"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 µm;

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 sporeshowing 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 resemblingstaphylococci

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

Small to medium gram positive bacilli with palisading and angulararrangements, the so-called Chinese letter appearance. Thismorphology is suggestive of cornybacteria or Listeria monocytogenes.

Pleomorphic large gram positive bacilli resembling Clostridium perfringens. Thisis a direct smear from an infected wound. The absence of spores is typical.

Red blood cells and large gram negative bacilli with roundedends in a blood culture. These are resemble enteric bacilli andPseudomonas.

Pleomorphic, variably staining gram negative bacilli with “pointedends” resembling Fusobacterium spp.

Intracellular gram negative diplococci in a gram stainof a urethral discharge from a male patient. This can be presumptively identified as Neisseria gononrrhoeae.

Gram stain of a stool sample showing leukocytes (PMN) andslightly curved, comma shaped, gull-wing appearing bacteria typicalof Campylobacter jejuni.

Wright’s stain of peripheral blood showing spiral bacteria typical ofBorrelia spp.

Gram positive

Gram negative

Electron micrograph of a thin section of the Gram-positive coccusshowing 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 1887by 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 GrowthGrowth 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

MeasurementsCell Mass (Weight, Light Scattering)Cell Numbers

Direct CountViable Count

Typical Bacterial Growth Curve in Batch Liquid Culture

Semi-Useless InformationA 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.

That’s 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

Antisepsis1864 -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 Beginning1875 – John Tyndall, EnglandQuestion: Are bacteria evenly dispersed or in “clouds”Experiment: A series of test tubes of broth exposed to

airObservation: 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, 1906Inoculation Department, St. Mary’s 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 asEngland’s foremost authorityon 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

1930’s – 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 miceInjected 4 mice with streptococcus + PCN

(all survived) and 4 with strep alone (all died)

1940 - findings extended to 3 different bacteria

First human patient treatedLesson: Mice & men are not so different.

Antibiotics: Innovative Strategies to Improve

Supply

Penicillin girlsReclaiming urineTreating 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 1940’s

US first use of penicillin about 5 weeks after initial publication of Oxford team in Lancet

Improved production developed following Florey’s 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

BeginningPenicillin

1932 Sulfapyridine1939 Polymixin1944 Streptomycin1945 Cephalosporin C1947 Chloramphenicol

Lesson: Run with a good thing!

Antibiotics: Penicillin Was Just the

Beginning1948 Tetracycline1950 Erythromycin1955 Vancomycin &

Lincomycin1959 Rifampin1960 Methicillin1962 Cephalothin

Lesson: Run with a good thing!

Scarlet Fever

Pre-antibiotic era:Feared infectionOverall mortality, 12 to 14%Mortality in those < 5 years, 20 to 30%

Now:An annoyance5 to 10 days of oral antibiotic therapy1 to 2 days of school missed

Appendicitis

Pre-antibiotic era:Outcome depends upon treatmentEarly operation, excellent prognosisWith advanced infection, 50% die

Now:Simple operation, short

hospitalizationWith perforation, antibiotics key Deaths, extremely uncommon

Cerebro-Spinal Fever (Meningitis)

Pre-antibiotic era:Horrible infectionInvariably fatal

Now:Serious infectionVaccine preventableHospitalization for IV antibiotics5-10% 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 infectionsTimely administrationHelp from the hostDevelopment 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, 1901-1982

Antibiotic Resistance Goes Way Back

1930’s & ‘40’s: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 & ResistanceIn 1965, 90% of P.aeruginosa isolates in a burn unit weresensitive to gentamicin. Following use of 0.7 tons of topicalgentamicin use in 1968, only 9% of isolates sensitive.

Annual use of erythromycin in Japan peaked at 170 tonsbetween 1974 and 1977; use decreased to 65 tons by 1984.In parallel, group A streptococcal resistance decreased from62% to 2%.

Data from Finland directly relates incidence of macrolidresistance 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 Persistent Bacterial Infections –– The The ProblemProblem

• 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 host’s 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

MultiplyThe 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 bacterium’s 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 tRNAsequences (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 1,448,60Urinary tract infections 470,20Cellulitis 335,30Septicemia 301,80Abdominal infections 282,60Upper respiratory tract infections 191,40Enteric infections 152,20Infections of prosthetic devices 73,50Postoperative infections 72,80Infections in pregnancy 56,00Osteomyelitis 48,10

Most Common Reported NotifiableInfectious Diseases in Adults (1997)

Gonorrhoeae 875,817AIDS 222,083Syphilis 71,397Tuberculosis 69,344Salmonellosis 55,229Hepatitis A 43,987Hepatitis B 35,627Shigellosis 22,328Lyme Disease 21,176Hepatitis C 14,393

A Different Kind of Bioterrorism

Infectious Diseases for Graduate Infectious Diseases for Graduate StudentsStudents

• 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 patient’s 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 An Example of a Syndrome ––Respiratory Respiratory Tract InfectionTract 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 patient’s 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