CLASS: 10:00 – 11:00 Scribe: Adam Baird
DATE: October 25, 2010 Proof:
PROFESSOR: Benjamin Gram-Negative Bacteria Page 8 of 8
I. MEDICAL MICROBIOLOGY: ENTEROBACTERIACEAE AND OTHER GRAM-NEGATIVE BACILLI [S1]
II. E. COLI O157:H7 SPINACH [S2]
a. 8/2006 – 10/2006
1. 26 states nationwide
2. 204 cases
3. 103 hospitalizations
4. 31 HUS
5. 3 Deaths
b. Since 1995, there have been 19 outbreaks of food borne E. coli O157:H7 presumably found in lettuce and leafy greens; found in cattle (the normal host of E. Coli O157:H7) one mile away and feral swine in the area.
c. Very infectious organism; can cause symptoms with very few organisms (as little as 1- 10 organisms).
III. SALMONELLA ON RESTAURANT TOMATOES [S3]
a. 9/14/2008 – 10/2/2008
1. 21 states nationwide
a. Highest reporting states: MA (50), CT (28), KY (19), MN (14), NH (14)
2. 183 cases
3. 22 hospitalizations
b. Salmonella enterica Typhimurium
c. Raw serrano and jalapeño peppers
d. Takes a higher dose of organisms to infect a host than E. Coli O157:H7
IV. CABURY CHOCOLATE: SALMONELLA ENTERICA MONTEVIDEO [S4]
a. 65 cases of salmonella enterica Montevideo in Great Britain
b. Salmonella can survive in chocolate for a long period of time
c. 6 months before cases occurred, Cadbury has a salmonella enterica Montevideo isolate from an ingredient
d. Leaking pipe at one of Cadbury’s main factories
e. Cadbury’s rational for not acting on the original isolate is that the numbers identified did not reach a recall “threshold”; the amount of possible salmonella in each chocolate was presumably low enough to not cause any harm. The waxy chocolate, though, acts as a protective barrier for the salmonella, so the salmonella wasn’t destroyed by stomach acid. Even a low dose of salmonella could cause symptoms.
V. COMMON CHARACTERISTICS OF ENTEROBACTERIACEAE [S5]
a. Both E. coli and salmonella are forms of enterobacteriaceae
b. Physiological characteristics of enterobacteriaceae:
1. Gram-negative rods
2. Rounded ends and straight parallel side
3. Colony morphology on sheep blood agar
a. ~2mm colonies (relatively large growth)
b. Usually dull gray
c. Other characteristics that might be present: hemoysis, mucoid, swarming, pigmented colonies, etc.
VI. PICTURE: SPUTUM GRAM STAIN [S6]
a. Notice the red gram-negative rods. (Blue would indicate gram-positive bacteria.)
b. However, just looking at this slide, it is not possible to identify which strain of enterobacteriaceae it is.
VII. PICTURE: E. COLI [S7]
a. Notice the nice, large colonies.
VIII. FIGURE: GRAM-POSITIVE CELL WALL VS. GRAM-NEGATIVE CELL WALL AND OUTER MEMBRANE [S8]
a. Left Figure: Gram-negative cell wall.
1. Relatively complex cell wall
2. Notice: Small peptidoglycan layer, outer membrane with lipopolysaccharide (the endotoxin, which is very important for gram-negative cells).
b. Right Figure: Gram-positive cell wall.
1. Relatively simple cell wall
2. Notice: Plasma membrane with outer peptidoglycan. Recall that the peptidoglycan is the structure that retains the blue coloration of gram staining, which is why gram-positive cells remain blue.
IX. FIGURE: NO TITLE [S9]
a. Gram-negative description of lipopolysaccharide function:
1. The lipopolysaccharide (endotoxin) stimulates a large number of the host’s cells (like granulocytes, macrophages, monocytes, vascular cells, B-cells, T-cells, and stem cells). The stimulation of these cells fires off defenses and triggers recruitment of additional mediators (like complement factors, clotting cascade, etc.) in the host in hopes to destroy the bacteria. This reaction, however, can also harm the host, leading to fever, hypotension, tachycardia, tachypnea, neutropenia, or potential multi-organ failure and death.
X. ENDOTOXIN RESISTANT MICE [S10]
a. Endotoxin resistant mice (lpsd): mice that spontaneously occurred during research; these mice, when given large doses of endotoxin, did not get sick (even though usually lethal doses up to 1 mg of lipopolysaccharide were given),
b. However, these endotoxin resistance mice (lpsd) were more susceptible to infection by salmonella (because their bodies didn’t recognize the infection and didn’t respond to it).
c. Two different spontaneous mutations seen:
1. One mutation seen in a single base pair mutation
2. One mutation seen in a large deletion
d. The endotoxin resistant mice (lpsd) mutation is “tlr4” (“toll-like receptor 4”, an endotoxin receptor gene that detects the lipopolysaccharide of gram-negative bacteria in mice)
XI. FIGURE: ANTIGENIC STRUCTURE OF ENTEROBACTERIACEAE [S11]
a. The antigenic structure is used to identify the organism (especially salmonella and shigella).
b. The lipopolysaccharide is made up of the o-antigen, the o-polysaccharide, and the lipid-a portion (which is common in all gram-negative bacteria). A host might be immune to one of the components of the lipopolysaccharide, but it won’t be immune to all of the components. The antigenic structure is very important.
XII. ANTIGENIC FORMULA OF ENTEROBACTERIACEAE [S12]
a. Escherichia coli typing
1. Example: E. Coli O157:H7 – The “O” portion of the disease results from the 157th o-antigen. The “H” portion of the disease results from the flagella (the motility organism of the bacteria).
2. Example: O75:K100:H5 – The “O” portion of the disease results from the 75th o-antigen. The “K” portion of the disease results from the capsule. The “H” portion of the disease results from the flagella.
b. Salmonella enterica serovar Typhi
1. O9, 12(Vi):Hd-
c. Salmonella enterica serovar Typhimrium
1. O1,4,512:Hi:1,2
d. There are 2400 different salmonella types.
e. Note: Dr. Benjamin did not discuss the antigenic formula for salmonella.
XIII. PHYSIOLOGY OF ENTEROBACTERIACEAE [S13]
a. Physiological characteristics of enterobacteriaceae:
1. Facultative anaerobes – They can grow aerobically or anaerobically, although they prefer to grow aerobically because they can get energy from sugar sources.
2. Ferment glucose
3. Oxidase negative
4. Nitrate positive
5. Catalase positive – For example, if peroxide drops were placed on enterobacteriaceae, it would be broken down to oxygen and hydrogen (displayed by bubbles).
6. Peritrichous flagella (multiple flagella) for motility
XIV. PICTURE: OXIDATION FERMENTATION TEST [S14]
a. Oxidation Fermentation Test determines if bacteria are facultative anaerobes.
b. Tubes contain glucose. If yellow = acid produced.
c. A plug can be added to the tube to exclude oxygen.
XV. IDENTIFICATION OF SPECIES AND GENERA [S15]
a. How are species and genera identified?
1. Biochemical Reactions
a. Fermentation of carbohydrates pH change
b. Decarboxylation of amino acids
c. Deamination of amino acids
2. Genetic Relatedness – Relatively expensive; might be more useful in future.
a. 16s RNA
b. DNA hybridization
XVI. PICTURE: MICROSCAN WALKAWAY [S16]
a. Microscan Walkaway holds 96 plates. Bacteria can be placed on plates in chamber overnight. The Microscan Walkaway will monitor each plate, recognize which chemical are broken down in each bacterial plate, compare it to a database, and record the name of the bacteria. It’s usually fairly accurate.
XVII. PICTURE: GRAM-NEGATIVE MICROSCAN PANEL [S17]
a. This is the plate that is used in the Microscan Walkaway.
b. Helps identify various drug resistances of bacteria
XVIII. ENTEROBACTERIACEAE HOST INTERACTIONS [S18]
a. Enterobacteriaceae interacts with:
1. Normal Flora – Which is nutritionally beneficial for humans. Normal flora provides competition and immunological stimulation in humans as well.
b. Enterobacteriaceae can lead to:
1. Opportunistic Infections (In Immunologically Compromised Individuals)
2. Infections (In Healthy Individuals)
a. Non-inflammatory diarrhea
b. Inflammatory invasive diarrhea
c. Invasive systemic infections (like typhoid and sepsis)
XIX. ESCHERICHIA COLI [S19]
a. E. Coli is normal flora found in mammals and birds
b. E. Coli is the most common enterobacteriaceae in the gut (although amount of E. Coli may fluctuate in the gut)
c. E. Coli coliform counts are used as an index of fecal pollution for food or water. If water, for example, has a high E. Coli coliform count, fecal matter may be the source of contamination, making it unsafe to drink.
d. Physiological Characteristics:
1. E. Coli is lactose positive.
2. E. Coli is indole positive.
XX. PICTURE: MACCONKEY AGAG PLATE [S20]
a. Picture showing lactose fermentation of E. Coli.
b. The E. Coli reacts with the lactose in the plate medium, producing acid. (Note the pink/purple color change of the active E. Coli and the lack of color change in the inactive E. Coli.)
XXI. PREDISPOSING FACTORS FOR URINARY TRACT INFECTIONS [S21]
a. E. Coli can cause urinary tract infections (URI). Predisposing factors for URI:
1. Female anatomy – Because females have a short urethra, E. Coli can easily get into the bladder.
2. Honeymoon cystitis in females – Cystitis is urinary bladder inflammation; can be caused by sexual intercourse, where the normal flora from female’s vagina and gut can pushed E. coli into the bladder.
3. Pregnancy and childbirth – Can cause bladder insufficiency and bladder problems, leading to cystitis.
4. Male prostatic hypertrophy – Enlarged prostate gland (typically in older males), which inhibits emptying of the bladder, allows bacteria to set up and potentially cause infection.
5. Catheterization or other mechanical manipulations
6. Failure to empty the bladder
XXII. OTHER OPPORTUNISTIC INFECTIONS [S22]
a. Other opportunistic infections that E. Coli causes:
1. Peritonitis (inflammation of the peritoneum) from ruptured gut
2. Septicemia (whole body inflammation) secondary to UTI or pneumonia
3. Wound infections
XXIII. ESCHERICHIA COLI [S23]
a. E. Coli can specifically cause diarrhea:
1. E. Coli causes an estimated 4% of diarrhea in US
2. Even higher percentages of diarrhea cause by E. Coli in developing countries
3. E. Coli causes diarrhea in 30-40% of visitors to Mexico (called “Travelers Diarrhea”)
XXIV. E. COLI ENTERIC PATHOGENS [S24]
a. E. Coli Pathogens:
1. Enteropathogenic E. Coli (EPEC) – Infantile diarrhea
2. Enteroaggregative E. Coli (EAEC) – Traveler’s diarrhea
3. Enterotoxigenic E. Coli (ETEC) – Traveler’s diarrhea
4. Enterohemorrhagic E. Coli (EHEC) – Hemolytic Uremic Syndrome
5. Enteroinvasive E. Coli – Bacillary dysentery
b. (It is not necessary to memorize these, but it’s important to be aware of the different types of E. Coli that can cause diarrhea)
XXV. KLEBSIELLA PNEUMONIAE [S25]
a. Another very common enterobacteriaceae.
b. Characteristics
1. Lactose positive
2. Does not cause diarrhea
3. Non-motile
4. Large capsule (77K antigens)
5. Beta-lactamase (resistant to ampicillin and carbenicillin; sensitive to cephalosporins)
XXVI. PICTURE: LACTOSE POSITIVE KLEBSIELLA SP [S26]
a. Klebsiella can cause large mucoid, slimy colonies.
b. Pink coloration (because it is lactose positive).
XXVII. PICTURE: KLEBSIELLA PNEUMONIAE PNEUMONIA [S27]
a. Gram stain of kelbsiella pneumoniae.
b. Notice the pink, gram-negative rods with large capsules (made of “sugary slime”)
XXVIII. PREDISPOSING FACTORS FOR ENTEROBACTERIACEAE PNEUMONIA [S28]
a. Predisposing factors for enterobacteriaceae pneumonia:
1. Hospitalization
2. Respirator
3. Increased age
4. Aspiration of oral secrections
5. Alcoholism
6. Diabetes mellitus
7. Chronic bronchopulmonary disease
XXIX. KLEBSIELLA PNEUMONIAE DISEASE [S29]
a. Klebsiella pneumoniae can cause various diseases:
1. Pneumonia (25-50% mortality rate; characteristics include thick non-purulent bloody sputum and necrosis and abscess formation)
2. Septicemia (Whole body inflammation)
3. Urinary Tract Infection (URI)
4. Meningitis
XXX. ENTEROBACTER CLOACAE E. AEROGENES [S30]
a. Physiological characteristics of enterobacter cloacae:
1. Lactose positive
2. Non-motile
3. UTI (often nosocomial, meaning that it results from hospitalization or other healthcare services)
4. Cephalosporinase (breaks down beta-lactam drugs; ampicillin and cephalothin resistant)
XXXI. PICTURE: DISC DIFFUSION ANTIMICROIAL SUSCEPTIBILITY TESTING [S31]
a. Sample organism spread all over the plate. Antibiotics discs are added to the plate. Each drug has a different zone size.
XXXII. PORTEUS MIRABILIS, P. VULAGRIS [S32]
a. Physiological characteristics of porteus mirabilis:
1. Urease positive
2. Highly motile (and will swarm on plate)
3. Bad smelling odor
4. H2S positive
b. Disease characteristics of porteus mirabilis:
1. Can lead to infections:
a. UTI (commonly aquired)
b. Wound infections
c. Pneumonia
d. Septicemia (whole body inflammation)
2. Tetracycline resistant (tetracycline is rarely used clinically though)
3. Ampicillin resistant
4. Cephalosporin resistant
XXXIII. PICTURE: UREASE TEST [S33]
a. Negative = no pH increase, no color change
b. Positive = ammonia is produced, pink color change
XXXIV. PICTURE: FOOD BORNE ILLNESS [S34]
XXXV. NORMAL ADULT [S35]
a. Normal adults have:
1. 9.0 liters of fluid entering the duodenum every day (saliva recirculation)
2. 1.5 liters crossing the ileocecal valve
3. 0.1 liters passed by the anal sphincter
b. Volume of diarrhea can help determine which level of the gut is infected.
XXXVI. OSMOTIC DIARRHEA [S36]
a. Osmotic diarrhea causes:
1. Increased intra-luminal osmotic pressure
2. Decreased absorption of fluid and solutes
3. Causes of osmotic diarrhea (may be caused by lactose intolerance, giardia lamblia)
4. Fasting stops osmotic diarrhea
XXXVII. SECRETORY DIARRHEA [S37]
a. Secretory diarrhea causes:
1. Malfunction of sodium absorption
2. Increased chlorine secretion
3. Mucosal cAMP increases as a result of the malfunction of sodium absorption and the subsequent increase of chlorine secretions
4. Bacterial toxins cause increases of cyclic nucleotides in mucosal ells
5. Fasting does not stop secretory diarrhea
XXXVIII. VIBRIO CHOLERAE [S38]
a. One of the most common causes of secretory diarrhea is vibrioi cholerae. (Currently, there is a cholera outbreak in Haiti.)
b. Physiological characteristics of vibrio cholerae:
1. Not an enterobacteriaceae.
2. Curved rod.
3. Oxidase positive.
c. Disease characteristics of vibrio cholerae:
1. 1-5 day incubation period
2. Profuse water diarrhea (about 20L a day)
3. Spread by contaminated water
4. Stomach acid offers protection (high dose is needed for infection; infection dose is 107 FCU)
5. Cholera toxin
a. B subunit binds intestinal cells
b. A subunit irreversibly activates adenyl cyclase
c. Results in water and electrolyte secretion
d. Treatment for vibrio cholerae:
1. Fluid rehydration (oral)