Advanced Diagnostic Microbiology

Course Overview

• The course title is Advanced Diagnostic Microbiology, a 3-credit hour course identified by code A1117403/ B1117403.
• There are no prerequisites for the course.
• The course covers a range of topics with focus on microbial diversity, metabolism, pathogenesis, host interactions, lab safety and antimicrobial resistance.
• Emphasis on clinically important bacterial agents like Gram-positive, Gram-negative, and acid-fast bacteria, with focus on laboratory diagnosis.
• The course involves biochemical tests, differential staining, microscopic examination, molecular testing, and diagnosis by organ system.
• Student learning is enriched through literature reviews.

Course Evaluation Breakdown

• Midterm Exam: 30%
• Evaluation: 30%(Average of 2 quizzes: 15%, Seminar, case studies, class participation: 15%)
• Final Exam: 40%

Course Learning Outcomes

• Understand and explain bacteriology-related aspects of taxonomy, metabolism, immune response, and safety.
• Classify and describe etiologies and mechanisms of pathogenesis in bacterial infections.
• Use and interpret diagnostic lab tests and antimicrobial susceptibility testing.
• Differentiate and discuss case studies of bacterial infections.
• Design diagnostic approaches and assess existing diagnostic methods.

Key Microbiology Concepts

• Understanding bacterial classification, staining techniques, and lab safety.
• Memorization of common pathogens and related diseases.
• Identification of standard culture media and biochemical tests.
• Explaining microbial growth patterns and metabolic pathways.
• Knowledge of antimicrobial resistance mechanisms.
• Interpret Gram stain results and differentiate bacterial structures.
• Application of lab techniques like PCR, MALDI-TOF, and antimicrobial susceptibility testing.
• Use of diagnostic algorithms for pathogen identification in patient samples.
• Practical application of quality control in lab testing.
• Ability to compare different diagnostic methods and assess clinical relevance.
• Interpretation of patient case studies.
• Differentiation between normal flora and pathogenic organisms.
• Critical review of lab reports and determination of clinical significance.
• Assessment of the reliability and limitations of different diagnostic techniques.
• Evaluation of patient care options based on microbiological findings.
• Creation of diagnostic approaches or research proposals.
• Development of lab accreditation and quality assurance protocols.
• Innovation in detecting emerging infectious diseases.

Taxonomy: Classifying Organisms

• Taxonomy facilitates consistent classification, naming, and identification.
• The major taxonomic domains include Bacteria, Archaea, and Eukarya.
• Bacteria or prokaryotes lack a nucleus and include both environmental and heterotrophic medically important types.
• Archaea are environmental isolates thriving in extreme conditions like high salinity, jet fuel, or temperature extremes.
• Eukarya, or eukaryotes, possess a true nucleus and include medically significant organisms like fungi and parasites.

Bacterial Taxonomy Sublevels

• Family includes multiple genera and shares common attributes.
• Family names use the suffix "-aceae". Examples: Streptococcaceae, Enterobacteriaceae.
• Genus contains related species with shared traits. Example: Streptococcus within Streptococcaceae.
• Species are collections of bacterial strains sharing physiology and genetic features, differing from other microbes.
• Example: Klebsiella pneumoniae and Klebsiella oxytoca within Klebsiella, family Enterobacteriaceae.
• Subspecies are distinct populations within a species.
• Alternate names include biotype, serotype, or genotype; for example: E. coli SE15.

Energy and Carbon Sources for Organisms

• Terms for energy and carbon sources combine to specify an organism's metabolic strategy.
• Photoautotrophs like plants, algae, cyanobacteria, and some sulfur bacteria use light for energy and CO2 as a carbon source.
• Photoheterotrophs, such as purple non-sulfur and green non-sulfur bacteria, utilize light and organic compounds instead of CO2.
• Chemoautotrophs, including nitrifying, hydrogen, iron, and sulfur bacteria, gain energy from chemicals, using CO2.
• Chemoheterotrophs use chemicals for energy along with organic sources other than CO2
• Most bacteria, fungi, protozoa, and all animals are chemoheterotrophs.
• All medically important bacteria are chemoheterotrophs.

Nomenclature

• Naming microorganisms follows rules by the International Code of Nomenclature of Bacteria (ICNB) or Bacteriological Code (BC).
• Provides labels by which organisms are universally recognized.
• Every organism gets a two-part scientific "label":
  • Genus name, with the first letter capitalized.
  • Species name, all lowercase.
• Names are usually printed in italics or underlined when handwritten.
• Examples: Streptococcus pneumoniae, Streptococcus pyogenes
• Names can be shortened using the capitalized first letter of the genus followed by a period.
• The species name is spelled out: S. pneumoniae, S. pyogenes Please underline when handwriting Streptococcus pneumoniae

Bacterial Genetics, Structure, and Metabolism

• Prokaryotes lack nucleus/ organelles, so hereditary material remains unenclosed.
• The bacterial chromosome contains essential genes for viability, existing as closed, circular double strand DNA
• Parasites and fungi have multiple chromosomes and are housed in membrane bound nucleus. Serves as major classification for bacteria/eukaryotes.
• Viruses have DNA or RNA within protein coat, rather than existing in a cell.
• Bacteria may include plasmids and transposable elements.
• Plasmids serve as code for specialized elements: antibiotic resistance, unique metabolic process
• Transposable elements are molecules of DNA which can move, coding for antimicrobial resistance
• Bacteria multiply via binary fission, which results in identical replica of parent genome

Microbial Genetic Expression and Diversity

• Bacteria are genetically changed by mutation, recombination, and genetic exchange.

Mutation

• Changes in DNA that occur spontaneously during DNA replication, or from environmental mutagens.
• Mutations alter the base sequence of mRNA during transcription.
• Such changes influence the proteins created during translation.
• Outcomes depend on the mutation site involved, potentially leading to:
  • Lethality.
  • No change in the organism.
  • An advantage and altered phenotype.

Clinical Relevance

• The survival advantage of a mutation is key to survival.
• It can lead to transfer of mutation via genetic exchange.
• This can cause increase in antibiotic resistance.
• Natural selection is survival of new gene variations .
• Mutation defined as permanent change in base sequence potentially leading to encoded product change.

Point Mutations

• Causes base pair substitution (silent, missense, nonsense)

Frameshift Mutations

• Insertion/deletion of nucleotide pairs

Types of Mutations

• In single base pairs there is a base substitution, and result is substituted pair.
• Occurs when no change in amino acids (redundancy)- also called silent mutation
• If change to amino acid occurs and alteration of protein is synthesized, it is Missense mutation.

More About Mutation

• Substitution prevents function. Fragments will create a nonsense codon
• Frameshift mutations cause insertion of nucleotides, shifting translational reading frame

Mutation Rate

• Mutations can occur spontaneously or via replication
• Environmental agents and radiation mutate.
• Mutagen increases rate by 10x-1000x

Genetic Recombination

• Bacterial genotypes are modified by recombination
• DNA transfers and exchanges to recipient genome with second bacterial cell.
• Referred to homologous recombination
• Main way bacteria evolve through illness

Genetic Exchange

• Organism must adapt to genetic exchange
• Exchanged via transformation transduction, conjunction

Transformation

• Recipient cells uptake free DNA
• DNA mixes and plays a major role in antibiotic resistance

Transduction

• DNA may transfer to bacteria via viruses/ bacteriophages, containing gens

Conjugation

• Requires contact of donors, in which chromosome DNA plasmids can be transferred.
• Gram neg is done via ‘sex pilus’

Cell Morphology

• Most species range from 0.25-1UM requiring microscopy
• Staining helps separate bacteria to gram positive or negative
• Distinct distinction is because constituent of cells wall allows differential dyes

Common Morphologies

• Cocci (circular), coccobacilli ovoid and bacili are rod like

Bacterial Cell Component: Cell Envelope

• Most outer part and comprises:
  • outer membrane found only in gram negative
  • Cell wall composed of petidogylcans and macromolecules
  • Peiplasm

Outer Membrane Function

• Found only in gram negative function as initial barrier
• Primary barrier that has hydrophilic parts
• Blayer structure- and gives negative bacteria charge
• Able to cause for disease

Inner vs Outer Membrane

• Inner: has protiens scattered
• Water controls solutes and antibiotics

Cellular Appendages, and Cellular Contents

-Celllular appendage is immediately exterior to murein -protect bacteria by human Immune system

Pilus or Flangella

-Adheasive that attaches to animal hosts -Flagella allows bacteria to move

Cell contents

-Cytoplasm is contents. thousands of enzymes are protein

• Nucleoid- bacterial chromosome with DNA
• Endospore can activate to form
• Sporulation allows resistantance in cells