Pub Date : 2025-02-04DOI: 10.1016/j.mimet.2025.107095
Jörg Böllmann, Ramona Riedel, Marion Martienssen
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 Pseudomonas aeruginosa 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.
{"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, Ramona Riedel, 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<sup>−1</sup>, 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}
Alpha-toxin of Staphylococcus aureus belongs to the pore-forming toxin (PFT) family, which can lyse red and white blood cells. In addition to the existence of the hla gene in the majority of S. aureus strains (about 95 %), higher expression exhibits enhanced pathogenicity to the bacteria. Various methods, such as antibodies and aptamers, could serve to detect this toxin. In the current study, for the first time, an improved sandwich aptamer-antibody-based method was developed using specific murine polyclonal antibodies and a specific aptamer to detect a wide range of α-toxin levels. Denatured recombinant α-toxin was administered to mice to trigger the production of specific antibodies, which were subsequently purified from immune sera. These antibodies served as capturers in the designed apta-qPCR assay, with an aptamer employed as a detector. The results showed that spiked α-toxin in the undiluted serum samples could detect α-toxin between 300 and 0.5 ng/mL with no cross-reactivity. The coefficient of variation (CV) percent of intra- and inter-assays were 0.84 and 1.06, respectively. Since in the apta-qPCR assay, a combination of specific polyclonal antibodies as capture and specific aptamer along with real-time PCR (qPCR) sensitivity is used, this robust method could be used in diagnostic laboratories to detect various levels of the toxin in human serum samples.
{"title":"Highly sensitive detection of Staphylococcus aureus α-hemolysin protein (Hla or α-toxin) by apta-qPCR","authors":"Abolfazl Jahangiri , Samira Dahaghin , Ehsan Malekara , Raheleh Halabian , Mahdieh Mahboobi , Elham Behzadi , Hamid Sedighian","doi":"10.1016/j.mimet.2024.107084","DOIUrl":"10.1016/j.mimet.2024.107084","url":null,"abstract":"<div><div>Alpha-toxin of <em>Staphylococcus aureus</em> belongs to the pore-forming toxin (PFT) family, which can lyse red and white blood cells. In addition to the existence of the <em>hla</em> gene in the majority of <em>S. aureus</em> strains (about 95 %), higher expression exhibits enhanced pathogenicity to the bacteria. Various methods, such as antibodies and aptamers, could serve to detect this toxin. In the current study, for the first time, an improved sandwich aptamer-antibody-based method was developed using specific murine polyclonal antibodies and a specific aptamer to detect a wide range of α-toxin levels. Denatured recombinant α-toxin was administered to mice to trigger the production of specific antibodies, which were subsequently purified from immune sera. These antibodies served as capturers in the designed apta-qPCR assay, with an aptamer employed as a detector. The results showed that spiked α-toxin in the undiluted serum samples could detect α-toxin between 300 and 0.5 ng/mL with no cross-reactivity. The coefficient of variation (CV) percent of intra- and inter-assays were 0.84 and 1.06, respectively. Since in the apta-qPCR assay, a combination of specific polyclonal antibodies as capture and specific aptamer along with real-time PCR (qPCR) sensitivity is used, this robust method could be used in diagnostic laboratories to detect various levels of the toxin in human serum samples.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"229 ","pages":"Article 107084"},"PeriodicalIF":1.7,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142914865","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 : 2025-02-01DOI: 10.1016/j.mimet.2025.107091
Abbey Saunders, Emily Johnstone, Adam Foster
The devastating plant pathogen Fusarium graminearum produces mycotoxins including the novel 3ANX toxin. To detect 3ANX-producing isolates, SYBR Green and locked nucleic acid probe assays were developed, targeting 3ANX Tri1 polymorphisms. Assays were efficient with R2 > 0.99 and specific to 3ANX, detecting as few as 2 copies/μL.
{"title":"Development of qPCR methods to detect and quantify the novel Fusarium graminearum 3ANX chemotype variant","authors":"Abbey Saunders, Emily Johnstone, Adam Foster","doi":"10.1016/j.mimet.2025.107091","DOIUrl":"10.1016/j.mimet.2025.107091","url":null,"abstract":"<div><div>The devastating plant pathogen <em>Fusarium graminearum</em> produces mycotoxins including the novel 3ANX toxin. To detect 3ANX-producing isolates, SYBR Green and locked nucleic acid probe assays were developed, targeting 3ANX <em>Tri1</em> polymorphisms. Assays were efficient with R<sup>2</sup> > 0.99 and specific to 3ANX, detecting as few as 2 copies/μL.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"229 ","pages":"Article 107091"},"PeriodicalIF":1.7,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143006974","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 : 2025-02-01DOI: 10.1016/j.mimet.2025.107093
Richard Martins Carrassai , Camila Mörschbächer Wilhelm , Kellen Figueira Tragnago , Aymê Duarte Echevarria , Afonso Luís Barth
Carbapenemase-producing Enterobacterales are a growing concern in public health. In order to rapidly determine the antimicrobial profile, the MALDI Biotyper - antibiotic susceptibility test rapid assay (MBT-ASTRA) was developed, based on the relative growth of bacteria in the presence of antibiotics. In this study, we added carbapenemase enzymatic inhibitors to the MBT-ASTRA and developed an adapted method named MALDI Biotyper - Phenotypic Identification Test Rapid Assay (MBT-PITRA) in order to perform a rapid and cost-effective phenotypic test to detect Klebsiella pneumoniae carbapenemase (KPC) and New Delhi metallo-beta-lactamase (NDM), including co-producers, in Enterobacterales. Fifty-nine clinical isolates of carbapenemase-producing Enterobacterales and 28 non-carbapenemase-producing isolates collected from 2013 to 2023 were assessed using this approach. The MBT-PITRA method involved incubating bacterial isolates with different solutions of meropenem plus enzymatic inhibitors, phenylboronic acid and/or ethylenediaminetetraacetic acid, followed by analysis using MALDI-TOF MS. Carbapenemase production was inferred based on relative growth (RG) values calculated from peak intensities in the presence and absence of enzymatic inhibitors. KPC-producing isolates presented 90 % (18/20) concordance, while isolates positive for NDM (16/16), KPC plus NDM (14/14) and negative (28/28) for carbapenemases were 100 % correctly identified. MBT-PITRA demonstrated to be a promising method for identification of carbapenemases.
Importance
The MBT-PITRA appears to be a promising method for the rapid detection of carbapenemases in Enterobacterales.
{"title":"MALDI biotyper - Phenotypic identification test rapid assay (MBT-PITRA): A new method to detect KPC and NDM in Enterobacterales","authors":"Richard Martins Carrassai , Camila Mörschbächer Wilhelm , Kellen Figueira Tragnago , Aymê Duarte Echevarria , Afonso Luís Barth","doi":"10.1016/j.mimet.2025.107093","DOIUrl":"10.1016/j.mimet.2025.107093","url":null,"abstract":"<div><div>Carbapenemase-producing <em>Enterobacterales</em> are a growing concern in public health. In order to rapidly determine the antimicrobial profile, the MALDI Biotyper - antibiotic susceptibility test rapid assay (MBT-ASTRA) was developed, based on the relative growth of bacteria in the presence of antibiotics. In this study, we added carbapenemase enzymatic inhibitors to the MBT-ASTRA and developed an adapted method named MALDI Biotyper - Phenotypic Identification Test Rapid Assay (MBT-PITRA) in order to perform a rapid and cost-effective phenotypic test to detect <em>Klebsiella pneumoniae</em> carbapenemase (KPC) and New Delhi metallo-beta-lactamase (NDM), including co-producers, in <em>Enterobacterales</em>. Fifty-nine clinical isolates of carbapenemase-producing <em>Enterobacterales</em> and 28 non-carbapenemase-producing isolates collected from 2013 to 2023 were assessed using this approach. The MBT-PITRA method involved incubating bacterial isolates with different solutions of meropenem plus enzymatic inhibitors, phenylboronic acid and/or ethylenediaminetetraacetic acid, followed by analysis using MALDI-TOF MS. Carbapenemase production was inferred based on relative growth (RG) values calculated from peak intensities in the presence and absence of enzymatic inhibitors. KPC-producing isolates presented 90 % (18/20) concordance, while isolates positive for NDM (16/16), KPC plus NDM (14/14) and negative (28/28) for carbapenemases were 100 % correctly identified. MBT-PITRA demonstrated to be a promising method for identification of carbapenemases.</div></div><div><h3>Importance</h3><div>The MBT-PITRA appears to be a promising method for the rapid detection of carbapenemases in <em>Enterobacterales</em>.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"229 ","pages":"Article 107093"},"PeriodicalIF":1.7,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143006975","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 : 2025-02-01DOI: 10.1016/j.mimet.2025.107094
Caroline Østergaard Klein , Martin Laage Kragh , Philip Junker Andersen , Niels Agersnap , Jesper Bryde-Jacobsen , Lisbeth Truelstrup Hansen
In the food industry, time-to-result is crucial for faster release of products, minimising recalls, mitigation of microbial contamination problems and, ultimately, food safety. Carrageenan is isolated from red seaweed (Rhodophyta) and applied in various foods and beverages as a gelling, thickening, texturing, or stabilising agent due to its hygroscopic properties. Currently, the standard industry plate count method entails a one-hundred-fold dilution of the sample before mixing with molten agar for assessing the level of microbial contamination in carrageenan samples prior to business-to-business shipment. However, even at this dilution, carrageenan swells, forms clumps, clogs pipettes, and leaves thick gel structures, bubbles, and debris in agar plates causing microbial enumeration to be challenging and subject to human error. Here, we report, for the first time, the application of mini agar plates monitored by the automated time-lapse microscopy IntuGrow solution to assess the microbiological quality in the challenging food ingredient. Without dilution of the food sample, the carrageenan powder is scattered between two layers of Plate Count Agar to enumerate bacteria within 12–20 h, while enumeration by traditional plate counts requires 72 h. A DELAY algorithm for optical time-lapse microscopy was developed and added to IntuGrow analysis software to suppress the effects of swelling and enhance detection of the presence of growing microbial colonies by normalising the background using previous images. Time-lapse microscopy image-based monitoring made it possible to obtain results from carrageenan samples that could not be obtained by traditional plate counts due to swarming bacteria. Comparison between the two methods showed a nearly perfect Demings slope of 0.96, while an observed bias of −0.33 log CFU/g indicated that IntuGrow counts were lower than traditional plate counts. This is likely due to carrageenan artefacts being counted as colonies in the latter plates. The ability of IntuGrow to enumerate bacteria in challenging food ingredients such as carrageenan implies that the technology should be easy to apply for easy-to-dilute samples or non-hydrocolloid powders. Further testing in an industrial setting by different operators should be used to validate the reproducibility of the method.
{"title":"Optical time-lapse microscopy for rapid assessment of microbial quality in hygroscopic food samples","authors":"Caroline Østergaard Klein , Martin Laage Kragh , Philip Junker Andersen , Niels Agersnap , Jesper Bryde-Jacobsen , Lisbeth Truelstrup Hansen","doi":"10.1016/j.mimet.2025.107094","DOIUrl":"10.1016/j.mimet.2025.107094","url":null,"abstract":"<div><div>In the food industry, time-to-result is crucial for faster release of products, minimising recalls, mitigation of microbial contamination problems and, ultimately, food safety. Carrageenan is isolated from red seaweed (<em>Rhodophyta</em>) and applied in various foods and beverages as a gelling, thickening, texturing, or stabilising agent due to its hygroscopic properties. Currently, the standard industry plate count method entails a one-hundred-fold dilution of the sample before mixing with molten agar for assessing the level of microbial contamination in carrageenan samples prior to business-to-business shipment. However, even at this dilution, carrageenan swells, forms clumps, clogs pipettes, and leaves thick gel structures, bubbles, and debris in agar plates causing microbial enumeration to be challenging and subject to human error. Here, we report, for the first time, the application of mini agar plates monitored by the automated time-lapse microscopy IntuGrow solution to assess the microbiological quality in the challenging food ingredient. Without dilution of the food sample, the carrageenan powder is scattered between two layers of Plate Count Agar to enumerate bacteria within 12–20 h, while enumeration by traditional plate counts requires 72 h. A DELAY algorithm for optical time-lapse microscopy was developed and added to IntuGrow analysis software to suppress the effects of swelling and enhance detection of the presence of growing microbial colonies by normalising the background using previous images. Time-lapse microscopy image-based monitoring made it possible to obtain results from carrageenan samples that could not be obtained by traditional plate counts due to swarming bacteria. Comparison between the two methods showed a nearly perfect Demings slope of 0.96, while an observed bias of −0.33 log CFU/g indicated that IntuGrow counts were lower than traditional plate counts. This is likely due to carrageenan artefacts being counted as colonies in the latter plates. The ability of IntuGrow to enumerate bacteria in challenging food ingredients such as carrageenan implies that the technology should be easy to apply for easy-to-dilute samples or non-hydrocolloid powders. Further testing in an industrial setting by different operators should be used to validate the reproducibility of the method.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"229 ","pages":"Article 107094"},"PeriodicalIF":1.7,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143066165","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}
In recent years, oxidoreductase enzymes such as laccases have received considerable attention for their ability to degrade and eliminate organic micropollutants from contaminated water in a process known as enzyme-based wastewater treatment. Thus, methods to produce high laccase activity in water are a point of focus, with white-rot fungi being highlighted as a tool in this context. This study, therefore, explored the applied approach of direct addition of mushroom spawn of the white-rot fungi Pleurotus ostreatus into water and its potential for laccase production under different conditions. Grain spawn was observed to be preferable to sawdust spawn, resulting in laccase activity of 53.9 ± 5.9 U/L and 4.8 ± 0.8 U/L, respectively. Laccase activity was induced by adding kraft lignin (4 g/L), and an eightfold increase to 446.3 ± 43.1 U/L was observed for grain spawn. Lignin accumulated in the spawn over time, resulting in brown pellets composed of spawn, mycelium and lignin. Our results demonstrated that high levels of laccase activity could be obtained in different types of water, including effluent municipal wastewater, using this method. No impact from the addition of inorganic nitrogen (ammonium nitrate, N-levels 14 mg/L, 140 mg/L) or organic nitrogen sources (urea, yeast extract, wheat bran, N-levels 14 mg/L, 140 mg/L) was observed for the treatment with grain spawn and lignin, suggesting that stable laccase activity can be expected under these nutritional conditions.
{"title":"Spawn-based pellets of Pleurotus ostreatus as an applied approach for the production of laccase in different types of water","authors":"Inoka Sanjeewani Ranamukha Hewage , Oksana Golovko , Malin Hultberg","doi":"10.1016/j.mimet.2025.107092","DOIUrl":"10.1016/j.mimet.2025.107092","url":null,"abstract":"<div><div>In recent years, oxidoreductase enzymes such as laccases have received considerable attention for their ability to degrade and eliminate organic micropollutants from contaminated water in a process known as enzyme-based wastewater treatment. Thus, methods to produce high laccase activity in water are a point of focus, with white-rot fungi being highlighted as a tool in this context. This study, therefore, explored the applied approach of direct addition of mushroom spawn of the white-rot fungi <em>Pleurotus ostreatus</em> into water and its potential for laccase production under different conditions. Grain spawn was observed to be preferable to sawdust spawn, resulting in laccase activity of 53.9 ± 5.9 U/L and 4.8 ± 0.8 U/L, respectively. Laccase activity was induced by adding kraft lignin (4 g/L), and an eightfold increase to 446.3 ± 43.1 U/L was observed for grain spawn. Lignin accumulated in the spawn over time, resulting in brown pellets composed of spawn, mycelium and lignin. Our results demonstrated that high levels of laccase activity could be obtained in different types of water, including effluent municipal wastewater, using this method. No impact from the addition of inorganic nitrogen (ammonium nitrate, N-levels 14 mg/L, 140 mg/L) or organic nitrogen sources (urea, yeast extract, wheat bran, N-levels 14 mg/L, 140 mg/L) was observed for the treatment with grain spawn and lignin, suggesting that stable laccase activity can be expected under these nutritional conditions.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"229 ","pages":"Article 107092"},"PeriodicalIF":1.7,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143006978","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 : 2025-01-01DOI: 10.1016/j.mimet.2024.107083
Giulio Camarlinghi , Eva Maria Parisio , Agostino Ognibene
This study evaluates the performance of I-dOne, the first CE-IVD marked software for microbial species identification based on Attenuated Total Reflection Fourier Transform Infrared Spectroscopy (ATR-FTIR) and compares its results with MALDI-TOF MS technology (Vitek MS, bioMérieux). A total of 410 clinical isolates were analyzed, spanning 45 species and 24 genera. I-dOne demonstrated a high agreement rate (97.3 %) with the Vitek MS, meeting CLSI standard for microbial identification accuracy. Additionally, this study explored the development of a novel algorithm within I-dOne to discriminate between Bacteroides fragilis and Bacteroides ovatus strains, overcoming the current limitations in species-level differentiation. Finally, the influence of ageing under prolonged aerobic exposure on ATR-FTIR profiles was investigated, highlighting no significant spectral changes in Bacteroides fragilis strains under prolonged aerobic exposure. These findings underscore the accuracy of I-dOne software in microbial identification, offering a reliable alternative to conventional methods.
{"title":"I-dOne: A diagnostic tool in the field of identification of clinically relevant microbial strains","authors":"Giulio Camarlinghi , Eva Maria Parisio , Agostino Ognibene","doi":"10.1016/j.mimet.2024.107083","DOIUrl":"10.1016/j.mimet.2024.107083","url":null,"abstract":"<div><div>This study evaluates the performance of I-dOne, the first CE-IVD marked software for microbial species identification based on Attenuated Total Reflection Fourier Transform Infrared Spectroscopy (ATR-FTIR) and compares its results with MALDI-TOF MS technology (Vitek MS, bioMérieux). A total of 410 clinical isolates were analyzed, spanning 45 species and 24 genera. I-dOne demonstrated a high agreement rate (97.3 %) with the Vitek MS, meeting CLSI standard for microbial identification accuracy. Additionally, this study explored the development of a novel algorithm within I-dOne to discriminate between <em>Bacteroides fragilis</em> and <em>Bacteroides ovatus</em> strains, overcoming the current limitations in species-level differentiation. Finally, the influence of ageing under prolonged aerobic exposure on ATR-FTIR profiles was investigated, highlighting no significant spectral changes in <em>Bacteroides fragilis</em> strains under prolonged aerobic exposure. These findings underscore the accuracy of I-dOne software in microbial identification, offering a reliable alternative to conventional methods.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"228 ","pages":"Article 107083"},"PeriodicalIF":1.7,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142895527","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 : 2025-01-01DOI: 10.1016/j.mimet.2024.107071
Max Sewell , Caitlin Farley , Edward A.R. Portal , Diane Lindsay , Maria Luisa Ricci , Sophie Jarraud , Maria Scaturro , Ghislaine Descours , Anne Vatland Krøvel , Rachael Barton , Ian Boostom , Roisin Ure , Darja Kese , Valeria Gaia , Matej Golob , Susanne Paukner , Christophe Ginevra , Baharak Afshar , Sendurann Nadarajah , Ingrid Wybo , Abdelrahim Zoued
Currently there is no detailed, internationally agreed protocol defined to evaluate antimicrobial susceptibility testing (AST) for Legionella pneumophila (required to establish epidemiological cut-off value or “ECOFF” boundaries); therefore, antimicrobial resistance in these isolates cannot be defined. AST methods utilising media containing activated charcoal as an ingredient, to enable Legionella growth, are unreliable as noted in an internationally authored opinion paper and a new gold standard is required. Here we define a detailed protocol for broth microdilution (BMD) using defined cell culture collection-deposited control reference strains (Philadelphia-1 and Knoxville-1) as well as two accessible reference strains with moderately (lpeAB-carrying) and markedly (23S rRNA mutation-carrying) elevated azithromycin minimum inhibitory concentration (MIC). The defined protocol enables up to eight L. pneumophila strains to be set up on a single 96-well plate per antimicrobial tested. Initial ranges to routinely capture an MIC for these reference strains using clinically relevant antimicrobials azithromycin (0.01–0.25 mg/L), levofloxacin (0.008–0.03 mg/L), lefamulin (0.01–2 mg/L), rifampicin (0.0002–0.0008 mg/L) and doxycycline (0.25–16 mg/L) following incubation for 48 h at 37 °C in a shaking incubator have been empirically determined. Establishment of this internationally agreed protocol sets the scene for the next step: validation and comparison of antimicrobial ranges between international Legionella reference laboratories to establish putative resistance cut-off thresholds for these clinically relevant antimicrobials.
{"title":"Broth microdilution protocol for determining antimicrobial susceptibility of Legionella pneumophila to clinically relevant antimicrobials","authors":"Max Sewell , Caitlin Farley , Edward A.R. Portal , Diane Lindsay , Maria Luisa Ricci , Sophie Jarraud , Maria Scaturro , Ghislaine Descours , Anne Vatland Krøvel , Rachael Barton , Ian Boostom , Roisin Ure , Darja Kese , Valeria Gaia , Matej Golob , Susanne Paukner , Christophe Ginevra , Baharak Afshar , Sendurann Nadarajah , Ingrid Wybo , Abdelrahim Zoued","doi":"10.1016/j.mimet.2024.107071","DOIUrl":"10.1016/j.mimet.2024.107071","url":null,"abstract":"<div><div>Currently there is no detailed, internationally agreed protocol defined to evaluate antimicrobial susceptibility testing (AST) for <em>Legionella pneumophila</em> (required to establish epidemiological cut-off value or “ECOFF” boundaries); therefore, antimicrobial resistance in these isolates cannot be defined. AST methods utilising media containing activated charcoal as an ingredient, to enable <em>Legionella</em> growth, are unreliable as noted in an internationally authored opinion paper and a new gold standard is required. Here we define a detailed protocol for broth microdilution (BMD) using defined cell culture collection-deposited control reference strains (Philadelphia-1 and Knoxville-1) as well as two accessible reference strains with moderately (<em>lpeAB</em>-carrying) and markedly (23S <em>rRNA</em> mutation-carrying) elevated azithromycin minimum inhibitory concentration (MIC). The defined protocol enables up to eight <em>L. pneumophila</em> strains to be set up on a single 96-well plate per antimicrobial tested. Initial ranges to routinely capture an MIC for these reference strains using clinically relevant antimicrobials azithromycin (0.01–0.25 mg/L), levofloxacin (0.008–0.03 mg/L), lefamulin (0.01–2 mg/L), rifampicin (0.0002–0.0008 mg/L) and doxycycline (0.25–16 mg/L) following incubation for 48 h at 37 °C in a shaking incubator have been empirically determined. Establishment of this internationally agreed protocol sets the scene for the next step: validation and comparison of antimicrobial ranges between international <em>Legionella</em> reference laboratories to establish putative resistance cut-off thresholds for these clinically relevant antimicrobials.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"228 ","pages":"Article 107071"},"PeriodicalIF":1.7,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142872025","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}
We constructed a simple genotyping method combining multiplex allele-specific PCR and DNA chromatography for Salmonella enterica subsp. enterica serovar Typhimurium and its monophasic variant. The developed method can be used to estimate the genetic background of isolates, and it facilitates easy identification of several epidemic clades among these serotypes.
{"title":"Multiplex allele-specific PCR and DNA chromatography to identify genotypes of Salmonella enterica serovar Typhimurium and its monophasic variants","authors":"Nobuo Arai , Yukino Tamamura-Andoh , Taketoshi Iwata , Ayako Watanabe-Yanai , Masahiro Kusumoto","doi":"10.1016/j.mimet.2024.107082","DOIUrl":"10.1016/j.mimet.2024.107082","url":null,"abstract":"<div><div>We constructed a simple genotyping method combining multiplex allele-specific PCR and DNA chromatography for <em>Salmonella enterica</em> subsp. <em>enterica</em> serovar Typhimurium and its monophasic variant. The developed method can be used to estimate the genetic background of isolates, and it facilitates easy identification of several epidemic clades among these serotypes.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"228 ","pages":"Article 107082"},"PeriodicalIF":1.7,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142876466","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}
Coconut wilt associated with phytoplasma presence is a serious disease that threatens the coconut plantations in South India. Symptoms progress rapidly and cause complete destruction of coconut palm which results in severe economic loss to farmers. Survey in the areas of Thanjavur and Coimbatore districts revealed disease incidence upto 2.5 % and the affected palms exhibited unique symptoms, which differ from the root wilt disease symptoms reported so far. Nested PCR with universal primers and multilocus characterization of tuf and certain rp genes confirmed the presence of phytoplasmas. The 16S rRNA ribosomal gene sequence-based identification assigned the coconut wilt phytoplasma to the ‘Candidatus Phytoplasma asteris’ species. To achieve timely management of the disease and also to check its spread, Loop Mediated Isothermal Amplification (LAMP) and real-time LAMP diagnostics by targeting the 16S rRNA gene, were established for rapid and specific detection of phytoplasma presence. PCR with LAMP outer primers was carried out and sequence analysis confirmed the amplification of the 16S rRNA gene of phytoplasma. LAMP assay positive samples showed the color shift from violet to blue and was further confirmed by the ladder-like bands produced during the amplification. Diseased samples also generated a unique annealing peak at 87 ± 0.5 °C in the real-time LAMP assay. The LAMP protocol devised will be useful for quick and sensitive detection of this phytoplasma and it has potential application to detect phytoplasma presence in suspected coconut palms and to allow screening of nursery seedlings to ensure disease free planting.
{"title":"Characterization of phytoplasma associated with wilt disease in coconut and approaches for its sensitive diagnostics","authors":"Natesan Boopathi , Gandhi Karthikeyan , Muthurajan Raveendran , Iruthayasamy Johnson , Subbaiyan Maruthasalam , Thulasy Srinivasan , Ramaswamy Manimekalai","doi":"10.1016/j.mimet.2024.107072","DOIUrl":"10.1016/j.mimet.2024.107072","url":null,"abstract":"<div><div>Coconut wilt associated with phytoplasma presence is a serious disease that threatens the coconut plantations in South India. Symptoms progress rapidly and cause complete destruction of coconut palm which results in severe economic loss to farmers. Survey in the areas of Thanjavur and Coimbatore districts revealed disease incidence upto 2.5 % and the affected palms exhibited unique symptoms, which differ from the root wilt disease symptoms reported so far. Nested PCR with universal primers and multilocus characterization of <em>tuf</em> and certain <em>rp</em> genes confirmed the presence of phytoplasmas. The 16S rRNA ribosomal gene sequence-based identification assigned the coconut wilt phytoplasma to the ‘<em>Candidatus</em> Phytoplasma asteris’ species. To achieve timely management of the disease and also to check its spread, Loop Mediated Isothermal Amplification (LAMP) and real-time LAMP diagnostics by targeting the 16S rRNA gene, were established for rapid and specific detection of phytoplasma presence. PCR with LAMP outer primers was carried out and sequence analysis confirmed the amplification of the 16S rRNA gene of phytoplasma. LAMP assay positive samples showed the color shift from violet to blue and was further confirmed by the ladder-like bands produced during the amplification. Diseased samples also generated a unique annealing peak at 87 ± 0.5 °C in the real-time LAMP assay. The LAMP protocol devised will be useful for quick and sensitive detection of this phytoplasma and it has potential application to detect phytoplasma presence in suspected coconut palms and to allow screening of nursery seedlings to ensure disease free planting.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"228 ","pages":"Article 107072"},"PeriodicalIF":1.7,"publicationDate":"2024-11-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142729702","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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