Dana W. Dunne, MD Course Director Assistant Professor, Department of Medicine
Section of Infectious DiseasesYale University School of Medicine
Microbiology and Infectious Diseases Physician Associate Program
Course Information
• [email protected]– (203) 737-4096
• Contact Information: – Alexandria Garino, PA-C– Assistant Director for Didactic Curriculum– Yale University Physician Associate Program– (203) 785-2860 E-mail:
Overview
• Medical microbiology– Morphology– Classification
• Clinical infectious diseases– Pathogenicity; virulence– Epidemiology– Clinical presentations– (Management)
Overview
• Required Textbook: Medical Microbiology.
6th edition. Murray PA et al. Mosby 2009.
• Lectures:– Objective based
• Interactive Seminars:– Clinical cases
• Exams:– Exam 1 (10/21): Bacteriology,
mycobacteria– Exam II (12/21): Fungi, parasites, viruses
Basic Principles and Definitions in Microbiology
1. List the 4 major groups of microbes
and their distinguishing features
2. Define colonization versus disease
3. List body systems colonized with
normal flora
Microbes
1.Viruses
2. Fungi
3. Parasites
4. Bacteria
Eukaryote vs Prokaryote
Eukaryote Prokaryote
Groups Algae, fungi, protozoa, plants, animals
Bacteria
Nucleus Classic membrane No nuclear membrane
Chromosomes Strands of DNA diploid Single, circular DNA haploid
Mitochondria/Golgi/ER Present Absent
Cell wall Present only fungi Complex of protein, lipids peptidoglycans
Cytoplasmic membrane
Contains sterols Does not contain sterols
Reproduction Sexual and asexual Asexual (binary fission)
Respiration Via mitochondria Via cytoplasmic membrane
Adapted from Murray , Medical Microbiology, 6th Ed.
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© 2005 Elsevier
Definition and Property of a Virus
• Filterable (smaller than bacteria)
• Obligate intracellular parasites (need the host
cell for replication)
• DNA or RNA + proteins + protein coat = capsid
• Naked or enveloped
• Range of diseases- acute to chronic
Fungi• Eukaryotic (true nucleus,
mitochondria, golgi bodies, ER)
• Yeast – Unicellular, asexual reproduction
• Molds – Filamentous, asexual or sexual
• Dimorphic
Parasites
• Eukaryotic
• Size range from tiny (1-2µm) to
tapeworm (10m)
• Complex structure and taxonomy
• Clinical syndromes + epidemiology
Bacteria
• Prokaryotic (no nuclear membrane, etc.)
• Ubiquitous
• Cell Wall- complex
• Classified based on size, shape, spatial arrangement (chains, clusters); phenotypic and genotypic properties
Bacterial Cell Wall
• Cell wall composition- one of the most important factors in bacterial species analysis and differentiation.– Gram positive- 90% of the Gram-positive cell
wall is comprised of • Peptidoglycan- THICK; essential for structure;
replication• teichoic acid- cross linked to pg• Lipoteichoic acid- lipid linked. Common on surface,
promote attachment, distinguishes serotypes
http://science.kennesaw.edu/biophys/biodiversity/eubacteria/eubpix.htm
Bacterial cell wall-cont.• Gram negative
• UNIQUEOuter plasma membrane: maintains structure
• lipopolysaccharide (LPS)- Endotoxin
• powerful immune stimulator; causes shock
• LPS structure is used to classify bacteria
• Lipid A – the subunit of LPS responsible for most of the pathogenic affects
• lipoproteins and the associated polysaccharides.
• Thinner peptidoglycan layer (5-10% of weight)
• Periplasmic space- transport systems
http://science.kennesaw.edu/biophys/biodiversity/eubacteria/eubpix.htm
Peptidoglycan structure
= NAG
= NAM
NAG= n, acetyl- glucosamine; NAM= N, acetyl muramic acid
A= d-alanine; G= d-glutamate; L- lysine
= pentaglycine peptide
A-(redder)-l-alanine
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© 2005 Elsevier
A- Gram Positive cell wall--Thick PG layer-teichoic acid-lipoteichoic acid
B- Gram Negative cell wall-Thin PG layer
outer membrane w/ LPSperiplasmic spacecytoplasmic membrane
Gram-staining
• Easy 4-step staining procedure that distinguishes the two major groups of bacteria:
– Heat fix specimen
#1- flood with Crystal violet – rinse
#2- flood with iodine (precipates crystal violet; binds PG)-rinse
#3- decolorize with acetone (gets rid of any unbound crystal
v.)
#4- counterstain with safranin- stains any decolorized cells
Gram stain- cont.
• Gram positive: the crystal-violet is still present in the cell, the counter stain is not incorporated, thus maintaining the cell's purple color.
• Gram negative: Because the cell wall is high in lipid content and low in peptidoglycan content, the primary crystal-violet escapes from the cell when the decolorizer is added – pick up counterstain color which is pink
Figure 2-3 Gram-stain morphology of bacteria. A, The crystal violet of Gram stain is precipitated by Gram iodine and is trapped in the thick peptidoglycan layer in gram-positive bacteria. The decolorizer disperses the gram-negative outer membrane and washes the crystal violet from the thin layer of peptidoglycan. Gram-negative
bacteria are visualized by the red counterstain. B, Bacterial morphologies.Downloaded from: StudentConsult (on 11 August 2009 07:26 PM)
© 2005 Elsevier
Gram-positive Cocci
• The Gram-positive cocci are grouped
together based on their Gram-stain reaction,
thick cell wall composition, and spherical
shape.
• Micrococcus and Staphylococcus
• Streptococcus and Enterococcus
Bacteria
Gram-Positive
http://www.slic2.wsu.edu:82/hurlbert/micro101/pages/101lab3.html
Practical Bacterial Taxonomy
Practical Taxonomy 2
Practical Taxonomy 3
Practical Taxonomy 4
Anaerobes
• Anaerobic organisms require an oxygen free environment to grow normally. – 5-10% of all clinical infections.
• Specific Characteristics: – foul smelling discharge – necrotic tissue – gas formation in tissues or discharge
World’s smelliest flower- Rafflesia
Definitions
• Colonization: presence of a microorganism in or on a body site- doesn’t interfere with normal body funtions– Transient– Permanent
• Disease: interaction leads to damage to the host– Organism replicating; toxin; immune response
Body Sites Normally Colonized- GI
• Esophagus – transiently colonized unless surgery or immunocompromised– Candida, Herpes simplex virus, Cytomegalovirus
• Stomach– Acidic – H. pylori
• Small Intestine: polymicrobial – anaerobes• Large Intestine: most heavily colonized
body site– Anaerobes predominate – Bacteroides – intra-abdominal dz– E. coli – extra-intestinal infections
Skin
• Coagulase negative Staphylococcus (CNS), Staphylococcus aureus
• Fungi (candida, malassezia)• Don’t expect many gram-negatives
(except acinetobacter), +/- streptococci• Clostridia perfringens - 20% of healthy
adults
Respiratory Tract/Head
• Mouth/oropharynx:– Anaerobes – peptostreptococci,
fusobacterium– Aerobes – streptococci, haemophilus,
neisseria, pneumococcus (Streptococcus pneumoniae)
• Ear: CNS, pneumococcus, pseudomonas
• Lower Resp tract: usually sterile, can be colonized (ie candida)
Genitourinary
• Mostly sterile except distal urethra and vagina– Lactobacilli, streptococci, staphylococci– Transient colonization with gram negative organisms,
candida• Can lead to urinary tract infections (UTI) if ascends to the
bladder
– Chlamydia, N. gonorrhea – always considered accociated with disease (even if no symptoms)
• Vagina: lactobacilli, varies with estrogen status
Diagnostic Methods
• Microscopy• Light (brightfield)• Darkfield- obliquely transmitted light• Phase-contrast• Fluorescent• Electron microscopy
• Transmission• scanning
Microscopy
• Staining• Wet mount/KOH – no stain (direct exam)• India ink – cryptococcus- helps identify capsule• Gram stain• Wright-Giemsa- malaria, other intracellular
infections• Acid Fast- mycobacteria• Fluorescent- direct or antibody labeled
Molecular Diagnosis-Detection of microbial genetic material
• DNA probes- bind to DNA sequence– Labeled (radioactive or chemical)– Tissue, fluids
• In-situ hybridization• Amplification of DNA/RNA- more sensitive
– Polymerase Chain Reaction (PCR), ligase chain rx– RT- PCR- use RT to convert RNA to DNA then
ampl.– Tissue, serum, body fluids
Serologic Diagnosis-Immunologic -detection of Antibody (Ab)/Antigen (Ag)
• Serology: detect type of Ab (ex: IgM; IgG) and titer (quantification of Ab strength) – Serum; CSF; other fluids
• Detect Ag on or within cells – Immunofluorescence (fluorescent labeled Ab)– EIA – enzyme immunoassay – labeled Ab with an
enzyme
• Detect Soluble Ag (done on serum/fluid)– ELISA (enzyme-linked immunosorbent assay) –
quantitate Ag– RIA (radiolabeled immunoassays)
Conclusions
• Know the microbial classes; gram stain and
morphology classifications; key chemical reactions
• Think about the organism – host interaction, the
likelihood of colonization vs. disease, the normal flora,
potentially pathogenic flora when in the clinical
setting
• Read the text, Come to class, Learn your lecture notes
and objectives