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Methods in Molecular Biology (Clifton,... 2024Genomic sequencing has revolutionized microbial typing methods and transformed high-throughput methods in reference, clinical, and research laboratories. The detection...
Genomic sequencing has revolutionized microbial typing methods and transformed high-throughput methods in reference, clinical, and research laboratories. The detection of antimicrobial-resistant (AMR) determinants using genomic methods can provide valuable information on the emergence of resistance. Here we describe an approach to detecting AMR determinants using an open access and freely available platform which does not require bioinformatic expertise.
Topics: Whole Genome Sequencing; Drug Resistance, Bacterial; Genome, Bacterial; Computational Biology; Humans; Anti-Bacterial Agents; Genomics; Software; Bacteria; High-Throughput Nucleotide Sequencing
PubMed: 38949713
DOI: 10.1007/978-1-0716-3981-8_19 -
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,... 2024Antibiotic resistance is a global challenge likely to cost trillions of dollars in excess costs in the health system and more importantly, millions of lives every year.... (Review)
Review
Antibiotic resistance is a global challenge likely to cost trillions of dollars in excess costs in the health system and more importantly, millions of lives every year. A major driver of resistance is the absence of susceptibility testing at the time a healthcare worker needs to prescribe an antimicrobial. The effect is that many prescriptions are unintentionally wasted and expose mutable organisms to antibiotics increasing the risk of resistance emerging. Often simplistic solutions are applied to this growing issue, such as a naïve drive to increase the speed of drug susceptibility testing. This puts a spotlight on a technological solution and there is a multiplicity of such candidate DST tests in development. Yet, if we do not define the necessary information and the speed at which it needs to be available in the clinical decision-making progress as well as the necessary integration into clinical pathways, then little progress will be made. In this chapter, we place the technological challenge in a clinical and systems context. Further, we will review the landscape of some promising technologies that are emerging and attempt to place them in the clinic where they will have to succeed.
Topics: Anti-Bacterial Agents; Microbial Sensitivity Tests; Humans; Drug Resistance, Bacterial; Bacteria
PubMed: 38949707
DOI: 10.1007/978-1-0716-3981-8_13 -
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,... 2024To model complex systems, individual-based models (IBMs), sometimes called "agent-based models" (ABMs), describe a simplification of the system through an adequate...
To model complex systems, individual-based models (IBMs), sometimes called "agent-based models" (ABMs), describe a simplification of the system through an adequate representation of the elements. IBMs simulate the actions and interaction of discrete individuals/agents within a system in order to discover the pattern of behavior that comes from these interactions. Examples of individuals/agents in biological systems are individual immune cells and bacteria that act independently with their own unique attributes defined by behavioral rules. In IBMs, each of these agents resides in a spatial environment and interactions are guided by predefined rules. These rules are often simple and can be easily implemented. It is expected that following the interaction guided by these rules we will have a better understanding of agent-agent interaction as well as agent-environment interaction. Stochasticity described by probability distributions must be accounted for. Events that seldom occur such as the accumulation of rare mutations can be easily modeled.Thus, IBMs are able to track the behavior of each individual/agent within the model while also obtaining information on the results of their collective behaviors. The influence of impact of one agent with another can be captured, thus allowing a full representation of both direct and indirect causation on the aggregate results. This means that important new insights can be gained and hypotheses tested.
Topics: Humans; Drug Resistance, Microbial; Anti-Bacterial Agents; Models, Theoretical; Bacteria; Host-Pathogen Interactions; Drug Resistance, Bacterial; Models, Biological; Computer Simulation
PubMed: 38949704
DOI: 10.1007/978-1-0716-3981-8_10 -
Methods in Molecular Biology (Clifton,... 2024Mathematical models have been used to study the spread of infectious diseases from person to person. More recently studies are developing within-host modeling which...
Mathematical models have been used to study the spread of infectious diseases from person to person. More recently studies are developing within-host modeling which provides an understanding of how pathogens-bacteria, fungi, parasites, or viruses-develop, spread, and evolve inside a single individual and their interaction with the host's immune system.Such models have the potential to provide a more detailed and complete description of the pathogenesis of diseases within-host and identify other influencing factors that may not be detected otherwise. Mathematical models can be used to aid understanding of the global antibiotic resistance (ABR) crisis and identify new ways of combating this threat.ABR occurs when bacteria respond to random or selective pressures and adapt to new environments through the acquisition of new genetic traits. This is usually through the acquisition of a piece of DNA from other bacteria, a process called horizontal gene transfer (HGT), the modification of a piece of DNA within a bacterium, or through. Bacteria have evolved mechanisms that enable them to respond to environmental threats by mutation, and horizontal gene transfer (HGT): conjugation; transduction; and transformation. A frequent mechanism of HGT responsible for spreading antibiotic resistance on the global scale is conjugation, as it allows the direct transfer of mobile genetic elements (MGEs). Although there are several MGEs, the most important MGEs which promote the development and rapid spread of antimicrobial resistance genes in bacterial populations are plasmids and transposons. Each of the resistance-spread-mechanisms mentioned above can be modeled allowing us to understand the process better and to define strategies to reduce resistance.
Topics: Bacteria; Humans; Gene Transfer, Horizontal; Drug Resistance, Microbial; Models, Theoretical; Drug Resistance, Bacterial; Anti-Bacterial Agents; Host-Pathogen Interactions
PubMed: 38949703
DOI: 10.1007/978-1-0716-3981-8_9 -
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,... 2024The use of animal models is still widespread in science but there is a movement away from this manner of experimentation. One option approved by the FDA for human-like...
The use of animal models is still widespread in science but there is a movement away from this manner of experimentation. One option approved by the FDA for human-like studies is the hollow fiber bioreactor (HFS). HFSs are highly controllable, self-contained systems that allow for the modeling of individual tissues and disease phenotypes. Oxygen, drug concentration & half-life, and immune cell invasion are all scalable to human and veterinary conditions using a HFS. There are drawbacks to the systems including cost and contamination so the use of these systems must be carefully managed.With these limitations in mind, the scope of the technology is great. Antimicrobial susceptibility testing (AST) is possible with greater accuracy and clinical validity than classical in vitro techniques making minimal inhibitory concentration (MIC) data generated on the bench more translatable to the clinic.In this chapter, we will outline the background of the HFS and some typical uses.
Topics: Humans; Microbial Sensitivity Tests; Bioreactors; Animals; Anti-Bacterial Agents; Drug Resistance, Bacterial
PubMed: 38949701
DOI: 10.1007/978-1-0716-3981-8_7 -
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