Meeting 6 Disease Manifestations Virulence Factors Antimicrobial resistance

Meeting 6

Disease ManifestationsVirulence Factors

Antimicrobial resistance

1 Aug 2009

Manifestations of infectious disease Classic

Signs• Fever, swelling, rashes, vomiting, diarrhea• Skin signs: lesions, erythema, papule (pimple), vesicles,

pustule, ulcer or erosion or abscess Symptoms

• Pain, headache, nausea, malais Inflammation

Result of Immune reactions Hematologic ( leukocytosis, anemia ) Cardiac (tachycardia to heart failure) Respiratory (hyperventilation) Renal Hepatic Upper GI bleeding

Manifestations of infectious disease

Classic Signs

• Fever, swelling, rashes, vomiting, diarrhea

• Skin signs: lesions, erythema, papule (pimple), vesicles, pustule, ulcer or erosion or abscess

Symptoms• Pain, headache,

nausea, malaise

Inflammation

Table 1 Types of skin lesions

Flat lesions (in the plane of the skin)

Elevated lesions (above the plane of the skin)

Depressed lesions (below the plane of the skin)

Macule IInfarct Sclerosis• Telangiectasis†

Vesicle and bulla Pustule Abscess‡ Cyst‡ Papule Wheal plague Nodule‡ Vegetation Keratosis Desquamation (scales) Exudate• (crusts) Lichenification

Atrophy§ Sclerosis§ Erosion Excoriation Scar† Ulcer Sinus‡ Gangrene§

• May also be below the plane of skin † May also be above the plane of skin ‡ May also be in or below the plane of skin § May also be in the plane of skin

Flat lesion MACULE

A macule is a change in the color of the skin. It is flat, if you were to close your eyes and run your fingers over the surface of a purely macular lesion, you could not detect it. A macule greater than 1 cm. may be referred to as a patch.

Elevated lesion

PAPULE is a solid raised lesion that has

distinct borders and is less than 1 cm in diameter. Papules may have a variety of shapes in profile (domed, flat-topped, umbilicated) and may be associated with secondary features such as crusts or scales.

Elevated lesion

TUMOR is a solid mass of the skin

or subcutaneous tissue; it is larger than a nodule.

Elevated lesion PAPULE

is a solid raised lesion that has distinct borders and is less than 1 cm in diameter. Papules may have a variety of shapes in profile (domed, flat-topped, umbilicated) and may be associated with secondary features such as crusts or scales.

Depressed lesion

ATROPHY Atrophy is thinning or

absence of the epidermis or subcutaneous fat.

Depressed lesion

EROSION Erosions are slightly

depressed areas of skin in which part or all of the epidermis has been lost

Depressed lesion

ULCERATION occur when there is

necrosis of the epidermis and dermis and sometimes of the underlying subcutaneous tissue.

FEVER

1. Fever increases the environmental temperature above the optimum growth temperature for many microorganisms. If the microorganisms are growing more slowly, the body's defenses have a better chance of removing them all.

2. Fever leads to the production of heat shock proteins that are recognized by some intraepithelial T-lymphocytes (delta gamma T-cells) resulting in the production of inflammation-promoting cytokines.

3. Fever elevates the temperature of the body increasing the rate of enzyme reactions, and speeding up metabolism within the body.

can increase the production and activity of phagocytes, speed up the multiplication of lymphocytes, increase the rate of antibody and cytokine production, increase the rate at which leukocytes are released from the bone marrow into the

bloodstream, and speed up tissue repair. Too high of a body temperature, however, may cause damage by denaturing the body's enzymes.

FEVER Fever may have certain signs in relation to its course.

Fever in infectious diseases usually is of short duration

Long duration (weeks or months) is always a very serious problem. If it is not possible to determine the cause of fever at the beginning, it is called the fever of unknown origin (FUO). This term is used to describe fever lasting at least 2 weeks, reaching temperatures above 38,2 , and the cause of the origin is uncertain.

Fever may last long in some infections with subacute or chronic course.

Stages in the Progression of Disease

1. No signs or symptoms2. Prodormal illness

Mild signs or symptoms Some chilling occur

3. Acute identifiable signs and symptoms Intermittent fever

4. Recovery Action of immune system Action of antibiotics

5. Convalescence

Stages in the Progression of Disease

Stage: Acute pyogenic: Chronic

granulomatous:

Encounter Often with symptomatic patient or after trauma.

Often impossible to pin down.

Entry/ Establishment

Obvious if associated with trauma. Adhesins and entry mechanisms well-studied.

Not well understood but uptake into macrophages a common feature.

Spread Often associated with damage.

Usually imperceptible.

Multiplication Rapid. Slow.

Damage Acute inflammation, toxins.

Cell mediated immunity.

Outcome Easy to treat with antibiotics.

Difficult.

Manifestations of infectious disease

Result of Immune reactions

Hematologic ( leukocytosis, anemia ) Cardiac (tachycardia to heart failure) Respiratory (hyperventilation) Renal Hepatic Upper GI bleeding

Case study 1

S. T. was seen by you in the Emergency Room in December

23 y/o medical student in Southern California c/o sudden onset of fever, chills, malaise,

headache, myalgia, sore throat, runny nose, sneezing

Room-mate has same symptoms Exam: erythematous, inflammed tonsils, no pharyngeal exudates Throat culture: Group A streptococci

Case study 1

erythematous, inflammed tonsils

Case study 2 P. A. is a patient in the Intensive Care Unit 65 y/o man Intubated, on respirator: good oxygenation Nurse says that he hasn’t had fever or purulent

sputum Exam: clear breath sounds, no rhonchi nor rales CXR: clear without infiltrates or effusions Sputum Gram stain: mixed flora with Gram positive

cocci; thin, long Gram negative rods Sputum culture: normal respiratory flora, 2+

Pseudomonas aeruginosa

Case study 3 T. M. was referred to you by the Public Health

Department 38 y/o woman Private cook in Manhattan In the past 10 years, 7 of the 8 families she

has worked for have had outbreaks of illness: Fever, malaise, headache, myalgia, maculopapular rash,

bradycardia, constipation, bloody diarrhea

T. M. denies h/o similar illness and denies current symptoms

“But, Doctor, I’m not sick!”

Case study 3

maculopapular rash

Disease state: complex interaction between pathogen and host

1. If a bacterium is present, is it causing disease? a. Normal flora b. Colonization c. Carrier state d. Infection:

• i. Asymptomatic• ii. Symptomatic

2. Is the bacterium capable of causing disease? a. Nonpathogen b. Opportunistic pathogen c. Primary pathogen

Case study 1: Viral pharyngitis + Group A strep normal respiratory

flora S. T. was seen by you in the Emergency

Room in December 23 y/o medical student in Southern California c/o sudden onset of fever, chills, malaise,

headache, myalgia, sore throat, runny nose, sneezing

Room-mate has same symptoms Exam: erythematous, inflammed tonsils, no pharyngeal exudates Throat culture: Group A streptococci

Case study 2: Colonization with Pseudomonas aeruginosa

P. A. is a patient in the Intensive Care Unit 65 y/o man Intubated, on respirator: good oxygenation Nurse says that he hasn’t had fever or purulent sputum Exam: clear breath sounds, no rhonchi nor rales CXR: clear without infiltrates or effusions Sputum Gram stain: mixed flora with Gram positive cocci;

thin, long Gram negative rods Sputum culture: normal respiratory flora, 2+

Pseudomonas aeruginosa

Case study 3: Typhoid MarySalmonella typhi carrier

T. M. was referred to you by the Public Health Department

38 y/o woman Private cook in Manhattan In the past 10 years, 7 of the 8 families she has

worked for have had outbreaks of illness: Fever, malaise, headache, myalgia, maculopapular rash, bradycardia,

constipation, bloody diarrhea

T. M. denies h/o similar illness and denies current symptoms

“But, Doctor, I’m not sick!”

Outbreak 2001

Virulence Factors that Promote Colonization and Invasion

Virulence factors that damage the host Exotoxins Endotoxins

Ability to resist innate immunity Ability to evade adaptive immunity Ability to induce autoimmune response


Basic requirements: receptor + adhesin Receptor

• Specific carbohydrate or peptide residues on the cell surface

Adhesin or adherence factor• A macromolecular component of the bacterial cell

surface interacting to the receptor

Surface proteins called adhesins in the bacterial cell wall bind to receptor molecules on the surface of a susceptible host cell enabling the bacterium to make intimate contact with the host cell, adhere, colonize, and resist flushing.


Specific adherence of bacteria to cell and tissue surfaces

Tissue tropism• Strep mutans in dental plaque but not in the tongue

Species specificity• Neisseria gonorrheae limited to humans

Genetic specificity within a species

Mechanism of specific adherence Reversible attachment – “docking” Non-reversible attachment – “anchoring”


(Tabulate according to the ff:

Bacterium:Adherence Factor:Receptor (optional):Attachment Site: Disease)

Streptococcus pyogenes: Protein F Aminoterminus of fibronectin Pharyngeal Epithelium Sore Throat

Virulence Factors that Promote Colonization and Invasion: Example

S. pyogenes

fibronectin

F-proteinlipoteichoic acid

Fimbriae Adhesins

Protein F Capsules Invasins

Cleaves secretory IgA (Glycosyl transferase) Spreading factors:

• Hyaluronidase – attacks the interstitial cement of connective tissue by depolymerizing hyaluronic acid

• Collagenase• Neuraminidase• Streptokinase and staphylokinase

Siderophores Low molecular weight compounds that chelate iron with very high affinity Competing for iron and other nutrients

Virulence Factors that Promote Colonization and Invasion:

AdhesionAdhesion

adhesinadhesin

EPITHELIUMEPITHELIUM

receptorreceptor

BACTERIUMBACTERIUM



Glycocalyx Polysaccharide

or peptide slime Capsule Slime layer

Functions: Resists

phagocytosis Enhanced

attachment


Shigella Passing Through the Mucous Membraneand Invading Mucosal Epithelial Cells


Properties Secreted during exponential growth Protein toxins High biological activity Exhibits specificity of action

Components A = active B = binding

Virulence Factors that Promote Damage to the Host: EXOTOXINS

A-B toxins

ActiveBinding

A

Cell surface

B

Binding and Entry of an A-B Toxin

A-B toxins consist of two parts, an A (active) component and a B (binding) component. The B component of the exotoxin binds to a receptor on the surface of a susceptible host cell. The exotoxin now enters

the host cell, in this case by endocytosis, and causes harm by inactivating a host cell target protein through ADP-ribosylation.

Based on structure A-B prototype

• Botulinum toxin, diphtheria toxin, shiga toxin, tetanus toxin

Membrane disrupting toxin• Lacks A & B• Pore forming• Phospholipase

Superantigens


Superantigens

1. Some exotoxins are superantigens

2. Produced by bacteria and viruses

3. Action: polyclonal stimulation of subset of lymphocytes to divide and produce cytokines

4. Best known example: Staph Toxic Shock Syndrome Toxin-1 (TSST-1)

5. Also Strep exotoxins

6. Pyrogenic toxins cause fever (unlike other exotoxins)

2-subunit A-B exotoxinNeurotoxin: Clostridium

botulinum

Murray, P.R. et al. Medical Microbiology, 3rd edition, 1998, p. 156

Botulism Exotoxin Blocking Acetylcholine Release

The botulism exotoxin binds to the presynaptic neuron and blocks its release of acetylcholine. This causes a flaccid paralysis, a weakening of the involved muscles.

• Proteases • Phospholipases• Detergent-like action

Membrane damaging exotoxins

Based on modes of action Breaks down cells

• Alpha toxins, hemolysins, leukocidins Enhance microbial spread

• Spreading factors: hyaluronidase, mucinase etc Interfere in cellular metabolism

• Tetanus toxin, botulinum toxin


3 ways this can contribute to the progression of disease:

Ingestion of preformed toxin Colonization of wound or mucosal surface

followed by exotoxin production Colonization of wound followed by local

exotoxin production


Neutralization of Exotoxins

Evasion of host immune response: IgA protease

1. Cleaves IgA, which is important for mucosal immunity

2. An enzyme produced by Neisseria gonorrhoeae, Neisseria meningitidis, Haemophilus influenzae, Streptococcus pneumoniae

Requirement for iron

1. Most iron in the body is intracellular In hemoglobin and myoglobin

2. There is very little free iron Extracellular iron is bound to transferrin (in

plasma) and lactoferrin (in milk and other secretions)

3. Some bacteria produce siderophores to capture iron e.g. Escherichia coli

Siderophores bind iron with high affinity

Virulence Factors that Promote Damage to the Host: ENDOTOXINS

Outer membrane of the gram negative cell wall Lipid A:polysaccharide:O antigen Toxicity is associated with Lipid A Immunogenicity is associated with the polysaccharide

component Less potent, less specific Does not form antitoxin Released when the bacterial cell is damaged

GRAM NEGATIVE CELL ENVELOPE

Cytoplasm

Inner (cytoplasmic) membrane

Outer Membrane(Major permeability barrier) LipopolysaccharidePorin

Braun lipoprotein

Periplasmic space Degradative enzyme

Periplasmic binding proteinPermease

• Septic shock • hypotension (tissue pooling of fluids)• disseminated intravascular coagulation• fever• lack of effective oxygenation• overall system failure

• Septicemia

Septic shock

Exotoxin vs Endotoxin

Exotoxin Endotoxin

Gram negative & positive Gram negative only

Secreted Liberated upon bacterial lysis

Highly toxic Not as toxic

Highly antigenic Less antigenic

Inactivated to form toxoid Heat stable

Anti-toxoid often protective No toxoid

No fever (except pyrogenic toxins)

Fever and septic shock

Bacterial Pathogenesis 1. Microbial pathogens and their hosts

have often co-evolved

2. Clinical disease a. Non-adapted host/pathogen b. May promote pathogen’s survival and

transmission c. Can result from host response to pathogen

3. Extracellular bacteria produce toxins and enzymes

Bacterial Pathogenesis 4. Bacteria secrete proteins into host cells to

modify host cell function

5. Intracellular bacteria must bind, enter and survive inside host cells

6. Pathogenic bacteria can evade the host immune response and have mechanisms for antibiotic resistance

7. Expression of virulence factors is often regulated in response to the environment and other signals

Bacterial Pathogenesis: Concepts 1. Virulence genes that encode virulence

factorsA virulence gene confers on a bacterium the ability to cause disease

Pathogenic strains have acquired virulence genes that allow them to exploit the host as an environmental niche

Examples of virulence factors are toxins, enzymes, type III secreted proteins, adhesins, siderophores

Virulence factors are obvious targets for prevention or treatment strategies

Bacterial Pathogenesis: Concepts

2. Bacteria have found many way to modulate host cell function

Exotoxins and type III secreted proteins are virulence factors with specific functions such as to kill host cells or induce the uptake of bacteria by the host cell

Knowledge of these mechanisms can help explain the clinical disease caused by a particular bacterium, and the basis for prevention and treatment strategies

Bacterial Pathogenesis: Concepts 3. Toxins vs. toxoids vs. antitoxin (antitoxoid)

Toxins are made by bacteria and have harmful effects on host cells

A toxoid is the inactivated form of an exotoxin that can be used as a vaccine to elicit an immune response

Toxoids were traditionally inactivated by heat or formaldehyde, but now can be genetically engineered

Antitoxin is the antisera produced in response to the toxoid (can be from animals or humans) and can be given as treatment

Bacterial Pathogenesis: Concepts

4. Mechanisms of pathogenesis are conserved among bacteria

Virulence genes that provide a selective advantage are spread among different bacterial genera and species by horizontal transfer

These virulence genes are often located on mobile DNA elements such as plasmids or bacteriophages

… refresh...

Antibiotics

BC Yang

Antibiotics and vaccines are among the biggest medical advances since 1000. (Culver Pictures)

For lecture only

A brief history of antibiotics 1495, mercury to treat syphilis. 1630, quinine (chinchona tree) for malarial fever by South

American Indians. 1889, Buillemin defined antibiosis. 1910, Paul Ehrlich developed arsenical compound (Salvarsan) for

syphilis, term: the chemical knife. 1929, Alexander Fleming found penicillin. 1935, Gerhard Domagk showed the value of sulfonamides. 1940, Ernst Chain and Howard Flory demonstrated the effect of

penicillin. 1940-1970, then searching for new antibiotics ~ recent year: modifying old drugs, finding new discipline in

antibacterial combats Early time in war: thanks penicillin, we can go home now Now a day……….Oh eh?!

Thanks to work by Alexander Fleming (1881-1955), Howard Florey ( 1898-1968) and Ernst Chain (1906-1979), penicillin was first produced on a large scale for human use in 1943. At this time, the development of a pill that could reliably kill bacteria was a remarkable development and many lives were saved during World War II because this medication was available.

E. Chain H. FloreyA. Fleming

A tale by A. Fleming He took a sample of the

mold from the contaminated plate. He found that it was from the penicillium family, later specified as Penicillium notatum. Fleming presented his findings in 1929, but they raised little interest. He published a report on penicillin and its potential uses in the British Journal of Experimental Pathology.

An ideal antibiotic

Broad-spectrum Does not induce resistance Selective toxicity, low side effects Preserve normal microbial flora

Definitions Antimicrobial

Inhibits growth of micro-organisms Antibacterial

Inhibits growth of bacteria Antibiotic

Inhibits growth of micro-organisms Made by other micro-organisms Usually extended to include synthetic

drugs

Susceptibility test

Tube dilution method Minimal inhibitory

concentration (MIC): the smallest amount of chemotherapeutic agent required to inhibit the growth of organism in vitro

Disk diffusion method Zone of inhibition (ZOI):

the correlation of ZOI and MIC has been established by FAD

ETest. This commercially-prepared strip creates a gradient of antibiotic concentration when placed on an agar plate

Bacteriostatic vs Bactericidal

Bacteriostatic Reversible inhibition of growth When the antibiotic is removed, almost all of

the bacteria can replicate Bactericidal

Irreversible inhibition of growth When the antibiotic is removed, almost none

of the bacteria (10-7 to 10-3) can replicate

Guidance of antimicrobial therapy

Minimum inhibitory concentration: lowest concentration of antibiotic that inhibits visible growth

Minimum bactericidal concentration: lowest concentration of antibiotic that kills 99.9% of the inoculum

Serum bactericidal title: dilution of serum that kills 99.9% of the inoculum

Synergy test: synergistic activity of multiple antibiotics

Use of antibiotics; is it properly applied?

Acute infections in outpatients

Acute infections in hospitalized patients

Chronic infection (tuberculosis, AIDS)

Agriculture/veterinary medicine

In vitro: Factors for optimal antibiotic action

pH of environment: Nitrofurantoin is more active in acid pH; sulfonamides and

aminoglycoside are more active in alkaline pH. Components of medium:

Anionic detergents inhibit aminoglycosides, serum proteins bind to penicillin in varying degrees.

Stability of drug: Aminoglycosides and chloramphenical are stable for long

period in vivo. Size of inoculums:

The larger the bacterial inoculum, the greater the chance for resistnat mutant to emerge.

Metablic activity of microorganisms: Actively and rapidly growing organisms are more

susceptible to drug action

BC Yang

Affecting factors in vivo

Abscess: circulation is blocked off.

Foreign bodies: obstruction of the

urinary, biliary or respiratory tracts

etc.

Immunity.

Sites of actionBC Yang

Modes of action (1)

Inhibitors of cell wall synthesis.Penicillins, cephalosporin, bacitracin, carbapenems and vancomycin.

Inhibitors of Cell Membrane.Polyenes - Amphotericin B, nystatin, and condicidin.Imidazole - Miconazole, ketoconazole and clotrimazole.Polymixin E and B.

Inhibitors of Protein Synthesis.Aminoglycosides - Streptomycin, gentamicin, neomycin and kanamycin.Tetracyclines - Chlortetracycline, oxytetracycline, doxycycline and minocycline.Erythromycin, lincomycin, chloramphenicol and clindamycin.

Amphotericin

Tetracyclines

Aminoglycosides

vancomycin

For lecture only BC Yang

Modes of action (2)

Inhibitors of metabolites

(Antimetabolites).Sulfonamides - Sulfanilamide, sulfadiazine silver

and sulfamethoxazole.

Trimethoprim, ethambutol, isoniazid.

Inhibitors of nucleic acids

(DNA/RNA polymerase).Quinolones - Nalidixic acid, norfloxacin and

ciprofloxacin.

Rifamycin and flucytosine.

rifamycin


Resistance

Natural (inherent) resistanceStructural barrelLack of targetTransport system

Acquired resistanceMutationGene exchange (conjugation in most)

Transferable antibiotic resistance in bacteria

Reduced uptake into cell (chloramphenicol) Active efflux from cell (tetracycline) Modification of antibiotic targets (b-lactam,

erythromycin) inactivation of antibiotic by anzymic

modification: hydrolysis (b-lactam, erythromycin); derivatization (aminoglycosides)

Sequestration of antibiotic by protein binding (b-lactam)

Metabolic bypass (sulfonamides) Overproduction of antibiotic target (titration:

sulfonamides)

Spread of resistanceIn most:

Day-care, nursing homes,

correctional facilities Sanitation, animal feeds (fecal-oral) Sexual/ Respiratory transmission International travel Immunosuppression

Some probable overuse/misuse of antibiotics

Prophylatic use before surgery Empiric use (blinded use) Increased use of broad spectrum agents Pediatric use for viral infections Patients who do not complete course

(chronic disease, eg. TB, AIDS) Antibiotics in animal feeds


Policy to deal drug resistance (1)

Ideally, bacteriological management of clinical infection should involve:

1. Identification of causative organism2. Sensitivity test3. Follow-up the drug effect4. Monitor antibiotic level to avoid toxicity.

In reality, most patients requiring antimicrobial therapy are treated empirically. In serious infections immediate chemotherapy may be life-saving.

Policy to deal drug resistance (2)

Periodic changes of antibiotics used might change selective pressure and thus avoid the emergence of resistance and retain the therapeutic value of antibiotics over a longer period.

The unnecessary prophylactic or animal feeds use should be discouraged.

Distribution of information on current/updated infectious microbes (consult microbiologists): use more targeted antibiotics

Patient education

New antibiotics development

Pharmaceutical industry putting resources

back into discovery Liaisons with university researches Discoveries in microbial physiology and

genetics offering new targets, new disciplines Combinational chemistry (mass screening)


Meeting 6 Disease Manifestations Virulence Factors Antimicrobial resistance

Documents

depressed areas of skin

skinelevated lesions

skindepressed lesions

solid raised lesion

papule pimple

production of inflammation

elevated lesionpapuleis

depressed lesionatrophyatrophy