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

• dana.dunne@yale.edu– (203) 737-4096

• Contact Information: – Alexandria Garino, PA-C– Assistant Director for Didactic Curriculum– Yale University Physician Associate Program– (203) 785-2860 E-mail:

Alexandria.garino@yale.edu

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