Medbac lecture 2 - Study Guide.pdf

The First Lines of Defense 1. Introduction to Infection Definition: Infection refers to the entry and invasion of foreign materials (e.g., bacteria, toxins) into the body. Symptoms vs. Signs: ○ Symptoms: What the patient complains about. ○ Signs: Observable...

The First Lines of Defense 1. Introduction to Infection Definition: Infection refers to the entry and invasion of foreign materials (e.g., bacteria, toxins) into the body. Symptoms vs. Signs: ○ Symptoms: What the patient complains about. ○ Signs: Observable evidence of infection by medical professionals. 2. Pathophysiology and Pathology Pathophysiology: Disturbance in the body's natural physiology due to infection. Pathology: Anatomical changes that occur with infection. Chemopathology: Chemical changes occurring with infection. 3. Non-Specific Immunity (First Line of Defense) Overview: Non-specific immunity, also known as innate immunity, targets pathogens in a general way without specific targeting. Categories: 1. Physical Defenses 2. Chemical Defenses 3. Cellular Defenses 4. Physical Defenses Barriers: Physical barriers like skin and mucosal linings. Mechanical Defenses: ○ Examples: Tears, urine flow. 5. Chemical Defenses Microbiomes: Populations on skin surfaces that inhibit pathogen growth. Body Fluids: ○ Enzymes: Hemokines, lysozymes. ○ Antimicrobial peptides: Granulozymes. Chemical Mediators: ○ Types: Antimicrobial peptides, complement components, cytokines, immunoglobulins. ○ Functions: Inhibit microbial colonization and infection. Cytokines: ○ Function: Cell signaling, immune modulation. ○ Types: Chemokines, interferons, interleukins, TNF.  ○ Production: Macrophages, T lymphocytes, and mast cells. Inflammation Mediators: ○ Examples: Histamine, prostaglandins, leukotrienes, free radicals, serotonin. 6. Cellular Defenses Granulocytes vs. Agranulocytes: ○ Granulocytes: Cells with granules involved in inflammation (e.g., mast cells, basophils, eosinophils, neutrophils). ○ Agranulocytes: Include natural killer cells, T cells, and B cells (adaptive immunity). 7. Detailed Structures and Functions Skin Structure: ○ Layers: Epidermis and dermis. ○ Cell Types: Flat pavement cells, cuboidal cells, columnar cells. ○ Functions: Dry, inhospitable environment for bacteria, high turnover rate. Mucosal Membranes: ○ Functions: Protect respiratory, gastrointestinal, and urogenital tracts. ○ Secretions: Mucus, antimicrobial peptides, lysozymes, surfactant. 8. Specific Chemical Defenses Lysozyme: Found in tears, saliva, milk, mucus; hydrolyzes bacterial cell walls. Lactoferrin: Binds iron, depriving bacteria of nutrients. Lactoperoxidase: Produces superoxide, destroys bacterial proteins. 9. Microbiome and Special Defenses Resident Microbiota: ○ Function: Prevent pathogen attachment and proliferation. ○ Examples: E. coli in the colon, competition with pathogenic bacteria. Special Defenses: ○ Gastrointestinal Tract: Acidic environment, bile, pancreatic enzymes. ○ Urinary Tract: Flushing action of urine, sphincters. 10. Consequences of Breaching Defenses Common Issues: ○ Catheters: Risk of biofilm formation. ○ Burns: Increased susceptibility to infections. ○ Contact Lenses: Corneal damage. ○ Respiratory Tubes: Risk of bypassing upper airway defenses. ○ Surgical Perforations: Release of bacteria into tissues. 11. Summary of Physical Defenses Physical Barriers: Skin, mucous membranes, endothelia. Mechanical Actions: Shedding of cells, mucociliary action, peristalsis. Antimicrobial Peptides: Defensins, cathelicidins, histatins. Resident Microbiota: Occupy binding sites, compete for nutrients. 12. Assignment Questions 1. Staphylococcus epidermidis Infections: ○ Explain how it can become pathogenic and what conditions contribute to its opportunistic nature. 2. Special Defenses: ○ Discuss the roles of resident microbiota, mucosal defenses, and physical barriers in maintaining health. Identifying Bacteria Through Genetic Testing Overview 1. Introduction ○ Purpose: Identifying bacteria is crucial for accurate diagnosis and treatment. This is akin to conducting a census, but for bacteria. ○ Traditional Methods: Historically, bacteria were identified using chemical tests on cultures grown in Petri dishes. ○ Modern Approach: Genetic testing, particularly focusing on ribosomal RNA, has become the preferred method due to its speed and accuracy. Structure of Bacteria 1. Bacterial Ribosomes ○ Components: Ribosomes in bacteria consist of two subunits: 50S (Large Subunit) 30S (Small Subunit) ○ Naming: The 'S' refers to Svedberg units, a measure of ribosomal size based on sedimentation rates. 2. Ribosomal RNA (rRNA) ○ Composition: 30S Subunit: Contains the 16S rRNA 50S Subunit: Completes the ribosomal structure 16S Ribosomal RNA Sequencing 1. Importance ○ Universal Presence: 16S rRNA is present in all bacterial species and is used for taxonomic classification. ○ Conserved Regions: Contains both highly conserved and variable regions. 2. Regions of Interest ○ Fixed Regions: Highly conserved, used for broad classification. ○ Hypervariable Regions: Subject to change among different species, crucial for precise identification. 3. Process ○ Sequencing: DNA is amplified and sequenced to compare with known bacterial sequences. ○ Library Comparison: Sequences are matched against a library of known bacterial DNA to identify species. Techniques in Sequencing 1. Traditional Methods ○ Sanger Sequencing Process: Uses chain termination to stop DNA replication at specific points, then separates fragments by size using gel electrophoresis. Limitations: Time-consuming and less effective for large-scale sequencing. 2. Advanced Methods ○ Pyrosequencing Method: Uses light emission to detect DNA synthesis in real-time. ○ Illumina Sequencing Technique: Uses sequencing by synthesis with millions of reads, allowing for large-scale and high-throughput sequencing. Modern Sequencing Workflow 1. Steps ○ DNA Isolation: Extract DNA from the sample. ○ Library Preparation: Amplify DNA using PCR, add sequencing adapters. ○ Sequencing: Perform sequencing using platforms like Illumina MiSeq. ○ Analysis: Use software to analyze the data and compare with existing databases. 2. Advantages ○ High Throughput: Can sequence many samples simultaneously.  ○ Accuracy: Provides detailed and precise bacterial identification. ○ Cost-Effective: More affordable than traditional methods. 3. Disadvantages ○ Variable Copy Numbers: Differences in rRNA gene copies per genome can affect results. ○ PCR Biases: Amplification steps can introduce biases. ○ Diversity Overestimation: Hypervariable regions can overinflate diversity estimates. ○ Resolution Limits: May not distinguish closely related species effectively. Practical Application 1. Emergency Situations: Rapid identification of bacterial pathogens in clinical settings. 2. Research: Studying microbial communities and their roles in health and disease. Further Reading and Videos 1. Video Content: ○ Basic 16S Sequencing: Overview of 16S rRNA sequencing, including techniques and applications. ○ Sanger Method: Detailed explanation of the Sanger sequencing technique. ○ Next-Generation Sequencing: Overview of modern sequencing technologies and their applications. 2. Recommended Review: ○ Slides and Video Lectures: Review provided materials on bacterial identification and genomics techniques. Summary Understanding bacterial identification through genetic sequencing involves knowledge of ribosomal RNA structure, the importance of the 16S rRNA gene, and the various sequencing methods available. Modern techniques offer more efficient and accurate results compared to traditional methods, making them essential for current microbiological studies and clinical diagnostics. 16S Ribosomal RNA Gene and Sequencing 1. Introduction to the 16S Ribosomal RNA Gene Description: ○ The 16S ribosomal RNA (rRNA) gene is part of the ribosomal RNA found in prokaryotes. ○ Woese and colleagues described bacterial rRNA genes as "molecular clocks" due to their universality, role in cellular functions, and conserved structure. Ribosomal RNA Types: ○ 23S rRNA: ~3,300 nucleotides ○ 16S rRNA: ~1,550 nucleotides ○ 5S rRNA: ~120 nucleotides Significance of the 16S rRNA Gene: ○ Standard for Bacterial Taxonomy: Due to ease of sequencing and phylogenetic information. ○ Structure: Consists of 8 highly conserved regions and 9 variable regions. ○ Conservation Levels: More conserved regions relate to higher taxonomy levels (e.g., phylum), while less conserved regions relate to lower levels (e.g., species). 2. 16S Ribosomal RNA Sequencing Purpose: 1. Microbial Classification: Commonly used for taxonomic identification at the species level. 2. Sequence Divergence: Typically uses a range of 0.5% to 1% sequence divergence to delineate species. Advantages: 1. Universally distributed across bacteria. 2. High abundance of 16S rRNA gene sequences. 3. Useful for measuring phylogenetic relationships. 4. Minimal issues with horizontal gene transfer. 5. Affordable amplification and sequencing costs. Disadvantages: 1. Variability in copy numbers per genome. 2. PCR amplification biases. 3. Overestimation of diversity. 4. Low resolution for closely related species. 5. Shift towards more comprehensive sequencing methods as costs drop (e.g., whole genome or shotgun metagenomics). 3. Sequencing Workflow 1. DNA Isolation: ○ Extract DNA from the microbial sample. 2. Library Preparation: ○ PCR Amplification: Use primers targeting the 16S rRNA gene. Common primers: 27F and 1492R. Full-length 16S rRNA gene: ~1,500 base pairs. Various sequencing platforms may require different primer pairs. 3. Sequencing: ○ Technologies: Sanger Sequencing Illumina MiSeq 454 Pyrosequencing PerkBioSmart Sequencing ○ Illumina MiSeq Workflow: Amplify V3 and V4 regions using limited cycle PCR. Purify, quantify, and pool PCR products. Add Illumina sequencing adapters and dual-index barcodes. Pool up to 96 libraries using Nextera XT indices. 4. Data Analysis: ○ Post-Sequencing Steps: Filter and trim sequences. Cluster into Operational Taxonomic Units (OTUs) based on 97% sequence identity. Perform species annotation, OTU phylogeny, diversity analysis, and other analyses. Sanger Method vs. Illumina Method for DNA Sequencing 1. Sanger Method (Chain Termination Method) Overview: The Sanger method is used to sequence DNA by determining the nucleotide order through chain termination. Steps: 1. Preparation: ○ Single-Stranded DNA: DNA must be in single-stranded form.  ○Reaction Mixture: Contains single-stranded DNA, DNA polymerase, four deoxyribonucleotides (A, T, G, C), and a short primer. Primer: Complementary to the three-prime end of the target region, necessary for DNA polymerase to start replication. 2. Chain Termination: ○ Tube Setup: Divide the reaction mixture into four tubes, each receiving a different dideoxyribonucleotide (ddNTP). ○ Replication: Deoxyribonucleotides: Allow replication to continue. Dideoxyribonucleotides: Cause termination of the chain when incorporated. 3. Electrophoresis: ○ Gel Transfer: Transfer the contents of each tube to four lanes of an electrophoresis gel. ○ Separation: Oligonucleotides are separated by size; shorter fragments move further down the gel. 4. Reading the Sequence: ○ Visualization: Read from bottom to top of the gel, one base at a time, to determine the nucleotide sequence. 2. Illumina Method (Next-Generation Sequencing) Overview: The Illumina method, particularly for 16S ribosomal RNA sequencing, allows for high-throughput, simultaneous identification of multiple bacterial species in a sample. 16S Ribosomal RNA Sequencing: 1. Gene Overview: ○ 16S rRNA Gene: Codes for part of the ribosome in bacterial cells. ○ Conservation and Variation: Sections are conserved across all bacteria; variations help differentiate species and infer relationships. 2. Traditional vs. Next-Generation Sequencing: ○ Traditional: Bacteria needed to be cultured individually and sequenced one at a time. ○ Next-Generation Sequencing (NGS): Allows simultaneous identification of multiple bacterial species from a complex sample without culture. 3. Procedure: ○ Sample Collection: Swab the sample (e.g., gut). ○ Sample Preparation: Perform sample prep and PCR amplification using primers specific to the 16S rRNA gene. ○ Sequencing:  Platform: Sequence using Illumina’s MiSeq for comprehensive microbial community analysis. 4. Data Analysis: ○ MiSeq Reporter Software: Includes a range of analysis pipelines, including a 16S metagenomics tool. ○ BaseSpace: Illumina’s cloud computing environment for data analysis. ○ Third-Party Software: Data can also be exported for analysis using external tools. 5. Experimental Workflow: ○ Step 1: Order Amplicon primers based on the region of interest. ○ Step 2: Generate Amplicon libraries by PCR, add Illumina sequencing and index adapters. ○ Step 3: Sequence on MiSeq. ○ Step 4: Analyze results using MiSeq reporter software or BaseSpace. 6. Applications: ○ American Gut Project: Uses next-generation sequencing to analyze gut microbiomes.