Microbiology and Infectious Diseases

Physician Associate Program

Dana W. Dunne, MD Course Director

Assistant Professor, Department of Medicine
Section of Infectious Diseases
Yale University School of Medicine
 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: [email protected]
 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, Bacteria
plants, animals
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
• UNIQUEOuter 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

= pentaglycine peptide

NAG= n, acetyl- glucosamine; NAM= N, acetyl muramic acid

A= d-alanine; G= d-glutamate; L- lysine A-(redder)-l-alanine

A- Gram Positive cell wall-
-Thick PG layer
-teichoic acid
-lipoteichoic acid

B- Gram Negative cell wall-

Thin PG layer
outer membrane w/ LPS
periplasmic space
cytoplasmic membrane

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© 2005 Elsevier
 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
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bacteria are visualized by the red counterstain. B, Bacterial morphologies. © 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