What are some characteristics of Clostridium?

• Gram +
• Spore forming
• Large rod
• Obligate anaerobe
• Produce neurotoxin (some of the most toxic to humans)
• Disease examples:  GI infection, soft tissue infection

What are some complications of Clostridium difficile - associated diarrhea (CDAD)?

• Antibiotic-associated diarrhea (C. d. is only one cause)
• CDAD - diarrhea + positive stool test
• C. d. colitis - layers of skin formed inside colon->enlarges colon->megacolon (can be asymptomatic infection).
• Pseudomembranous colitis - endoscopic demonstration of exudative lesions.
• Toxic megacolon: radiologic and surgical diagnosis

What are some ways to diagnose CDAD?

• Endoscopy (pseudomembranous colitis)
• Anaerobic culture
• Cell culture cytotoxin test
• EIA toxin test
• PCR toxin gene detection

In CDAD diagnosis, what are the facts on doing the anaerobic culture?

• Must be in a selective and differential medium (CCFA)
• Very sensitive
• Doesn't differentiate between toxin and non-toxin strains
• Must add a toxin test to increase specificity
• Essential for epidemiologic studies
• Cost issue, no longer routine

In diagnosis CDAD, what are the specifics of the cell culture cytotoxin test?

• Stool filtrate added to mammalian cell line - with and without toxin-neutralizing Ab.
• Sample w/o neutralizing Ab shows cytopathic effect (dead clumps of rounded cells)
• Sample w/ neutralizing Ab shows intact cell monolayer.

In diagnosis of CDAD, what are some specs. on EIA toxin tests (which are not as important)?

• Can detect toxin A or B or both
• Rapid, cheap, and specific
• Less sensitive than cytotoxin test
• Toxin tests for A will miss rare C. difficile isolates that produce toxin B only.

How is CDAD treated?

• Discontinue/modify offending agent (antibiotic, get new agent)
• Replace fluids and electrolytes
• Do not treat asymptomatic C. d. colonization
• Don't treat nosocomial diarrhea empirically w/o testing, since even during outbreaks, <30% have CDAD.

What are some facts about CDAD relapses?

• May occur in 1/5 of CDAD patients
• Usually occurs after 1-3 wks. after termination of Rx
• Relapse is recurrent disease with same strain

What bacteria cause gas gangrene?

• Obligate anaerobe
• Gram +
• Spore-forming bacilli
• Genus Clostridium, including C. perfringenes

What is gas gangrene?

• Acute and fatal type of tissue death from bacterial infection
• Infections occur with impaired blood supply secondary to trauma, surgery, foreign bodies, or malignancy
• Clostridium can originate from external environment or from host's natural flora

What are the three major types of clostridial wound infections?

1. Gas gangrene (fatal form)
2. Anaerobic cellulitis
3. Superficial contamination

What are the 3 types of gas gangrene?

1. Posttraumatic (auto accident)
2. Postoperative (recent GI or biliary tract surgery)
3. Spontaneous (often have blood vessel disease, diabetes, or colon cancer)

What are the characteristics of gas gangrene?

• Myonecrosis (soft muscle/tissue destroyed - no O₂) - rapid onset
• Muscle swelling
• Severe pain
• Gas production (H₂, N₂, CO₂,O₂)- foul smelling
• Sepsis (spread to blood and organs)

What is treatment for gas gangrene?

• Antibiotics
• Surgical debridement
• Mortality possible - depends on severity, age, and time required to find medical assistance

What type of toxin does gas gangrene (C. perfringens) produce?

Alpha toxin (a phospholipase C) is the most dangerous:

• Destroys cell membranes
• Suppresses myocardial contractility
• Helps to cause hypotension
• Irreversibly decrease blood flow to area by increasing the freely moving aggregates that form blood clots
• Oxygen deprivation occurs and bacteria spreads and destroys tissue
• Can be dangerous to RBCs, WBCs, platelets, fibroblasts, and muscle cells.

What is the virulence factor of Clostridium perfringens?

At least 12 toxins and enzymes, with Alpha-Toxin (a phospholipase C) being the most important.

What are the virulence factors for Clostridium difficile?

• Enterotoxin (toxin A)
• Cytotoxin (toxin B)
• Adhesin factor
• Hyaluronidase
• Spore formation

What are the virulence factors of Clostridium botulinum?

• Spore formation
• Botulinum toxin (prevents release of Acetylcholine)
• Binary toxin

What are the virulence factors for Clostridium tetani?

• Spore formation
• Tetanospasmin (heat-labile neurotoxin; blocks release of neurotransmitters for inhibitory synapses)
• Tetanolysin (heat-stable hemolysin of unknown significance)

What are the characteristics of Clostridium botulinum?

• Gram +
• Rod-shaped
• Toxin producing
• Anaerobic
• Serotypes of toxin
• Can form spores

What are some diseases caused by Clostridium botulinum?

• Food-borne botulism
• Infant botulism
• Wound botulism
• Undetermined botulism

What is Infant Botulism?

• Discovered in 1976
• In infants under one, stomach not acidic enough, spores survive and grow in s. i.  Can't cry or drink, then eventually can't breathe.
• From spores in corn syrup or honey.
• 70% of reported cases.

What is wound botulism?

• First discovered in 1950s
• Assoc. with illegal drug use
• 30-40% of reported cases

How can you be exposed to C. botulinum?

• Ingestion of preformed toxin
• Inhalation of preformed toxin
• Local production of toxin in g. i. tract (grows in deep tissue or in g. i. where there is no O₂)
• Local production of toxin by organisms at site of wound.

What are the differences between C. botulinum and C. tetani?

• C. botulinum's toxin blocks the release of ACH from vesicles in nerve-muscle transmission.  Stimulation is blocked, producing paralysis of muscles, leading to failure of airway/breathing muscles.
• C. tetani's neurotoxin inactivates proteins that regulate release of the inhibitory neurotransmitters glycine and GABA, leading to continuous stimulation by excitatory neurotransmitter.

What are some specifics on Streptococcus spp.?

• Gram + cocci
• Can be a chain, diplococci, or quad - but not dispersed, very rare
• Prefer high CO₂ for growth
• Most infectious to humans, causes more disease than any.
• Can ID by: biochemical activity (type of sugar fermented, etc.), hemolysis (how they react to RBCs), and Lancefield group (molecules on surface)

What are the important features of group A streptococci?

• Most important - causes many diseases
• Beta hemolytic
• Facultative anaerobe
• Normal flora-found in mouth, throat, resp. tract
• Mild to life-threatening diseases:  pharyngitis (strep throat), tonsilitis, scarlet fever, rheumatic fever, impetigo, cellulitis, erysipelas, bacteremia, necrotizing fasciitis, and TSS.

What are the main virulence factors possessed by Streptococci?

• Attachment via pili to epithelial cells
• M protein - adhesive protein, antiphagocytic
• Hyaluronidase - anti-clotting factor
• Streptolysins - kill RBCs, WBCs, platelets
• Leipoteichoic acid - allows colony formation
• Streptokinase - anti-clotting factor, allows spread
• Pyrogenic exotoxin
• System infection

What are the important features of Strepococcus pneumoniae?

• Alpha hemolytic
• Resembles viridans group of streptococci; alpha hemolytic
• Mainly an invasive organism
• Invasion and host response are more important than toxins in causing disease - mere presence is a problem.

What are the virulence factors of Strepococcus pneumoniae?

• Capsules are major virulence determinant - prevents phagocytosis
• Ab to capsule is protective

What are the important features of pneumococcal pneumonia?

• Bacteria multiply in alveolar spaces
• Onset is abrupt
• Shaking chill and fever of 39 - 41 degrees C.
• Often viral infection before onset (inhibits mucociliary escalator)
• Productive cough with blood-tinged sputum
• Common to have chest pain
• Other predisposing factors:  cardiac failure, alcohol addiction, anything interfering with the cough or epiclottal reflex or ciliary action.

Name a disease that can be caused by both Streptococci.

Bacterial meningitis can be caused by S. agalactiae (beta hemolytic) or by S. pneumoniae (alpha hemolytic)

What are the important features of TSS?

Caused by Staphylococcus aureus

• Gram + cocci arranged in clusters
• Facultative anaerobe
• Capsule and slime layer
• Initiated with the localized growth of toxin-producing strains of S. aureus in the vagina or a wound, followed by release of toxin into blood.
• Fever, hypotension, diffuse macular erythematous rash
• Multiple organs affected
• High mortality without prompt anibiotic therapy and elimination of focus of infection.

What are the general characteristics of group of Enterobacteriaceae?

• Small, Gram - rods
• Motile with peritrichous flagella (or non motile)
• Facultative bacteria that ferment glucose with acid +/- gas
• Group includes:  soil and water inhabitants, normal intestinal flora, intestinal pathogens

E. coli

What are members and diseases caused by Enterobacteriaceae?

• Gastroenteritis: E. coli, Salmonella, Shigella, Yersinia, Campylobacter
• Septicemia: E. coli, Salmonella, Yersinia pestis
• Enteric fevers:  Salmonella
• Pneumonia: Klebsiella pneumoniae, Yersinia pestis
• UTI:  E. coli, Klebsiella, Proteus mirabilis

What is a selective media used for Enterobacteriaceae detection from extraintestinal samples - blood cultures, wounds, sputum, etc.?

MacConkey agar - both selective and differential.  Differentiates one G (-) from another (pH indicator shows fermenters vs. non-fermenters)

Name 3 organisms that cause UTI.  Which one is the most frequent cause?

1. E. Coli (most frequent)
2. Klebsiella pneumonia
3. Proteus mirabilis

• Endotoxin
• Capsule
• Antigenic phase variation
• Sequestration of growth factors
• Resistance to serum killing
• Antimicrobial resistance

What are some common virulence factors associated with

Escherichia coli?

Adhesions

• Type 1 pili
• Colonization factors I and II
• K88 Ag

Toxins

• In ETEC E. coli, Heat labile toxins LT-I and LT-II:  LT-I is functionally and structurally similar to cholera toxin, leads to watery diarrhea.  LT-II is not associated with human disease. 
• In ETEC E. coli, Heat stable toxins STa and STb:  Only STa is associated with human disease, leads to hypersecretion of fluids.
• In EHEC E. coli, Shiga-like toxins SLT-I and SLT-II:  Both destroy intestinal villi.  SLT-II destroys glomerular endothelial cells.
• Hemolysin: lyses erythrocytes and other cell types, leading to inflammatory response in UTI infections.

With Gastroenteritis, what are the major groups of E. coli that have been recognized?

• EPEC - Enteropathogenic
• EIEC - Enteroinvasive
• EAEC - Enteroaggregative
• DAEC - Diffuse aggregative

What is the function of adenyl cyclase?  Name the exotoxins that affect this enzyme.

It is an enzyme that catalyzes the conversion of adenosine triphosphate (ATP) to cyclic adenosine monophosphate (cAMP) and inorganic pyrophosphate (PPi).

The exotoxins that affect this enzyme are LT toxins in ETEC.

What are specifics for Shigella spp.?

• Virulence factors: Endotoxin, capsule, anitgenic phase variation, sequestration of growth factors, resistance to serum killing, animicrobial resistance, Hemolytic colitis (HC) and Hemolytic uremic syndrome (HUS), Permeability  barrier of outer membrane
• ID50:  Low ~200 bacteria
• Vaccine/Treatment:  Antibiotic therapy, fluoroquinolone or trimethoprim-sulfamethoxazole initially, prevention with hand washing and disposal of soiled linens.

What are characteristics of typhoid fever and extraintestinal diseases caused by Salmonella?

Typhoid fever

• Bacteria go from intestinal wall and to regional lymph nodes (cause constipation, headache, and general symptoms)
• Bacteria spread through blood stream to body. 
• Bacteria phagocytized, not killed;intracellular multiplication occurs.
• Re-enter bloodstream; prolonged, severe septicemia may result (fever, shock from endotoxin - LPS)
• Bacteria may re-enter intestinal tract, cause diarrhea and may infect gall bladder
• Severe, freq. fatal disease results if not treated.
• Treat with antibiotics.
• Vaccination can reduce risk of disease for travelers in endemic areas.  Produces anti-O and anti-Vi antibodies.

Name a disease caused by Klebsiella pneumoniae.  What is its major virulence factor?

UTI, and pneumonia

Major v. factor:  Capsules

What are the ID50 specifics for Salmonella spp.?

• ID50: Low for S. typhi, but high for symptomatic disease with other Salmonella spp.

What are the specifics of Yersinia pestis?

• Virulence factors:  Capsule, resistant to serum killing, Lipid A, etc. (pg. 334)
• Vaccine:  Yes for plague
• Treatment:  antibiotics, but penicillin not effective
• ID50:
• G -, facultatively anaerobic rods

What diseases discussed in class are zoonotic dieseases?

• Plague
• Anthrax
• Campylobacter (poultry)

Name and describe the mechanism by which some organisms may resist phagocytosis.

• In the blood stream, Fraction 1 (F1) gene, coding for an antiphagocytic protein capsule - goes through phase variation type III.  There is a chemical produced by the organism that inhibits the phagocytic cells to kill the organism.
• In macrophages, expression of outer membrane proteins (hi temp., low calcium) allow survival inside macrophage.

What are the specifics on Vibrio?

• G -
• Oxidase (+) curved bacilli
• Many found in sea and estuarine waters - require salt for growth.
• Vibrio cholerae O1 and O139 produce cholera toxin. 
• Virulence factor of V. c. O1 and O139:  enterotoxin = major virulence determinant, cholera toxin, toxin co-regulated pilus, accessory cholera enterotoxin, zonnula occludens toxin, colonization factor, neuraminidase.
• ID50:  high, more than 10⁸, 50% less with buffer(bicarbonate)
• Treatment: for Cholera, vaccine provides limited protection; immunity not long lasting; antibiotics; fluid and electrolyte replacement.  No vaccine for other Vibrio spp.
• Cholera A1 fragment enzymatically catalyzes the ADP-ribosylation of a regulatory protein (raises cAMP)-->ions secreted by intestinal cells and water stays in intestine--> up to 20 liters diarrhea/day

What are the specifics on Vibrio cholerae non-O1 or 139?

• Not assoc. w/ epidemics
• In estuarine waters
• Presence related to salinity not fecal pollution
• Enterotoxin can cause intestinal disease
• Assoc. w/ eating raw oysters

What are specifics on Vibrio parahaemolyticus?

• Similar to V. cholerae (morphology and biochem.)
• Needs 2-7% NaCl for growth
• Causes mild to severe intestinal disease (50% of g. i. disease in Japan at times)
• Usu. produce hemolysis on blood agar + NaCl (Wagatsuma agar); a (+) Kanagawa test

What are the specifics on Vibrio vulnificus?

• Halophilic, estuarine inhabitant
• Routes of infection:  1) wounds exposed to estuarine water, shellfish, or crabs-->swelling, pain-->necrosis; 2) septicemia after ingestion of seafood, raw oysters-->fever, chills, prostration; g. i. disease less common
• ID50: High, but iron can lower the ID50

• G -
• Slender, curved rod
• Flagella (1 or 2)
• Microaerophilic
• Non-spore forming
• ID50: Low - 400-500 organisms
• Virulence factors: attachment (involves lipids in cell membrane of host cells), toxins - damage tight junctions between epithelial intestinal cells
• More infections from C. jejuni than from Shigella and Salmonella combined
• Watery or sticky diarrhea - can become bloody, fever, headache, nausea, abdom. pain (resembles typhoid fever)
• 1/1000 infections will result in Guillain-Barre Syndrome (self-attack on peripheral myelin-->paralysis-->not permanent)
• From ingestion of contaminated poultry, unpasteurized milk, untreated water
• Treatment:  fluid and electrolyte replacement; antibiotics
• Can be confused with IBS or other gut disorders - need correct diagnosis - antibiotic resist. strains 

What are the specifics of Bacillus anthracis?

• Rod-shaped
• G +
• Facultative anaerobe
• 6 micrometers in size
• Zoonosis disease: domestic and wild animals (eat animal with disease, or exposed to high level of spores)
• Main virulence factor: capsule; others are 3 exotoxins that combine to form edema toxin (liquid in lung) and lethal toxin (TNF alpha-->cell death)
• Septicemia and death result
• 3 main routes to contract: 1) Cutaneous - through skin abrasion; 2) Inhalation; 3) Intestinal
• Treatment:  Oral or IV antibiotics, may fail to treat inhalation anthrax once bacteria releases large amts. of toxin
• Vaccination: of animal herds and people in endemic areas can control; human vaccines have limited usefulness

What are some specifics of TB (Mycobacterium tuberculosis)?

• Slender, rod-shaped bacilli
• Aerobic
• G +
• Non-spore forming
• Non-motile
• Virulence: Capable of intracellular growth in unactivated alveolar macrophages; disease primarily from host response to infection
• Hydrophobic
• Primary infection to lung, but can spread (tubercle ruptures and releases bacteria into blood)

What is the significance of the cell wall in TB?

• The lipid-rich cell wall makes the organism resistant to disinfectants, detergents, common antibacterial antibiotics, and traditional stains.

What are the main clinical manifestations of TB?

• Productive, persistent cough
• Weight loss
• Night sweats

Also,

• Fever
• Fatigue
• Hemoptysis
• Dyspnea
• Orthopnea

What is the mechanism of pathogenicity of TB?

M. tuberculosis prevents fusion of the phagosome with lysosomes, and multiply within the phagosome.

What are the treatment options for TB patients?

• Multi-drug regimen, take meds., including Isoniazid and Rifamptin.  Drug regimen takes 9-12 months.
• Treatment individualized.
• Depends on active vs. chronic infection
• Cured when X-ray OK and sputum sample OK.
• Vaccine is Bacille Calmette-Guerin (BCG) - only if epidemic

What are the characteristics of TB skin test?  What material is used for the test?

• PPD (purified protein derivitive, from M. bovis) is used
• Intracutaneous injection, read 48-72 hrs. afterwards
• Results vary:  Positive if - active infection, previous infection, been vaccinated, low-grade infection, chronic infection

What are the specifics of leprosy(Hansen's disease): Mycobacterium leprae?

• G +
• Bacillus
• Virulence: Obligate intracellular growth (main factor); disease primarily from host response to infection
• 2 forms of disease: 1) Tuberculoid form - causes high CMI; milder; T cells invade perineurium and destroy Schwann cells and axons; infectivity is low  2) Lepromatous form - causes high AFB - strong antibody response - bacilli invade Schwann cells; degenerative myelination and axon degeneration.  More live bacteria - disfigures skin - highly infectious.
• Treatment:  Tuberculoid form treated with rifampicin and dapsone; Add clofazimine for treatment of lepromatous form.
• Spread by direct contact or infectious aerosol inhalation
• Vaccine: none
• No cure

What are some specifics on Chlamydia trachomatis?

• Virulence: Intracellular replication; Prevents fusion of phagosome with cellular lysosomes; Pathologic effects of trachoma caused by repeated infections
• Resemble virus in lack of cell wall - no peptidoglycan layer, low energy production (RNA/DNA ratio of elementary body is ~1), and both are intracellular parasites.
• Passes through a 0.45 micrometer filter
• Small, G - rods
• Treatment: tetracycline or erythromycin usually effective.
• Detection:  Cell culture (only certain cell lines):  McCoy, Buffalo green monkey kidney [BGM]); Direct specimen tests - PCR, ELISA, FA 

What are the specifics for Neisseria spp.?

• G -
• cocci (pairs are common)
• Non-motile, aerobic
• 2 main pathogens: N. meningitidis, and N. gonorrhoeae
• Easily transmitted
• Chocolate blood agar needed for isolation with blood and CSF cultures
• A selective medium (Thayer-Martin) is needed for cervical, nasal, or rectal cultures
• Need high CO₂

What are specifics for Neisseria meningitidis?

• Transmission: aerosols
• Virulence: Polysaccharide capsule, endotoxin (LOS - lipooligosaccharide) is overproduced and shed, IgA protease, transferrin
• Carrier state persists for days - months
• Causes mild fever, pharyngitis, septicemia with fever, shock because of LOS, extensive inflammation of brain and spinal cord, skin infections--> ecchymosis, and petechiae.
• Mortality ~85% if untreated
• Treatment: vigorous antibiotic therapy and chloramphenicol.  Rifampin can eliminate carrier state.  Penicillin can cross blood-brain barrier.
• Vaccine:  Available for some serogroups.
• Overall, mortality rate ~10%
• A deficiency in complement leads to a 5,000 to 10,00 fold increase in sensitivity to disease.  Extracellular killing is reduced.

What are some specifics for Neisseria gonorrhoeae?

• Causes gonorrhea
• Not resistant to environmental stress (light, heat, cold)
• Determinants of virulence:  multiple - pili (types 1 and 2 colonies more virulent than 3 and 4); LOS - had lipid A and endotoxin activity; Opa proteins - mediate binding to cells; Tbps - transferrin binding proteins; Lbp - lactoferrin binding proteins
• Disease in females:  bacteria are engulfed and transported to the base of the cell into the submucosal area-->epithelial destruction.  No fusion with lysosomes.  Causes PID. Skin  and joints may become involved-->arthritis
• Vaccine: none, antigenic variability (pili) is high
• Treatment:  Not penicillin (resistance common, and beta-lactamase bacteria more common), Quinolones effective