Journal of microbiological methods最新文献

{"title":"A TaqMan real-time PCR assay for detection of qacEΔ1 gene in Gram-negative bacteria","authors":"","doi":"10.1016/j.mimet.2024.107054","DOIUrl":"10.1016/j.mimet.2024.107054","url":null,"abstract":"<div><div>The transfer of biocide and antibiotic resistance genes by mobile genetic elements is the most common mechanism for rapidly acquiring and spreading resistance among bacteria. The <em>qacEΔ1</em> gene confers the resistance to quaternary ammonium compounds (QACs). It has also been considered a genetic marker for the presence of class 1 integrons associated with multidrug-resistant (MDR) phenotypes in Gram-negative bacteria. In this study, a TaqMan real-time PCR assay was developed to detect the <em>qacEΔ1</em> gene in Gram-negative bacteria. The assay has a detection limit of 80 copies of the <em>qacEΔ1</em> gene per reaction. No false-positive or false-negative results have been observed. Simultaneous amplification and detection of the 16S rRNA gene is performed as an endogenous internal amplification control (IAC). The TaqMan real-time PCR assay developed is a rapid, sensitive, and specific method that could be used to monitor resistance to QACs, the spread of class 1 integrons, and the prediction of associated MDR phenotypes in Gram-negative bacteria.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":null,"pages":null},"PeriodicalIF":1.7,"publicationDate":"2024-10-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142441261","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Mycobacterium tuberculosis complex sample processing by mechanical lysis, an essential step for reliable whole genome sequencing","authors":"","doi":"10.1016/j.mimet.2024.107053","DOIUrl":"10.1016/j.mimet.2024.107053","url":null,"abstract":"<div><div><em>Mycobacterium tuberculosis</em> complex (MTBC) whole genome sequencing (WGS) turnaround time and WGS success rates are highly influenced by DNA extraction protocols even from cultures. Efficient mycobacterial lysis is crucial for obtaining sufficient DNA from cultures to facilitate reliable genomic drug susceptibility prediction and accurate genotyping with WGS. We compared four DNA extraction protocols from BD BACTEC™ Mycobacterial Growth Indicator Tubes (MGIT) for WGS with a focus on the lysis step: protocol A) column-based protocol without mechanical lysis; protocol B) an adapted protocol including a bead beating step; protocol C) DNA extraction from primary received cultures using bead beating: and protocol D) DNA extraction from pre MGIT-positive (enriched) cultures. Protocol B increased DNA yield approximately 60-fold, and significantly improved the sequencing success rate. The increased yield also allowed DNA extraction from primary cultures with high success rates (protocol C). Additionally, by using pre-positive enriched MGIT cultures, we demonstrated that bead beating opens the possibility of reliable WGS up to five days before a MGIT tube would be flagged positive (protocol D). The most optimal bead beating-based DNA extraction was also evaluated for Nanopore sequencing. Shortening bead beating duration to 15 s resulted in longer read lengths (N50 from 1.4 kb to 2.6 kb) while still providing efficient lysis. Furthermore, AmpureXP bead beating-based DNA capture / purification proved to be as efficient as Qiagen column-based DNA extraction, further simplifying and shortening the DNA extraction protocol. Adding a mechanical lysis step to our routine MTBC DNA extraction protocol has allowed us to reduce the turnaround time while maintaining DNA quality sequencing success rates.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":null,"pages":null},"PeriodicalIF":1.7,"publicationDate":"2024-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142441309","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Evaluation of protein extraction protocols for MALDI-TOF Biotyper analysis of mycobacteria","authors":"","doi":"10.1016/j.mimet.2024.107052","DOIUrl":"10.1016/j.mimet.2024.107052","url":null,"abstract":"<div><div>Infections caused by <em>Mycobacterium tuberculosis</em> and nontuberculous mycobacteria represent a significant global threat and medical concern. Therefore, accurate and reliable methods must be employed to identify mycobacteria rapidly. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a technique that compares the cellular protein profiles of unknown isolates with reference mass spectra in a database to identify microorganisms. However, the thick and waxy lipid layer, which is rich in mycolic acids and is present in mycobacterial cells, makes protein extraction challenging. To identify the optimal protocol for correctly identifying bacilli using MALDI-TOF mass spectrometry, this study compared four different cellular protein extraction methods. Four strains of <em>M. bovis</em> BCG were selected as representatives of slow-growing mycobacteria, while three strains of fast-growing mycobacteria were also included: <em>M. peregrinum</em>, <em>M. smegmatis,</em> and <em>M. farcinogenes</em>. The extraction method that proved most effective was the extraction of inactivated cells with chloroform and methanol, which partially delipidates the cells. These cells were then extracted with formic acid, as is standard practice for protein extraction. The advantage of this method is that it allows the parallel analysis of cellular lipids and proteins from a single sample. It is therefore important to optimize mycobacterial protein extraction for MALDI-TOF MS analysis in clinical microbiology laboratories.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":null,"pages":null},"PeriodicalIF":1.7,"publicationDate":"2024-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142391168","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Whole genome sequencing approaches for taxonomic profiling and evaluation of wastewater quality","authors":"","doi":"10.1016/j.mimet.2024.107051","DOIUrl":"10.1016/j.mimet.2024.107051","url":null,"abstract":"<div><div>Tracking metagenomic abundance in wastewater is undoubtedly a powerful tool to detect emerging variants and improve community health. However, there are a few factors that limit environmental water-based genomic monitoring: sampling variability, incomplete coverage, genetic fragmentation, degradation, data analysis and interpretation. The decreasing costs of high-throughput sequencing and high-end supercomputers have increased the use and accuracy of genomic data for microbial detection and monitoring in wastewater samples within any given region. To better understand the microbial dynamics and to determine the target sequencing throughput required to establish taxa that may pose as bio-indicators of an epidemiological outbreak, wastewater samples were collected from distinct locations within the Emirate of Abu Dhabi, United Arab Emirates using appropriate sampling methods. A reference database of ∼27,000 known species was developed and used for further analysis. The results showed that 15 % of data in each sample matched any of ∼27,000 known bacterial, viral, fungal, or protozoan species. Despite the high fraction of unclassified data (85 %), more than 2000 species from &gt;800 genera across &gt;30 phyla were detected in each sample. Both 5 Gb and 10 Gb of sequenced data detected the top ∼2000 species with highest abundance. Doubling the target sequencing throughput (i.e., 10 Gb vs 5 Gb) detected ∼500 additional low-abundance species per sample however it did not affect the overall sample composition or translate into higher per-sample species diversity captured. There was a marginal increase in the number of species detected in each sample beyond 0.20 Gb of classified data. Overall, the results indicate that sequencing to a 3 Gb throughput detects nearly 95 % of all species in the samples.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":null,"pages":null},"PeriodicalIF":1.7,"publicationDate":"2024-10-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142391169","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Fermentation dynamics of bile salt hydrolase production in Heyndrickxia coagulans ATCC 7050 and Lactiplantibacillus plantarum ATCC 10012: Addressing ninhydrin assay limitations with a novel HPTLC–MS method","authors":"","doi":"10.1016/j.mimet.2024.107050","DOIUrl":"10.1016/j.mimet.2024.107050","url":null,"abstract":"<div><div>Bile salt hydrolase (BSH), a pivotal enzyme in cholesterol management, holds significant promise in both human and animal subjects. This study investigated the effect of fermentation dynamics in <em>Heyndrickxia coagulans</em> ATCC 7050 and <em>Lactiplantibacillus plantarum</em> ATCC 10012 to enhance BSH production. Cultivation of cultures in MRS and M17 media revealed that MRS medium enhanced BSH production by 235.98 % in <em>H. coagulans</em> ATCC 7050 and 147.37 % in <em>L. plantarum</em> ATCC 10012, compared to M 17 medium. Additionally, varying oxygen concentration levels indicated that <em>H. coagulans</em> ATCC 7050 exhibited its minimum doubling time of 79.8 ± 0.64 min in anaerobic conditions, whereas <em>L.</em> <em>plantarum</em> ATCC 10012 demonstrated its minimum doubling time of 85.5 ± 1.2 min under microaerophilic conditions. However, their highest BSH activity was observed during the stationary phase under anaerobic conditions, yielding 17.14 ± 0.78 U/mL by <em>H. coagulans</em> ATCC 7050 and 19.04 ± 0.81 U/mL by <em>L.</em> <em>plantarum</em> ATCC 10012. Furthermore, it was observed that both organisms did not retain BSH within their cells. BSH activity was assessed using ninhydrin assay that detected free taurine liberated from sodium taurocholate. However, ninhydrin can yield false–positive results owing to its interaction with other free amino acids. To subjugate this limitation, the study introduced a novel and sensitive HPTLC–MS method capable of accurately detecting taurine. By comprehending fermentation dynamics and selecting appropriate conditions, BSH production increased 2.1–fold in both organisms. These findings illuminate critical insights, offering a pathway for novel strategies to enhance the BSH–producing capabilities of these LAB strains.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":null,"pages":null},"PeriodicalIF":1.7,"publicationDate":"2024-09-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142365454","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Campylobacter fetus subspecies specific PCR assays inferred from comparative genomic analysis for accurate subspecies identification","authors":"","doi":"10.1016/j.mimet.2024.107049","DOIUrl":"10.1016/j.mimet.2024.107049","url":null,"abstract":"<div><div>Bovine Genital Campylobacteriosis (BGC) is caused by <em>Campylobacter fetus</em> subsp. <em>venerealis</em> and is a notifiable disease to the WOAH (World Organisation for Animal Health). For an effective BGC control program, the reliable differentiation of <em>Campylobacter fetus</em> subsp. <em>venerealis</em> (Cfv) from the closely related <em>Campylobacter fetus</em> subsp. <em>fetus</em> (Cff) is required<em>.</em> However, the available molecular <em>C. fetus</em> subspecies identification assays lack sensitivity and specificity to differentiate <em>C. fetus</em> isolates based on their phenotypic or genotypic differences. Furthermore, the current biochemical subspecies identification is not fully congruent with the genomic differentiation of <em>C. fetus</em> strains.</div><div>In this study, the genome sequences of 41<em>C. fetus</em> strains with well identified subspecies, were analyzed with the large-scale BLAST score ratio (LS-BSR) pipeline to identify Cff and Cfv specific sequences. With this analysis, the <em>asd</em> gene encoding an aspartate-semialdehyde dehydrogenase was identified, which contained a 6-bp Cff-specific sequence, and this 6-bp sequence was absent in the <em>asd</em> gene of Cfv strains. This sequence was used for the development of PCR assays to differentiate Cff and Cfv strains. The <em>C. fetus</em> subspecies identification of the developed <em>asd</em> PCR assays was in full congruence with the genomic classification of strains and are recommended for molecular identification of <em>C. fetus</em> subspecies in BGC control programs.</div><div>The <em>asd</em> PCR can be assessed on sequenced genomes using a web interface containing the Cfvcatch tool, which includes placement of the tested genome in a phylogenetic tree with reference <em>C. fetus</em> genomes to distinguish the two subspecies and to detect antimicrobial resistance genes.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":null,"pages":null},"PeriodicalIF":1.7,"publicationDate":"2024-09-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142348448","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Optimized protocol for collecting root canal biofilms for in vitro studies","authors":"","doi":"10.1016/j.mimet.2024.107048","DOIUrl":"10.1016/j.mimet.2024.107048","url":null,"abstract":"<div><div>Endodontic retreatment is often necessitated by several factors, including the persistence of microorganisms in the root canal system (RCS). Their complex organization in biofilms increases their pathogenic potential, necessitating new disinfection strategies. This study aimed to standardize a new <em>in vitro</em> protocol for collecting biofilm from the RCS. Thirty-four bovine incisors were used in the study, divided into two experimental groups with two collection steps each: (a) biofilm collection protocol and (b) absorbent paper points protocol. Twelve specimens from each group were selected for counting colony-forming units (CFUs), while eight specimens were prepared for scanning electron microscopy (SEM). Two additional specimens served as sterilization controls to ensure that experiments were free of contamination. The coronal region was removed and standardized at 15 mm. After preparation with ProTaper up to F5, the apical foramen was sealed with composite resin, and the roots were stabilized with acrylic resin in 1.5-mL Eppendorf tubes. The specimens were sterilized and inoculated with <em>Enterococcus faecalis</em> NTCT 775 every 24 h for 21 days. After this period, each group underwent biofilm collection protocols, and CFU and scanning electron microscopy (SEM) data were analyzed. The Shapiro–Wilk test was performed to assess the normality of log-transformed data, and the results indicated a normal distribution for all groups, allowing parametric testing. The Levene test was used to evaluate the equality of variances. The proposed biofilm collection method yielded significantly higher CFU counts compared with the absorbent paper points method, particularly when analyzed on a log₁₀ scale. An independent samples <em>t</em>-test confirmed a statistically significant difference between the two methods (<em>p</em> &lt; 0.0001). The proposed protocol achieved an efficiency rate of 95.85 % ± 1.15 %, whereas the absorbent paper points protocol yielded a lower efficiency of 5.46 % ± 1.37 %. Therefore, the biofilm collection protocol proposed in this study proved to be more effective for biofilm removal from the RCS.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":null,"pages":null},"PeriodicalIF":1.7,"publicationDate":"2024-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142348449","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A comparative study of a rapid phenotypic antimicrobial susceptibility testing system directly from positive blood cultures to the disk diffusion and VITEK 2 methods","authors":"","doi":"10.1016/j.mimet.2024.107046","DOIUrl":"10.1016/j.mimet.2024.107046","url":null,"abstract":"<div><h3>Background</h3><div>Sepsis is a life-threatening condition that impacts 49 million people annually and causes 11 million deaths worldwide. Surviving bloodstream infections (BSIs) depends on the rapid administration of effective antimicrobial treatment, underscoring a need for rapid antimicrobial susceptibility testing (AST).</div></div><div><h3>Aim</h3><div>To evaluate the performance of Quantamatrix's dRAST v2.5 system (Seoul, South Korea) for AST directly from positive blood cultures as compared to the Disk-Diffusion (DD) and VITEK 2 methods.</div></div><div><h3>Methods</h3><div>The study included 191 positive blood cultures from clinical samples and spiked blood culture bottles. Following Gram staining and species-level identification, AST was performed by VITEK 2 and standard DD methods using CLSI (2021) interpretation.</div></div><div><h3>Results</h3><div>dRAST demonstrated very good AST performance for a Gram-negative isolate, and good performance for Gram-positive isolates, meeting CLSI criteria for the acceptance of a new method. Antimicrobials that were not considered verified compared to VITEK 2 and DD were cefazolin, ceftazidime, meropenem, and trimethoprim/sulfamethoxazole for Gram-negatives and clindamycin, erythromycin, penicillin, and oxacillin for Gram-positives. dRAST ESBL detection results were strongly correlated with the ESBL phenotypes obtained with other methods. Additional resistance mechanisms were in concordance with traditional tests.</div></div><div><h3>Conclusions</h3><div>dRAST demonstrated good AST performance, meeting CLSI criteria for most relevant antibiotics. dRAST was associated with a significant reduction in time-to-results, labor, and the subjectivity of result analyses, making it a valuable addition to efforts supporting the treatment of patients with bacteremia.</div><div>AST (antimicrobial susceptibility test), blood culture, dRAST, rapid methods, sepsis, turnaround time (TAT).</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":null,"pages":null},"PeriodicalIF":1.7,"publicationDate":"2024-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142289361","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"“Curing of common plasmids in gram-negative bacteria using a Cas9-based conjugative vector”","authors":"","doi":"10.1016/j.mimet.2024.107047","DOIUrl":"10.1016/j.mimet.2024.107047","url":null,"abstract":"<div><div>We report the creation of 17 <em>Escherichia coli</em> strains harboring the conjugative plasmid pLCasCureT with a CRISPR-Cas9 system to surgically “cure” the most common plasmids among <em>Enterobacterales</em> species. This approach can create isogenic pairs of strains to study host-plasmid interactions, correlate plasmid genotype and phenotype, and create plasmid-free cloning strains.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":null,"pages":null},"PeriodicalIF":1.7,"publicationDate":"2024-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142289363","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A simple culture medium for phenotypic characterization and long-term storage of medically relevant fusarioid fungi","authors":"","doi":"10.1016/j.mimet.2024.107042","DOIUrl":"10.1016/j.mimet.2024.107042","url":null,"abstract":"<div><div>Fusarioid fungi, particularly <em>Neocosmospora solani</em> and <em>Fusarium oxysporum</em>, are emerging as significant human pathogens, causing infections ranging from localized mycoses to life-threatening systemic diseases. Accurate identification and preservation of these fungi in clinical laboratories remain challenging because of their diverse morphologies and specific growth requirements. This study evaluated a novel milk-honey and malt agar (MHM) against conventional media for cultivating and preserving 60 clinical fusarioid isolates, including <em>Neocosmospora</em> spp. (<em>n</em> = 47), <em>Bisifusarium</em> spp. (<em>n</em> = 5), and <em>Fusarium</em> spp. (<em>n</em> = 8). Compared with Sabouraud dextrose 2 % agar (SDA) and malt extract agar (ME2), MHM significantly increased conidia production (<em>p</em> &lt; 0.0001, mean = 3.4 × 10³, standard deviation (SD) = ±1.3 × 10³), with results similar to those of carnation leaf agar (CLA). MHM facilitated superior preservation of fusarioid viability for up to one year at room temperature on slant cultures and over two years on swabs in Amies gel with charcoal, outperforming current methods such as Castellani (water) or cryopreservation. Morphological characterization of fusarioid fungi grown on MHM revealed distinct growth patterns and conidial structures for <em>Neocosmospora</em>, <em>Bisifusarium</em>, and <em>Fusarium</em> species, aiding in identifying these genera. The superior performance of MHM in stimulating conidiation, maintaining viability, and preserving morphology underscore its potential as a reference medium for medically relevant fusarioid fungi, with broad implications for clinical mycology laboratories and resource-limited settings.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":null,"pages":null},"PeriodicalIF":1.7,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142289362","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}