"You can observe a lot just by watching.

"

Typical shapes and arrangements of bacterial cells.

Freeze-fractured Bacteriodes cell showing typical major convex fracture

faces through the inner (im) and outer (om) membranes. Bars = 1 m;

Electron micrograph of a thin section of Neisseria gonorrhoeae

showing the organizational features of prokaryotic cells. Note
the electron-transparent nuclear region (n) packed with DNA fibrils,
the dense distribution of ribosomal particles in the cytoplasm, and
the absence of intracellular membranous organelles

Typical arrangements of bacterial flagella

Cross section of a Bacillus spore. The events involved in sporulation of

vegetative cells and in germination of spores are complex and are influenced by
factors such as temperature, pH, and the availability of certain divalent cations and
carbon- and nitrogen-containing compounds. Spores formed under different
conditions have different stabilities and degrees of resistance to heat, radiation,
chemicals, desiccation, and other hostile conditions

Electron micrograph of a thin section of a Bacillus megaterium spore

showing the thick spore coat (SC), germinal groove (G) in the spore coat, outer
cortex layer (OCL) and cortex (Cx) germinal cell wall layer (GCW), underlying
spore protoplast membrane (PM), and regions where the nucleoid (n) is visible.

1884 -Hans Christian Gram

A Danish Bacteriologist developed a staining
method, now called the Gram stain to
distinguish between different types of
bacteria.

Gram positive cocci in pairs, clusters and tetrads resembling

staphylococci

Red blood cells and gram positive cocci in pairs, short and long chains
and clusters suggestive of a mixed staphylococci and streptococci.
Staphylococci often appear as perfect spheres while Streptococci
often are slightly elongated.

Small to medium gram positive bacilli with palisading and angular

arrangements, the so-called Chinese letter appearance. This
morphology is suggestive of cornybacteria or Listeria monocytogenes.

Pleomorphic large gram positive bacilli resembling Clostridium perfringens. This

is a direct smear from an infected wound. The absence of spores is typical.

Red blood cells and large gram negative bacilli with rounded
ends in a blood culture. These are resemble enteric bacilli and
Pseudomonas.

Pleomorphic, variably staining gram negative bacilli with pointed

ends resembling Fusobacterium spp.

Intracellular gram negative diplococci in a gram stain

of a urethral discharge from a male patient. This can be
presumptively identified as Neisseria gononrrhoeae.

Gram stain of a stool sample showing leukocytes (PMN) and

slightly curved, comma shaped, gull-wing appearing bacteria typical
of Campylobacter jejuni.

Wrights stain of peripheral blood showing spiral bacteria typical of

Borrelia spp.

Gram positive

Gram negative

Electron micrograph of a thin section of the Gram-positive coccus

showing the thick peptidoglycan cell wall (cw), underlying
cytoplasmic (plasma) membrane (cm), mesome (m), and nucleus (n).

Robert Koch (1843 -1910) The

Father of Medical Microbiology

Robert Koch (1843 -1910) The Father of

Medical Microbiology
German country physician who
developed microbiology into a science.
Developed Pure culture techniques
Used Potato slices -first found individual
bacterial colonies growing with different
appearance.
-microscopic examination revealed cells
within a single colony were similar pure
culture may be isolated fom a diseased
organ or normally sterile site of the body

Development of solidifying
agents and complex media
Koch wanted to culture pathogens so he
used something similar to the body
tissue -meat extracts.
Initially, Gelatin was used to solidify
beef extract broth.
But, (1) many organisms can digest
gelatin and (2) it melts at 37 C, the
favored incubation temperature for
most pathogens.

Development of solidifying
agents
Agar-was suggested by Fannie Hesse wife of
Walther Hesse working in Koch lab.
Walther was working with gelatin plates in summer
and was having troubles.
Asked wife Why do your jellies and pudding stay
solid in warm weather?
Fannie learned to use AGAR-AGAR from a Dutch
neighbor in New York who spent time in Asia.
AGAR-AGAR had been used as a gelling agent in
Asia for centuries.

Petri Dish
Petri Dish was invented in 1887
by another student of Koch, R.J. Petri
They are shallow glass plates.

Koch Develops Pure Culture as

the Key to Studying Microbes as
the Etiologic Agents of Disease
Definition: Pure culture is a population
of organisms, all of which are the
progeny of a single organism.
In nature, microbes almost never occur as
pure cultures but as mixed populations.
-But during infection a pure culture may be
isolated fom a diseased organ or normally
sterile site of the body

E. coli API strip

Salmonella API strip

Lac-, forms H2S, Produces gas during glucose fermentation.

Bacterial identification

A given level of similarity can be equated with relatedness at the genus,

species, and, sometimes, subspecies levels. For instance, strains of a
given species may cluster at a 90% similarity level, species within a
given genus may cluster at the 70 percent level, and different genera in
the same family may cluster at the 50 percent or lower level

Bacterial Growth

Growth is the orderly increase in the quantity

of ALL components of the bacterial cell.
Hence, growth of bacteria is measured in
terms of an increase in cell numbers rather
than a significan increase in the size of an
individual organism
Measurements
Cell Mass (Weight, Light Scattering)
Cell Numbers
Direct Count
Viable Count

Typical Bacterial Growth Curve in Batch Liquid Culture

Semi-Useless Information
A bacterial cell doubling every 20 min will yield 10
generations and 109 cells in 10 hours.
A single cell on an agar plate yields a visible mass (108
cells) overnight
The volume of an average bacterial cell is 10-12 cm3. The
volume of the earth is equivalent to 4 X 1039 average
sized bacteria.
A single cell doubling every 20 min without restraint
would reach the volume of the earth in 45 hours. In 48
hours the total weight of the bacterial cells would be
4000X (more or less) the weight of the earth.
Thats a lot of bacteria

Self-Contained Anaerobic Chamber

Normal teeth just after brushing (left). A few minutes later after the additioh of
an oxidation-reduction (Eh) indicator (right). The dark discoloration denotes an
anerobic environment

Antisepsis
1864 -Joseph Lister, a surgeon, found
survival rate of his surgical patients
increased if he reduced chance of
infection during surgery.
--sterilized his instruments --used
disinfectant during surgery -mist of
carbolic acid.

Ways to Think About Microbial

Death
Sterilization Eliminate ALL microbes
Disinfection Destroy Potential Infectivity
Sanitize Lower microbial count to an
acceptable level
Antisepsis Topical application of chemicals to
body surface to kill or inhibit pathogens
Bactericidal Kill bacteria
Bacetristatic Restrain or prevent bacterial
growth

Antibiotics: The Real Beginning

1875 John Tyndall, England
Question: Are bacteria evenly dispersed or in clouds
Experiment: A series of test tubes of broth exposed to
air
Observation: Some tubes with rich bacterial growth,
others with less bacteria amid exquisitely beautiful
Penicillium mold
Conclusion: Bacteria are not evenly dispersed

Lesson: Timing is everything!

Antibiotics: Enter Fleming

Alexander Fleming MD, 1906
Inoculation Department, St. Marys Hospital
1928 Mold
contamination of petri
dish incubating
Staphylococcus while
Fleming off on vacation

Lesson: Luck is key!

Antibiotics: Early In Vitro Testing

Active substance in broth on which mold
grew called penicillin
Canals in agar plate experiments
conducted against a broad range of
bacteria, but not the spirochete that
causes syphilis *
* Despite being recognized as
Englands foremost authority
on therapy of syphilis

Lesson: Do not ignore what is closest to

you!

Antibiotics: Constrained Thinking

The chemical, salvarsan, injected for
years as the therapy of choice for
syphilis, yet
Not conceivable that bacterial infections
could be treated by ingested or
injected drugs

Lesson: Be willing to relinquish old

paradigms!

Antibiotics: Limited Collaboration

Development compromised by
instability of penicillin and failure
to engage biochemisty expertise

Lesson: Collaboration may be key to

success!

Antibiotics: Moving Forward

1930s Paine (bacteriologist) uses broth
culture of Penicillium to treat eyes of 4
babies and 1 adult with dramatic clearing
of infection in 4/5
1935 Domagk cured systemic
streptococcal infection with the injection of
prontosil, overcoming the notion that
injected drugs not useful in treating
infections

Lesson: Just Do It!

Antibiotics: Back to Penicillin

Florey & Chain (Oxford):
Created stable form of penicillin and
studied kinetics in mice
Injected 4 mice with streptococcus + PCN
(all survived) and 4 with strep alone (all
died)
1940 - findings extended to 3 different
bacteria
Lesson:
Mice
& men
are not so
First human
patient
treated

different.

Antibiotics:
Innovative Strategies to Improve
Supply
Penicillin girls
Reclaiming urine
Treating children vs. larger
adults
Lesson: Ingenuity comes in many
forms.

Antibiotics: Increasing
Experience
Experience bolstered during World War 2,
with results leading to government
approval in early 1940s
US first use of penicillin about 5 weeks after
initial publication of Oxford team in
Lancet
Improved production developed following
Floreys meetings with pharmaceutical
companies in USA & Canada
Subsequent patent, royalty and political
disputes between US & UK

Lesson: Profit fuels conflict!

Antibiotics:
Penicillin Was Just the
Beginning
Penicillin
1932
Sulfapyridine
1939
Polymixin
1944
Streptomycin
1945
Cephalosporin C
1947
Chloramphenicol

Lesson: Run with a good thing!

Antibiotics:
Penicillin Was Just the
Beginning
1948
1950
1955
1959
1960
1962

Tetracycline
Erythromycin
Vancomycin &
Lincomycin
Rifampin
Methicillin
Cephalothin

Lesson: Run with a good thing!

Scarlet Fever
Pre-antibiotic era:

Feared infection
Overall mortality, 12 to 14%
Mortality in those < 5 years, 20 to
30%
Now:
An annoyance
5 to 10 days of oral antibiotic therapy
1 to 2 days of school missed

Appendicitis
Pre-antibiotic era:
Outcome depends upon treatment
Early operation, excellent prognosis
With advanced infection, 50% die

Now:
Simple operation, short
hospitalization
With perforation, antibiotics key
Deaths, extremely uncommon

Cerebro-Spinal Fever
(Meningitis)
Pre-antibiotic era:
Horrible infection
Invariably fatal

Now:
Serious infection
Vaccine preventable
Hospitalization for IV antibiotics
5-10% mortality rate

Antibiotics: What are They?

A chemical substance that is important
in the treatment of infectious diseases
Produced either by a microorganism or
semi-synthetically
Having the capacity in dilute solutions to
either kill or inhibit the growth of
certain other harmful microorganisms

Antibiotics: How Do They Work?

Using Antibiotics Wisely

Antibiotics: Limitations
Bacterial infections
Timely administration
Help from the host
Development of resistance

The Battle Between Bugs & Drugs

To regard any form of life as

slave or foe will one day be
considered poor philosophy,
for all living things constitute an
integral part of the cosmic
order.
Rene Dubos, 19011982

Antibiotic Resistance Goes Way

Back
1930s & 40s:
Avery reported in vitro production of sulfa
resistant pneumococci by progressive
selection (training) in increasing drug
concentrations.
Fleming recommended bold dosing of
sulfa, so that infection could be dealt
with before the cocci had a chance to
become fast.

Antibiotic Resistance;
Why Is It Increasing?
Worldwide overutilization of
antibiotics.
Increasing use of broader-spectrum,
more expensive antibiotics
Release of enormous quantities of
antibiotics in agriculture, fisheries, &
animal husbandry.
JAMA 1995;273:214-219

Antibiotics Overuse & Resistance

In 1965, 90% of P.aeruginosa isolates in a burn unit were
sensitive to gentamicin. Following use of 0.7 tons of topical
gentamicin use in 1968, only 9% of isolates sensitive.
Annual use of erythromycin in Japan peaked at 170 tons
between 1974 and 1977; use decreased to 65 tons by 1984.
In parallel, group A streptococcal resistance decreased from
62% to 2%.
Data from Finland directly relates incidence of macrolid
resistance among GAS to country-wide consumption of drug.

The Time Frame of Human Generations

Bacterial Interactions with the Human Host

Transients just passing through
Commensals literally to eat from the same
plate. Some are present from the cradle to the
grave
Primary Pathogens A primary pathogen
regulalrly causes disease in an immunologically
normal host
Opportunistic Pathogens cause disease only in
immunolgically compromisd hosts. Many
commensals can be opportunists. An opportunist
in one host may be a primary pathogen in another
host.

Attributes Shared by Bacterial Pathogens

and Commensal Species of a Host
Enter the Host
Attain a Unique Niche
Avoid, Subvert, Circumvent or Manipulate the
Immune Defenses of the Host Including
Competition From Resident Microflora.
Multiply Sufficiently To Either Persist Or Be
Transmitted To A New Susceptible Host.
Exit

Optional Attributes of
Pathogenicity
Breach Cellular and Anatomic Barriers of
the Host
Cause Overt Disease
Persist in ordinarily sterile body sites
Avoid the Adaptive Immune Response of
the Host

Persistent Bacterial Infections The

Problem
Why do some organisms cause acute infection
while others cause a persistent infection?
How long after the infective event occurs is the
transition to persistent infection?
How does the organism change during this
transition?
How does the host change during this transition?
When does the relationship become obligate for
the microbe?
Does the relationship ever become symbiotic?

Bacterial Pathogencity
Enter the Host
Attain a Unique Niche
Avoid, Subvert, Circumvent or Manipulate
the Immune Defenses of the Host
Multiply
Exit

Entry into Host

Each of the nine portals of humans open to
the environment serve as a point of entry
for one or more pathogenic microbes.
Entry is not a random event but rather has
been selectively evolved by microbes to
exploit the hosts needs to breathe, eat, see,
hear, eliminate wastes and reproduce.

Attain a Unique Niche

All pathogens have evolved a specific
means of association with at least one
unique host cellular target shortly after
entry.
The specificity of this initial molecular
interaction may dictate the subsequent fate
of the microbe for hours or even days
afterwards

The Pathogenic Signature

(Most pathogens have a distinct personality)
Avoid
arthropod bite, enter cells

Circumvent
capsules, IgA protease

Subvert
ADP ribosylation, host enzyme activation or repression

Manipulate
Exploit the host response for persistence, transmission
or nature of the host cell immune response

Multiply
The Definitive Goal of the
Pathogenic Strategy is to Produce
Sufficient Number of Cells to either
Persist in the Host or be Transmitted
to a New Susceptible Host
(Every bacteriums wish is to be
bacteria)

Exit the Host

In many cases virulence is the consequence
of selection for determinants maximizing
transmission from host-to-host.
It is likely that microbes have specialized
determinants for leaving the host, existing
in between hosts and preparing for
subsequent entry in a new host.

The Corollaries of Pathogenicity

Pathogenicity Islands are:

An Ancient Form of Microbial Genetic Internet
(Horizontal Gene Transfer).
Seen as Insertions of DNA of Varying Size in the
Bacterial Chromosome.
Possess an Alien Signature Since the Island Often
Displays a Base Composition Distinct from Most Other
Chromosomal Genes and Even Different Preferred
Codon Usage.
Often are found adjacent to genes encoding tRNA
sequences (at least in enteric species).
Usually Provide Some Degree of Selective Advantage
Over Other Related Microbes

The Contemporary Ambivalence

about Infectious Disease

Most Common Infections That

Cause Hospitalizations in USA
(1994)
Lower respiratory tract infections
Urinary tract infections
Cellulitis
Septicemia
Abdominal infections
Upper respiratory tract infections
Enteric infections
Infections of prosthetic devices
Postoperative infections
Infections in pregnancy
Osteomyelitis

1,448,60
470,20
335,30
301,80
282,60
191,40
152,20
73,50
72,80
56,00
48,10

Most Common Reported Notifiable

Infectious Diseases in Adults (1997)
Gonorrhoeae
AIDS
Syphilis
Tuberculosis
Salmonellosis
Hepatitis A
Hepatitis B
Shigellosis
Lyme Disease
Hepatitis C

875,817
222,083
71,397
69,344
55,229
43,987
35,627
22,328
21,176
14,393

A Different Kind of Bioterrorism

Infectious Diseases for Graduate

Students
Infection and disease for biologists begins with the
microbe
Infectious Diseases for physicians begins with a
patient complaining of a constellation of
symptoms a syndrome.
A syndrome can be caused by more than one
microbial agent.
Therefore Infectious diseases are usually first
characterized by the dominant organ system which
the patients symptoms reveal by symptoms.

Recognize the Probable Site of

Infection

Site & Signs of Infection Predict

Cause

The Type of Host Helps Predict

Cause

An Example of a Syndrome Respiratory

Tract Infection
Streptococcus pneumoniae is the most common cause of
adult community-acquired pneumonia.
The patient does not present with complaining of
pneumococcal pneumonia.
Rather, the patient complains of fever, cough, shortness of
breath and chest pain , perhaps with chills.
Similar symptoms can be caused by viral agents and any
different bacterial agents, fungi and even large parasites.
The differential diagnosis is made by and by careful
attention to the patients history coupled with physical and
laboratory examination.

Some Terms Used to Describe Infectious

Diseases
Infection is growth and reproduction of a
microorganism on or within a host
Disease is is the overt clinical damage done to the
host by infection
Pathology refers to the anatomic and tissue
abnormalities induced by infection.
Pathogenesis refers to the events induced by the
microbe causing the pathology.
A Pathogen is a microbe capable of causing
disease.
Virulence is a quantitative assessment of
pathogenicity.

Infectious Syndromes
Skin and soft tissue
Respiratory
Upper Respiratory
Lower Respiratory

Gastrointestinal
Urogenital Tract
Nervous System
Generalized or Systemic