictures.
{"title":"Objectification of evaluation criteria in microscopic agglutination test using deep learning","authors":"Risa Nakano , Yuji Oyamada , Ryo Ozuru , Michinobu Yoshimura , Kenji Hiromatsu","doi":"10.1016/j.mimet.2024.106955","DOIUrl":"https://doi.org/10.1016/j.mimet.2024.106955","url":null,"abstract":"<div><p>We aim to objectify the evaluation criteria of agglutination rate estimation in the Microscopic Agglutination Test (MAT). This study proposes a deep learning method that extracts free leptospires from dark-field microscopic images and calculates the agglutination rate. The experiments show the effect of objectification with real pictures.</p></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"222 ","pages":"Article 106955"},"PeriodicalIF":2.2,"publicationDate":"2024-05-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140951213","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 : 2024-05-14DOI: 10.1016/j.mimet.2024.106953
Sakina Bombaywala , Abhay Bajaj , Nishant A. Dafale
The microbial composition and stress molecules are main drivers influencing the development and spread of antibiotic resistance bacteria (ARBs) and genes (ARGs) in the environment. A reliable and rapid method for identifying associations between microbiome composition and resistome remains challenging. In the present study, secondary metagenome data of sewage and hospital wastewaters were assessed for differential taxonomic and ARG profiling. Subsequently, Random Forest (RF)-based ML models were used to predict ARG profiles based on taxonomic composition and model validation on hospital wastewaters. Total ARG abundance was significantly higher in hospital wastewaters (15 ppm) than sewage (5 ppm), while the resistance towards methicillin, carbapenem, and fluoroquinolone were predominant. Although, Pseudomonas constituted major fraction, Streptomyces, Enterobacter, and Klebsiella were characteristic of hospital wastewaters. Prediction modeling showed that the relative abundance of pathogenic genera Escherichia, Vibrio, and Pseudomonas contributed most towards variations in total ARG count. Moreover, the model was able to identify host-specific patterns for contributing taxa and related ARGs with >90% accuracy in predicting the ARG subtype abundance. More than >80% accuracy was obtained for hospital wastewaters, demonstrating that the model can be validly extrapolated to different types of wastewater systems. Findings from the study showed that the ML approach could identify ARG profile based on bacterial composition including 16S rDNA amplicon data, and can serve as a viable alternative to metagenomic binning for identification of potential hosts of ARGs. Overall, this study demonstrates the promising application of ML techniques for predicting the spread of ARGs and provides guidance for early warning of ARBs emergence.
{"title":"Meta-analysis of wastewater microbiome for antibiotic resistance profiling","authors":"Sakina Bombaywala , Abhay Bajaj , Nishant A. Dafale","doi":"10.1016/j.mimet.2024.106953","DOIUrl":"10.1016/j.mimet.2024.106953","url":null,"abstract":"<div><p>The microbial composition and stress molecules are main drivers influencing the development and spread of antibiotic resistance bacteria (ARBs) and genes (ARGs) in the environment. A reliable and rapid method for identifying associations between microbiome composition and resistome remains challenging. In the present study, secondary metagenome data of sewage and hospital wastewaters were assessed for differential taxonomic and ARG profiling. Subsequently, Random Forest (RF)-based ML models were used to predict ARG profiles based on taxonomic composition and model validation on hospital wastewaters. Total ARG abundance was significantly higher in hospital wastewaters (15 ppm) than sewage (5 ppm), while the resistance towards methicillin, carbapenem, and fluoroquinolone were predominant. Although, <em>Pseudomonas</em> constituted major fraction, <em>Streptomyces, Enterobacter</em>, and <em>Klebsiella</em> were characteristic of hospital wastewaters. Prediction modeling showed that the relative abundance of pathogenic genera <em>Escherichia, Vibrio,</em> and <em>Pseudomonas</em> contributed most towards variations in total ARG count. Moreover, the model was able to identify host-specific patterns for contributing taxa and related ARGs with >90% accuracy in predicting the ARG subtype abundance. More than >80% accuracy was obtained for hospital wastewaters, demonstrating that the model can be validly extrapolated to different types of wastewater systems. Findings from the study showed that the ML approach could identify ARG profile based on bacterial composition including 16S rDNA amplicon data, and can serve as a viable alternative to metagenomic binning for identification of potential hosts of ARGs. Overall, this study demonstrates the promising application of ML techniques for predicting the spread of ARGs and provides guidance for early warning of ARBs emergence.</p></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"223 ","pages":"Article 106953"},"PeriodicalIF":2.2,"publicationDate":"2024-05-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140957586","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 : 2024-05-11DOI: 10.1016/j.mimet.2024.106952
Akashdeep , Suman Kumari , Neeraj Rani
The present study was carried out to valorise cereal (rice and wheat) bran for the development of low-cost liquid consortium bioformulation. Different concentrations of bran-based liquid media formulations were evaluated for the growth of consortium biofertilizer cultures (Azotobacter chroococcum, Bacillus subtilis and Pseudomonas sp.). Among the bran-based formulations, wheat bran-based formulation WB5, exhibited the highest viable cell of 10.68 ± 0.09 Log10 CFU/ml and 12.63 ± 0.04 Log10 CFU/ml for Azotobacter chroococcum and Bacillus subtilis whereas for Pseudomonas sp., rice bran based bioformulation RB5 recorded maximum viability (12.71 ± 0.05 Log10 CFU/ml) after 72 h of incubation. RB51 and WB52liquid formulations were further optimized for enhanced shelf life using 5, 10 and 15 mM of trehalose, 0.05 and 0.1% carboxymethyl cellulose, and 0.5 and 1.0% glycerol. Following the peak growth at 72 h of incubation, a gradual decrease in the viable population of consortium biofertilizer cultures was observed in all the liquid formulations. The WB5 and RB5 formulations with 15 mM trehalose and 0.1% CMC, not only recorded significantly highest cell count of consortium biofertilizer cultures, but also maximally supported multi-functional traits i.e., phosphate and zinc solubilization, ammonia and IAA production up to 150 days. Further evaluation of seedling emergence and growth of wheat (PBW 826) under axenic conditions recorded WB5 amended with 15 mM trehalose-based consortium bioformulation to exhibit maximum emergence and growth of wheat seedlings. This low-cost liquid formulation can be used for large-scale biofertilizer production as a cost-effective liquid biofertilizer production technology.
{"title":"Novel cereal bran based low-cost liquid medium for enhanced growth, multifunctional traits and shelf life of consortium biofertilizer containing Azotobacter chroococcum, Bacillus subtilis and Pseudomonas sp.","authors":"Akashdeep , Suman Kumari , Neeraj Rani","doi":"10.1016/j.mimet.2024.106952","DOIUrl":"10.1016/j.mimet.2024.106952","url":null,"abstract":"<div><p>The present study was carried out to valorise cereal (rice and wheat) bran for the development of low-cost liquid consortium bioformulation. Different concentrations of bran-based liquid media formulations were evaluated for the growth of consortium biofertilizer cultures (<em>Azotobacter chroococcum</em>, <em>Bacillus subtilis</em> and <em>Pseudomonas</em> sp.). Among the bran-based formulations, wheat bran-based formulation WB5, exhibited the highest viable cell of 10.68 ± 0.09 Log<sub>10</sub> CFU/ml and 12.63 ± 0.04 Log<sub>10</sub> CFU/ml for <em>Azotobacter chroococcum</em> and <em>Bacillus subtilis</em> whereas for <em>Pseudomonas</em> sp., rice bran based bioformulation RB5 recorded maximum viability (12.71 ± 0.05 Log<sub>10</sub> CFU/ml) after 72 h of incubation. RB5<span><sup>1</sup></span> and WB5<span><sup>2</sup></span>liquid formulations were further optimized for enhanced shelf life using 5, 10 and 15 mM of trehalose, 0.05 and 0.1% carboxymethyl cellulose, and 0.5 and 1.0% glycerol. Following the peak growth at 72 h of incubation, a gradual decrease in the viable population of consortium biofertilizer cultures was observed in all the liquid formulations. The WB5 and RB5 formulations with 15 mM trehalose and 0.1% CMC, not only recorded significantly highest cell count of consortium biofertilizer cultures, but also maximally supported multi-functional traits i.e., phosphate and zinc solubilization, ammonia and IAA production up to 150 days. Further evaluation of seedling emergence and growth of wheat (PBW 826) under axenic conditions recorded WB5 amended with 15 mM trehalose-based consortium bioformulation to exhibit maximum emergence and growth of wheat seedlings. This low-cost liquid formulation can be used for large-scale biofertilizer production as a cost-effective liquid biofertilizer production technology.</p></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"222 ","pages":"Article 106952"},"PeriodicalIF":2.2,"publicationDate":"2024-05-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140916804","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 : 2024-05-08DOI: 10.1016/j.mimet.2024.106945
Takuma Kozawa , Hideki Aoyagi
Tolerance to human gastrointestinal stressors is crucial for probiotics to exhibit their health benefits; however, there is no standardised method for screening their stress tolerance. In this study, we proposed a novel method for screening probiotic candidates tolerant to human gastrointestinal stress—gastrointestinal tolerance assay and culture (GTA-C) method—using black polyethylene terephthalate (PET) non-woven fabric as a scaffold to modify the specialized cellulose film (SCF) method. The modified SCF method showed excellent pH-based diffusion of medium components, had minimal effect on the growth of Escherichia coli K12, and improved the visibility of the colonies. Analysis of kimchi samples cultured using the SCF and modified SCF methods revealed that the modified method diversified the cultured bacteria. GTA in a simulated human fasting state using the modified SCF method showed that acid stress significantly affected the growth of four bacteria used as probiotics and that tolerance to acid stress may be species-dependent. Screening of probiotics in kimchi samples resulted in the identification of lactic acid bacteria tolerant to human gastrointestinal stress during fasting. Our results indicate that the modified SCF method (GTA-C method) is useful for screening probiotics resistant to the gastrointestinal environment during fasting.
{"title":"Novel method for screening probiotic candidates tolerant to human gastrointestinal stress","authors":"Takuma Kozawa , Hideki Aoyagi","doi":"10.1016/j.mimet.2024.106945","DOIUrl":"10.1016/j.mimet.2024.106945","url":null,"abstract":"<div><p>Tolerance to human gastrointestinal stressors is crucial for probiotics to exhibit their health benefits; however, there is no standardised method for screening their stress tolerance. In this study, we proposed a novel method for screening probiotic candidates tolerant to human gastrointestinal stress—gastrointestinal tolerance assay and culture (GTA-C) method—using black polyethylene terephthalate (PET) non-woven fabric as a scaffold to modify the specialized cellulose film (SCF) method. The modified SCF method showed excellent pH-based diffusion of medium components, had minimal effect on the growth of <em>Escherichia coli</em> K12<em>,</em> and improved the visibility of the colonies. Analysis of kimchi samples cultured using the SCF and modified SCF methods revealed that the modified method diversified the cultured bacteria. GTA in a simulated human fasting state using the modified SCF method showed that acid stress significantly affected the growth of four bacteria used as probiotics and that tolerance to acid stress may be species-dependent. Screening of probiotics in kimchi samples resulted in the identification of lactic acid bacteria tolerant to human gastrointestinal stress during fasting. Our results indicate that the modified SCF method (GTA-C method) is useful for screening probiotics resistant to the gastrointestinal environment during fasting.</p></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"222 ","pages":"Article 106945"},"PeriodicalIF":2.2,"publicationDate":"2024-05-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140904627","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}
Reliable detection of bacteria belonging to the Borrelia burgdorferi sensu lato species complex in vertebrate reservoirs, tick vectors, and patients is key to answer questions regarding Lyme borreliosis epidemiology. Nevertheless, the description of characteristics of qPCRs for the detection of B. burgdorferi s. l. are often limited. This study covers the development and validation of two duplex taqman qPCR assays used to target four markers on the chromosome of genospecies of B. burgdorferi s. l.
Analytical specificity was determined with a panel of spirochete strains. qPCR characteristics were specified using water or tick DNA spiked with controlled quantities of the targeted DNA sequences of B. afzelii, B. burgdorferi sensu stricto or B. bavariensis. The effectiveness of detection results was finally evaluated using DNA extracted from ticks and biopsies from mammals whose infectious status had been determined by other detection assays.
The developed qPCR assays allow exclusive detection of B. burgdorferi s. l. with the exception of the M16 marker which also detect relapsing fever Borreliae. The limit of detection is between 10 and 40 copies per qPCR reaction depending on the sample type, the B. burgdorferi genospecies and the targeted marker. Detection tests performed on various kind of samples illustrated the accuracy and robustness of our qPCR assays.
Within the defined limits, this multi-target qPCR method allows a versatile detection of B. burgdorferi s. l., regardless of the genospecies and the sample material analyzed, with a sensitivity that would be compatible with most applications and a reproducibility of 100% under measurement conditions of limits of detection, thereby limiting result ambiguities.
可靠地检测脊椎动物蓄水池、蜱虫媒介和患者体内属于广义鲍曼不动杆菌的细菌是回答莱姆病流行病学问题的关键。然而,对用于检测枸椽酸杆菌的 qPCRs 特性的描述往往很有限。本研究涵盖了针对 B. burgdorferi s. l 基因种染色体上四个标记的两种双联 taqman qPCR 检测方法的开发和验证。最后,使用从蜱虫和哺乳动物活体组织中提取的 DNA 对检测结果的有效性进行了评估,这些哺乳动物的感染状况已由其他检测方法确定。所开发的 qPCR 检测方法只能检测 B. burgdorferi s.l.,但 M16 标记除外,它还能检测复发性热鲍里氏菌。每个 qPCR 反应的检测限在 10 到 40 个拷贝之间,具体取决于样品类型、布氏杆菌基因种和目标标记。对各种样品进行的检测测试表明,我们的 qPCR 检测方法准确可靠。在规定的限度内,这种多靶标 qPCR 方法可以对 B. burgdorferi s. l.进行多种检测,而不受基因种属和所分析样本材料的影响,其灵敏度符合大多数应用的要求,在检测限度的测量条件下,重现性达到 100%,从而限制了结果的不确定性。
{"title":"Development and validation of a multi-target TaqMan qPCR method for detection of Borrelia burgdorferi sensu lato","authors":"Sébastien Masséglia , Magalie René-Martellet , Maxime Rates , Cecilia Hizo-Teufel , Volker Fingerle , Gabriele Margos , Xavier Bailly","doi":"10.1016/j.mimet.2024.106941","DOIUrl":"10.1016/j.mimet.2024.106941","url":null,"abstract":"<div><p>Reliable detection of bacteria belonging to the <em>Borrelia burgdorferi</em> sensu lato species complex in vertebrate reservoirs, tick vectors, and patients is key to answer questions regarding Lyme borreliosis epidemiology. Nevertheless, the description of characteristics of qPCRs for the detection of <em>B. burgdorferi</em> s. l. are often limited. This study covers the development and validation of two duplex taqman qPCR assays used to target four markers on the chromosome of genospecies of <em>B. burgdorferi</em> s. l.</p><p>Analytical specificity was determined with a panel of spirochete strains. qPCR characteristics were specified using water or tick DNA spiked with controlled quantities of the targeted DNA sequences of <em>B. afzelii</em>, <em>B. burgdorferi</em> sensu stricto or <em>B. bavariensis</em>. The effectiveness of detection results was finally evaluated using DNA extracted from ticks and biopsies from mammals whose infectious status had been determined by other detection assays.</p><p>The developed qPCR assays allow exclusive detection of <em>B. burgdorferi</em> s. l. with the exception of the M16 marker which also detect relapsing fever <em>Borreliae</em>. The limit of detection is between 10 and 40 copies per qPCR reaction depending on the sample type, the <em>B. burgdorferi</em> genospecies and the targeted marker. Detection tests performed on various kind of samples illustrated the accuracy and robustness of our qPCR assays.</p><p>Within the defined limits, this multi-target qPCR method allows a versatile detection of <em>B. burgdorferi</em> s. l., regardless of the genospecies and the sample material analyzed, with a sensitivity that would be compatible with most applications and a reproducibility of 100% under measurement conditions of limits of detection, thereby limiting result ambiguities.</p></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"222 ","pages":"Article 106941"},"PeriodicalIF":2.2,"publicationDate":"2024-05-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S0167701224000538/pdfft?md5=ad306daec5af9aebe13a7a368dae4ebd&pid=1-s2.0-S0167701224000538-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140876642","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}
Pub Date : 2024-05-04DOI: 10.1016/j.mimet.2024.106944
Xiangyu Xi , Binghua Wang , Ruimei Zhang , Chunhua Ling
Objective
To analyse the expression profiles of serum exosome tRFs/tiRNAs and to explore their diagnostic value in tuberculosis (TB) activity.
Methods
The serum exosome tRF/tiRNA profile was analysed using high-throughput sequencing technology in 5 active tuberculosis (ATB) patients, 5 latent tuberculosis infection (LTBI) patients and 5 healthy controls (HCs). Then, serum exosome tRFs/tiRNAs were validated by quantitative real-time polymerase chain reaction (qRT–PCR), and their diagnostic value was evaluated by receiver operating characteristic curve (ROC) and area under the curve (AUC). Finally, bioinformatics analysis was performed to explore and identify the potential biological pathways induced by tRFs/tiRNAs.
Results
The sequencing results revealed that serum exosome tRF/tiRNA expression profiles were different among ATB patients, LTBI patients and HCs. Three tRFs (tRF-56:75-Trp-CCA-4, tRF-1:22-chrM.Ser-GCT and tRF-56:76-Val-TAC-1-M2) were selected for qRT–PCR validation. The results demonstrated that the expression level of tRF-1-22-chrM.Ser-GCT was upregulated in ATB patients, while tRF-56-75-Trp-CCA-4 was downregulated, which was consistent with the sequencing data. The AUCs of tRF-56:75-Trp-CCA-4 and tRF-1:22-chrM. Ser-GCT were 0.824 and 1.000, respectively, which have significant values in the diagnosis of ATB patients. Moreover, the expression levels of tRF-56:75-Trp-CCA-4 and tRF-1:22-chrM.Ser-GCT and tRF-56:76-Val-TAC-1-M2 in ATB patients and LTBI were different, which indicated that these three tRFs could effectively distinguish ATB patients and LTBI patients.
Conclusion
Our findings indicate that serum exosome tRFs can be used as potential markers for the diagnosis of ATB and LTBI.
{"title":"Serum exosome tRFs as a promising biomarker for active tuberculosis and latent tuberculosis infection","authors":"Xiangyu Xi , Binghua Wang , Ruimei Zhang , Chunhua Ling","doi":"10.1016/j.mimet.2024.106944","DOIUrl":"10.1016/j.mimet.2024.106944","url":null,"abstract":"<div><h3>Objective</h3><p>To analyse the expression profiles of serum exosome tRFs/tiRNAs and to explore their diagnostic value in tuberculosis (TB) activity.</p></div><div><h3>Methods</h3><p>The serum exosome tRF/tiRNA profile was analysed using high-throughput sequencing technology in 5 active tuberculosis (ATB) patients, 5 latent tuberculosis infection (LTBI) patients and 5 healthy controls (HCs). Then, serum exosome tRFs/tiRNAs were validated by quantitative real-time polymerase chain reaction (qRT–PCR), and their diagnostic value was evaluated by receiver operating characteristic curve (ROC) and area under the curve (AUC). Finally, bioinformatics analysis was performed to explore and identify the potential biological pathways induced by tRFs/tiRNAs.</p></div><div><h3>Results</h3><p>The sequencing results revealed that serum exosome tRF/tiRNA expression profiles were different among ATB patients, LTBI patients and HCs. Three tRFs (tRF-56:75-Trp-CCA-4, tRF-1:22-chrM.Ser-GCT and tRF-56:76-Val-TAC-1-M2) were selected for qRT–PCR validation. The results demonstrated that the expression level of tRF-1-22-chrM.Ser-GCT was upregulated in ATB patients, while tRF-56-75-Trp-CCA-4 was downregulated, which was consistent with the sequencing data. The AUCs of tRF-56:75-Trp-CCA-4 and tRF-1:22-chrM. Ser-GCT were 0.824 and 1.000, respectively, which have significant values in the diagnosis of ATB patients. Moreover, the expression levels of tRF-56:75-Trp-CCA-4 and tRF-1:22-chrM.Ser-GCT and tRF-56:76-Val-TAC-1-M2 in ATB patients and LTBI were different, which indicated that these three tRFs could effectively distinguish ATB patients and LTBI patients.</p></div><div><h3>Conclusion</h3><p>Our findings indicate that serum exosome tRFs can be used as potential markers for the diagnosis of ATB and LTBI.</p></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"222 ","pages":"Article 106944"},"PeriodicalIF":2.2,"publicationDate":"2024-05-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140858516","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}