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Scientific Reports Jun 2024Bovine alveolar macrophages (AMs) defend the lungs against pathogens such as Mycobacterium bovis (M. bovis), the causative agent of bovine tuberculosis. However, little...
Bovine alveolar macrophages (AMs) defend the lungs against pathogens such as Mycobacterium bovis (M. bovis), the causative agent of bovine tuberculosis. However, little is known about the surface molecules expressed by bovine AMs and whether there is heterogeneity within the population. The purpose of this study was to characterise the bovine AM cell surface phenotype using flow cytometry. Bronchoalveolar lavage samples from four different calves were stained with a combination of antibodies against immune cell molecules prior to flow cytometric analysis. To assess the degree of expression, we considered the distribution and relative intensities of stained and unstained cells. We demonstrated that bovine AMs have high expression of CD172a, ADGRE1, CD206, and CD14, moderate expression of CD80, MHC II, CD1b, and CD40, low expression of CX3CR1 and CD86, and little or no expression of CD16 and CD26. Two distinct subsets of bovine AMs were identified based on CD163 expression. Subsequent analysis showed that the CD163 subset had greater expression of other typical macrophage molecules compared to the CD163 subset, suggesting that these cells may perform different roles during infection. The characterisation of the uninfected bovine AM phenotype will provide a foundation for the examination of M. bovis-infected AMs.
Topics: Animals; Cattle; Macrophages, Alveolar; Antigens, Differentiation, Myelomonocytic; Antigens, CD; Receptors, Cell Surface; Phenotype; Mycobacterium bovis; Flow Cytometry; Tuberculosis, Bovine; Immunophenotyping; Bronchoalveolar Lavage Fluid
PubMed: 38951667
DOI: 10.1038/s41598-024-65868-7 -
Methods in Molecular Biology (Clifton,... 2024Whole genome sequencing of Mycobacterium tuberculosis complex (MTBC) isolates has been shown to provide accurate predictions for resistance and susceptibility for many...
Whole genome sequencing of Mycobacterium tuberculosis complex (MTBC) isolates has been shown to provide accurate predictions for resistance and susceptibility for many first- and second-line anti-tuberculosis drugs. However, bioinformatic pipelines and mutation catalogs to predict antimicrobial resistances in MTBC isolates are often customized and detailed protocols are difficult to access. Here, we provide a step-by-step workflow for the processing and interpretation of short-read sequencing data and give an overview of available analysis pipelines.
Topics: Mycobacterium tuberculosis; Whole Genome Sequencing; Microbial Sensitivity Tests; Humans; Antitubercular Agents; Computational Biology; Genome, Bacterial; Drug Resistance, Bacterial; Mutation; Tuberculosis
PubMed: 38949712
DOI: 10.1007/978-1-0716-3981-8_18 -
Methods in Molecular Biology (Clifton,... 2024Whole genome sequencing (WGS) is becoming an important diagnostic tool for antimicrobial susceptibility testing of Mycobacterium tuberculosis complex (MTBC) isolates in...
Whole genome sequencing (WGS) is becoming an important diagnostic tool for antimicrobial susceptibility testing of Mycobacterium tuberculosis complex (MTBC) isolates in many countries. WGS protocols usually start with the preparation of a DNA-library: the critical first step in the process. A DNA-library represents the genomic content of a DNA sample and consists of unique short DNA fragments. Although available DNA-library protocols come with manufacturer instructions, details of the entire process, including quality controls, instrument parameters, and run evaluations, often need to be developed and customized by each laboratory to implement WGS technology effectively. Here, we provide a detailed workflow for a DNA-library preparation based on an adapted Illumina protocol optimized for the reduction of reagent costs.
Topics: Mycobacterium tuberculosis; Whole Genome Sequencing; Microbial Sensitivity Tests; Genome, Bacterial; Humans; Antitubercular Agents; Gene Library; DNA, Bacterial; Tuberculosis; High-Throughput Nucleotide Sequencing
PubMed: 38949711
DOI: 10.1007/978-1-0716-3981-8_17 -
Methods in Molecular Biology (Clifton,... 2024The diagnosis and monitoring of tuberculosis treatment is difficult as many patients are unable to produce sputum. This means that many patients are treated on the basis...
The diagnosis and monitoring of tuberculosis treatment is difficult as many patients are unable to produce sputum. This means that many patients are treated on the basis of clinical findings and consequently some will be exposed to anti-tuberculosis drugs unnecessarily. Moreover, for those appropriately on treatment and unable to produce a sputum sample, it will be impossible to monitor the response to treatment. We have shown that stool is a potential alternative sample type for diagnosis of tuberculosis. Currently, available protocols like the Xpert MTB/RIF use DNA as a target to detect Mycobacterium tuberculosis in stool but DNA survives long after the organism is dead so it is not certain whether a positive test is from an old or a partially treated infection. The TB MBLA only detects live organisms and thus, can be used to follow the response to treatment. In this chapter, we describe a protocol for TB-MBLA, an RNA-based assay, and apply it to quantify TB bacteria in stool.
Topics: Feces; Mycobacterium tuberculosis; Humans; Bacterial Load; Tuberculosis; Antitubercular Agents; DNA, Bacterial; Sputum
PubMed: 38949709
DOI: 10.1007/978-1-0716-3981-8_15 -
Methods in Molecular Biology (Clifton,... 2024Mycobacterium tuberculosis is an infectious pathogen that requires biosafety level-3 laboratory for handling. The risk of transmission is high to laboratory staff, and...
Mycobacterium tuberculosis is an infectious pathogen that requires biosafety level-3 laboratory for handling. The risk of transmission is high to laboratory staff, and to manage the organism safely, it is necessary to construct high containment laboratory facilities at great expense. This limits the application of tuberculosis diagnostics to areas where there is insufficient capital to invest in laboratory infrastructure. In this method, we describe a process of inactivating sputum samples by either heat or guanidine thiocyanate (GTC) that renders them safe without affecting the quantification of viable bacteria. This method eliminates the need for level 3 containment laboratory for the tuberculosis molecular bacterial load assay (TB-MBLA) and is applicable in low- and middle-income countries.
Topics: Mycobacterium tuberculosis; Humans; Thiocyanates; Containment of Biohazards; Sputum; Bacterial Load; Tuberculosis; Guanidines; Hot Temperature; Microbial Viability
PubMed: 38949708
DOI: 10.1007/978-1-0716-3981-8_14 -
Methods in Molecular Biology (Clifton,... 2024Going back in time through a phylogenetic tree makes it possible to evaluate ancestral genomes and assess their potential to acquire key polymorphisms of interest over...
Going back in time through a phylogenetic tree makes it possible to evaluate ancestral genomes and assess their potential to acquire key polymorphisms of interest over evolutionary time. Knowledge of this kind may allow for the emergence of key traits to be predicted and pre-empted from currently circulating strains in the future. Here, we present a novel genome-wide survival analysis and use the emergence of drug resistance in Mycobacterium tuberculosis as an example to demonstrate the potential and utility of the technique.
Topics: Phylogeny; Mycobacterium tuberculosis; Genome, Bacterial; Humans; Evolution, Molecular; Drug Resistance, Bacterial; Tuberculosis
PubMed: 38949706
DOI: 10.1007/978-1-0716-3981-8_12 -
Methods in Molecular Biology (Clifton,... 2024Tuberculosis (TB) is the most common cause of death from an infectious disease. Although treatment has been available for more than 70 years, it still takes too long and...
Tuberculosis (TB) is the most common cause of death from an infectious disease. Although treatment has been available for more than 70 years, it still takes too long and many patients default risking relapse and the emergence of resistance. It is known that lipid-rich, phenotypically antibiotic-tolerant, bacteria are more resistant to antibiotics and may be responsible for relapse necessitating extended therapy. Using a microfluidic system that acoustically traps live mycobacteria, M. smegmatis, a model organism for M. tuberculosis we can perform optical analysis in the form of wavelength-modulated Raman spectroscopy (WMRS) on the trapped organisms. This system can allow observations of the mycobacteria for up to 8 h. By adding antibiotics, it is possible to study the effect of antibiotics in real-time by comparing the Raman fingerprints in comparison to the unstressed condition. This microfluidic platform may be used to study any microorganism and to dynamically monitor its response to many conditions including antibiotic stress, and changes in the growth media. This opens the possibility of understanding better the stimuli that trigger the lipid-rich downregulated and phenotypically antibiotic-resistant cell state.
Topics: Spectrum Analysis, Raman; Mycobacterium smegmatis; Microfluidics; Anti-Bacterial Agents; Acoustics; Lab-On-A-Chip Devices; Microfluidic Analytical Techniques; Humans
PubMed: 38949705
DOI: 10.1007/978-1-0716-3981-8_11 -
Methods in Molecular Biology (Clifton,... 2024Pyrazinamide (PZA) is a key component of chemotherapy for the treatment of drug-susceptible tuberculosis (TB) and is likely to continue to be included in new drug...
Pyrazinamide (PZA) is a key component of chemotherapy for the treatment of drug-susceptible tuberculosis (TB) and is likely to continue to be included in new drug combinations. Potentiation of PZA could be used to reduce the emergence of resistance, shorten treatment times, and lead to a reduction in the quantity of PZA consumed by patients, thereby reducing the toxic effects. Acidified medium is required for the activity of PZA against Mycobacterium tuberculosis. In vitro assessments of pyrazinamide activity are often avoided because of the lack of standardization, which has led to a lack of effective in vitro tools for assessing and/or enhancing PZA activity.We have developed and optimized a novel, robust, and reproducible, microtiter plate assay, that centers around acidity levels that are low enough for PZA activity. The assay can be applied to the evaluation of novel compounds for the identification of potentiators that enhance PZA activity. In this assay, potentiation of PZA is demonstrated to be statistically significant with the addition of rifampicin (RIF), which can, therefore, be used as a positive control. Conversely, norfloxacin demonstrates no potentiating activity with PZA and can be used as a negative control. The method, and the associated considerations, described here, can be adapted in the search for potentiators of other antimicrobials.
Topics: Pyrazinamide; Mycobacterium tuberculosis; Antitubercular Agents; Hydrogen-Ion Concentration; Microbial Sensitivity Tests; Drug Synergism; Rifampin; Humans
PubMed: 38949702
DOI: 10.1007/978-1-0716-3981-8_8 -
Methods in Molecular Biology (Clifton,... 2024Current clinical practice assumes that a single antibiotic given as a bolus or as a course will successfully treat most infections. In modern medicine, this is becoming...
Current clinical practice assumes that a single antibiotic given as a bolus or as a course will successfully treat most infections. In modern medicine, this is becoming less and less true with drug-resistant, multi-drug-resistant, extensively drug-resistant, and untreatable infections becoming more common. Where single-drug therapy (monotherapy) fails, we will turn to multi-drug therapy. Alternatively, combination therapy could be useful to prevent the emergence of resistance. Multi-drug therapy is already standard for some multi-drug resistant infections and is the standard for the treatment of some pathogens such as Mycobacterium tuberculosis.The use of combination therapy for everyday infections could be a clear course out of the current AMR crisis we are facing. With every additional drug added to a combination (n + 1) the likelihood of the pathogen evolving resistance drops exponentially.Many generic antibiotics are cheap to manufacture as they have fallen out of patent protection but are less effective at pharmacologically effective doses due to overuse in the past. Combination therapy can combine these generic compounds into cocktails that can not only treat susceptible and resistant infections but can also reduce the risk of new resistances arising and can resuscitate the use of antimicrobials once thought defunct.In this chapter, we will summarize theory behind combination therapy and standard in vitro methodologies used.
Topics: Humans; Drug Therapy, Combination; Anti-Bacterial Agents; Microbial Sensitivity Tests; Anti-Infective Agents; Drug Resistance, Multiple, Bacterial
PubMed: 38949699
DOI: 10.1007/978-1-0716-3981-8_5 -
Methods in Molecular Biology (Clifton,... 2024Mycobacterium tuberculosis is the main causative agent of tuberculosis (TB)-an ancient yet widespread global infectious disease to which 1.6 million people lost their...
Mycobacterium tuberculosis is the main causative agent of tuberculosis (TB)-an ancient yet widespread global infectious disease to which 1.6 million people lost their lives in 2021. Antimicrobial resistance (AMR) has been an ongoing crisis for decades; 4.95 million deaths were associated with antibiotic resistance in 2019. While AMR is a multi-faceted problem, drug discovery is an urgent part of the solution and is at the forefront of modern research.The landscape of drug discovery for TB has undoubtedly been transformed by the development of high-throughput gene-silencing techniques that enable interrogation of every gene in the genome, and their relative contribution to fitness, virulence, and AMR. A recent advance in this area is CRISPR interference (CRISPRi). The application of this technique to antimicrobial susceptibility testing (AST) is the subject of ongoing research in basic science.CRISPRi technology can be used in conjunction with the high-throughput SPOT-culture growth inhibition assay (HT-SPOTi) to rapidly evaluate and assess gene essentiality including non-essential, conditionally essential (by using appropriate culture conditions), and essential genes. In addition, the HT-SPOTi method can develop drug susceptibility and drug resistance profiles.This technology is further useful for drug discovery groups who have designed target-based inhibitors rationally and wish to validate the primary mechanisms of their novel compounds' antibiotic action against the proposed target.
Topics: Microbial Sensitivity Tests; Mycobacterium tuberculosis; Drug Discovery; Gene Silencing; Humans; CRISPR-Cas Systems; Antitubercular Agents; Anti-Bacterial Agents; High-Throughput Screening Assays; Drug Resistance, Bacterial; Tuberculosis
PubMed: 38949697
DOI: 10.1007/978-1-0716-3981-8_3