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Family Mycobacteriaceae
Mycobacterium
Mycobacterium
1) Acid-fast slightly bent rods (neither Gram positive or Gram negative).

2) Obligate Aerobes.

3) Non-spore forming.

4) Non-Motile.

Cell walls rich in lipid content. Granular appearance after staining.
Mycobacterium Consist of
1) Slow growers.

2) Rapid growers.

3) Strains that have special growth requirements.
Slow Growers
Visible colonies after 7 days or more.

1) M. tuberculosis: Have the ability to survive in macrophages. Cell wall damages tissues. Non-pigmented colonies.

2) M. bovis

3) M.avium-intracellulare
Rapid Growers
Visible colonies in less than 7 days.

1) M. smegmatis
Organisms with Special Growth Requirements
Usually cannot be cultivated in vitro. They are cultivated in mouse foot pads and in the armadillo.

1) M. leprae:
Antibiotic Resistance of Tuberculosis
1) MDR TB: Multi-drug resistant form of M. tuberculosis. Resistant to 2 or more primary drugs (isoniazid and rifampin).

2) XDR TB: Extensively drug resistant form of M. tuberculosis. Resistant to isoniazid, rifampin, fluroquinolone and one of the 3 injectable drugs (kanamycin).

3) TDR: Total Drug Resistance.
Who is at Risk for Tuberculosis
Persons in close contact with people with TB. Persons with HIV.
Mycobacterium tuberculosis
Grow in long strands called serpentine cords due to the cord factor (trehalose dimycolate) which correlate to the virulence of the strain. Non-virulent strains do not produce the cord factor. Lesions seen with the disease are dependent on the presence of the microbe and the host’s response.

No exotoxins.
No endotoxins in their cell wall.
No histolytic enzymes.
Identification of M. tuberculosis
1) Pigment: Negative
2) Niacin (vitamin B3) free Niacin to Niacin Ribonucleotide: Positive
3) Nitrate Reduction: Positive
4) Tween 80 Hydrolysis: Negative
5) 68C for 20 minutes Catalase Activity: Negative
6) Urease Activity: Positive
7) Growth in Egg-Based Media Containing 5% NaCl: Negative initially. Positive in 4-6 weeks
Characteristics of Mycobacterium tuberculosis
1) Slow grower (18 hour doubling time).
2) Habitat: Man and primates.
3) Cell walls contain several complex lipids:
1) Mycolic Acids: C78-C90
2) Wax D: High molecular weight peptidoglycolipid. Stimulates antibody response.
3) Phosphatides: Play a role in necrosis.
2 types of Lesions of Mycobacterium tuberculosis
1) Exudative Lesion: Acute Inflammatory Response. At the primary site.

2) Granulomatous Lesion
Granulomatous Lesion
Macrophages become giant cells with live TB inside. Aggregations of macrophages that are transformed into epithelium-like cells.

The granuloma is referred to as a tubercle. This is characterized by central caseous necrosis, which is rare in other granulomatous diseases.

➢ Non-caseating Tubercle (Granuloma Prototype): A focus of epithelial cells rimmed by fibroblasts, lymphocytes and giant cells.
➢ Caseating Tubercle: Central granular debris, loss of cellular detail, acid-fast bacilli.
Pathogenesis of M. tuberculosis:
Pathogenicity due to its ability to escape killing by macrophages (Listeria can also live inside of macrophages) and induce a delayed type of hypersensitivity.
Pathogenic Factors of M. tuberculosis
1) Cord factor: Surface glycolipid that prevents macrophage activation.

2) Sulfatides: Surface glycopeptides that prevent activation of mΦ

3) LAM: Heteropolysaccharide. Prevents macrophage activation.

4) Heat shock protein

5) Cell-surface antigen that prevents macrophage killing

6) Granulomatous Response: Develop tubercles.
Primary Tuberculosis
Begins with inhalation of the strain and ends with a T-cell mediated response that induces hypersensitivity to the microbe. The microbe is phagocytized by macrophages and transported to lymph nodes. The macrophages are unable to destroy the microbe, which then multiples and spreads through the blood and other parts of the lung. The primary lesion of TB usually occurs in the lungs, therefore the exudative lesion and the lymph nodes it drains into are together called a Ghon Complex.
Secondary Tuberculosis
Some individuals become re-infected (reactivate-dormant disease). Granulomas of secondary tuberculosis occur in the lungs but may disseminate to the kidneys. These granulomas fail to contain the spread of TB.

Special feature of secondary TB are caesous necrosis (cheese-like consistency) and cavities that rupture into blood vessels resulting in release.
Complications of TB
1: Erosion of blood vessels.

2: Miliary tuberculosis is characterized by disseminated lesions in all organs. The host fails to mount a CMI response.

3: Reactivation tuberculosis seen in well-oxygenated sites of the kidney, brain and bone. Usually in immune-compromised patients.
Transmission of M. tuberculosis
1) Exogenous

2) Endogenous
Enxogenous TB Transmission
Transmission of the organism in children and adults by droplet nuclei, from a case resulting in primary pulmonary or first infection type of tuberculosis.

Crowded conditions and impaired host- resistance enhance the spread.
Endogenous TB Transmission
Activation of the disease occurs due to a waning acquired resistance and reactivation of quiescent primary foci within the respiratory tract.

Factors that influence reactivation: malnutrition, AIDS, alcoholism, and diabetes.
Immunity to TB
After recovery from the primary infection, resistance to the organism is acquired through cellular immunity. Circulating antibodies form but they play no role in resistance.
Hypersensitivity to TB
Tuberculin Skin Test: Prior infection detected by a positive tuberculin skin test, which due to a delayed hypersensitivity reaction (PPD) is used.

PPD is a purified cell wall protein from M. tuberculosis. Positive result indicates infection of the microbe but not necessarily active disease.
Treatment of TB
1) Rifampin: Inhibits RNA synthesis.

2) Ethambutol: Interferes with RNA synthesis and inhibits mycolic acid.

3) Isoniazid: Inhibits mycolic acid synthesis.
TB Vaccine
BCG Vaccine. An avirulent strain of M.bovis called BCG has been used as a TB vaccine. Sometimes works, sometimes doesn't.
Acid-Fast Stain
Due to the cell wall structure of the mycobacteria. Acid fast is a term used to describe bacteria that resist decolorization with acidified alcohol once they are stained with carbolfuchsin. Acid-fastness is due to mycobacteria’s thick waxy wall. The capsule is softened with heat to allow the carbolfuchsin to penetrate it. Mycobacteria are red and background is blue.
4 Mycobacterium Groups
Group 1: Photochromogens

Group 2: Scotochromogens

Group 3: Nonphotochromogens

Group 4: Rapid Growers
Group 1: Photochromogens
1) M. kansasii:

Slow growth rate.

Pigment produced in the light +

Pigment produced in the dark -

Colonies between fully rough and fully smooth.

Long, thick bacilli. Seen in people that are 50 and olde and in miners/painters. Causes persistent sub-acute cough and low-grade fever. Chronic infection in the lungs and skin lesions are seen. Light TB. AIDS- associated (#2 after M. tuberculosis). Treat with standard anti-TB drugs.
Group 2: Scotochromogens
1) M. scrofulaceum:

Slow growth rate.

Pigmentation in the light +

Pigmentation in the dark +

Colonies are smooth and buttery in consistency.
Infection in children (18 months-7 years). Associated with a break in the oral mucosal membrane (cutting molar teeth). Treat with surgical drainage. No drugs.
Group 3: Nonphotochromogens
1) M. avium-intracellulare (Battey's bacillus)

Slow growth rate

Pigmentation in the light –

Pigmentation in the dark –

3 colonial variants, 2 smooth and 1 rough

Most common antimicrobial infection associated with AIDS after TB. Pulmonary. Seen in 15-40% of AIDS patients

Treat with regular TB drugs. Known as Lady Windermere Syndrome. Seen in older ladies.
Group 4: Rapid Growers
1) M. fortuitum-chelonae

Rapid growers

Pigmentation in the light –

Pigmentation in the dark –

Colonies appear smooth.

Found in water supplies. Contaminates it and is a problem in reagents. Wash solutions in hospitals also contaminated by it.

3) M. Smegmatis: Also a rapid growing bacterium.
Mycobacterium leprae
Causes Leprosy (Hansen’s Bacillus). The microbe is harbored in the nasal secretions, ulcerative lesions and sputum of patients. Obligate intracellular parasite.

1) Unable to grow in the laboratory, on artificial media or in cell culture.

2) The microbe is grown in the mouse foot pad or the 9 banded armadillo.

3) Humans are the natural host. Grows normally at temperatures lower than body temperature, skin, superficial nerves, nose and ears. Therefore an obligate parasite of humans and the armadillo.
Transmission of M. leprae
Exogenous. Portals of entry are respiratory and cutaneous. Sometimes multiple cases appear in one family suggesting genetic linkage. Disease progression is slow (1 year before symptoms arise). 95% of people expose don’t develop disease.
Pathogenesis of M. leprae
This bacterium replicates intracellularly in skin histocytes and nerve cells. 3 intermediate stages.

2 forms of leprosy:
1) Tuberculoid (Paucibacilliary) Leprosy
2) Lepromatous (Multibacillary) Leprosy
Tuberculoid (Paucibacilliary) Leprosy
Cell-mediated response to the bacterium limits its growth. Lepromin skin-test positive. Mild severity. Skin lesions and sensation loss due to invasion of the peripheral sensory nerves. Can spontaneously recover. Infected patient can still mount good CMI (cell mediate immunity) and the infection is localized. Non-infectious. Good recovery odds. Non-Acid Fast. Individuals exhibit large flattened patches with raised or elevated red edges and dry pale, hairless centers on any body surface.
Lepromatous (Multibacillary) Leprosy
Cell-mediated response is poor. Skin and mucous membranes contain large numbers of bacteria. Lepromin skin-test negative. Extremely severe. Without treatment, patient dies. Has Lion-like face. Nasal and cartilage damage which leads to blindness. Can’t mount CMI. Usually infectious. Can be cured. Acid Fast. Individuals exhibit extensive skin involvement, with thickening of looser skin parts of the lips, forehead and ears. Extensive penetration of the microbe in the body may cause severe body damage resulting in the loss of bones, fingers and toes.
Tuberculoid Leprosy
M. leprae multiplies at the site of entry (skin) and invades/colonizes Schwann cells.

The microbe induces T helper lymphocytes, epithelioid cell, and giant cell infiltration of the skin. Strong CMI accounts for the absence of M.leprae recovered within lesions. Key clinicals are the macule at the cutaneous entry and loss of pain sensation.
Lepromatous Leprosy
The microbe proliferates within the macrophages (foam cells) at the entry site. The organism also grows in the epithelial tissues of the face and ear lobes. Suppressor T cells are numerous, but epitheloid and giant cells are rare or absent. CMI is impaired, large numbers of the organisms appear in the macrophages. Papules appear at the entry site with marked folding of the skin.
Immunity to M. leprae
The pathogenesis of the microbe relates to the patients CMI status.
Laboratory Diagnosis of M. leprae
Demonstration of acid-fast bacilli within phagocytic foam cells observed in lepromatous leprosy, but difficult in the tuberculoid type. M. leprae can survive for years in nasal secretions, dried sputum and relatively resistant to disinfecting agents (because some are acid fast). Incubation period in humans 3-5 years.
Treatment of M. leprae
Treatment:
Dapsone: Given for 2 years or lifetime. Combine Dapsone, rifampin and clofazimine if resistance develops.

Tuberculoid: 6 months to cure.
Leprotomous: 2 years to cure

1) Dapsone: Sulfonamide. Competes against PADA. Prevents folic acid synthesis in the microbe.
2) Rifampin: Inhibits bacterial RNA synthesis. Binds to DNA-dependent RNA polymerase. Blocks transcription. Most potent.
3) Clofazimine: Binds to mycobacterial DNA.
Characteristics of M. leprae
1) Microscopic Morphology: Acid-fast, non-motile, non-encapsulated rods. They are straight or curved.

2) Antigenic Structure: Components not defined.

3) Extracellular Products: No virulence factors.
Mycobacterium avium subsp. paratuberculosis
Map causes a Crohn’s-like gastrointestinal disease in cattle (Johne’s disease) and can infect humans. Symptoms include diarrhea, weight loss and periods of remission. Treating patients with drugs that target mycobacteria help these patients. Survives pasteurization. 25 % of Crohn’s patients have a mutation is NO2D2 gene which codes for a receptor that recognizes invading bacteria and triggers an immune response.
Lab Tests to Identify Mycobacteria
1) Pigmentation: Photoreactivity. Color develops upon incubation in the dark (scotochromogenic) or is stimulated only after exposure to light (photochromogenic).
Family Enterobacteriaceae
Includes 31 genera, many tribes and 139 species.

Contains: 4 species that result in diarrhea and intestinal infections, 7 species associated with nosocomial infections and 40 species that occur in clinical specimens.
The Enterobacteriaceae Consist of 7 Tribes
Tribe I: Eschericheae
Tribe II: Edwardsielleae
Tribe III: Salmonelleae
Tribe IV: Citrobactereae
Tribe V: Klebsielleae (Butanediol)
Tribe VI: Proteeae
Tribe VII: Yersinieae (Plage bacillus Yersinia perstis)
The Enterobacteriaceae
1) Small gram-negative rods.
2) Motile via peritrichious flagella or non-motile.
3) Non-spore forming.
4) Grow on peptone-based media without additional NaCl.
5) Grow well on MacConkey Agar.
6) Facultative anaerobes.
7) Ferment glucose often with gas production.
8) Catalase +
9) Oxidase –
10) Reduce nitrate to nitrite.
11) Hemolysis: Variable.
12) Carbohydrate Utilization:
1) Mixed Acid Fermentation: Glucose is converted into acetic, lactic and formic acids. Basis of Methyl Red Test (pH below 4.4).

2) Butanediol Fermentation: Results in the production of acetyl-methyl carbinol (acetoin). Basis of the Voges Proskauer Test (Acidic to Neutral).
Habitats of the Enterobacteriaceae
Found in nature, plants, soil, water and some can have a narrow niche. Lives in the GI tracts of: humans, animals, fish and insects.

1) Salmonella typhi: Only in humans.
2) Klebsiella pneumoniae: Found in nature and causes intestinal, urinary or respiratory infections.
IMViC (Indole, Methyl Red, Voges-Proskauer, Citrate)
E. coli + + - -
Enterobacter aerogenes - - + +
Urease Production
Proteus, Morganella and Providencia are positive. E. coli is negative.
Phenylalanine Deaminase Production
Proteus, Morganella and Providencia are positive. E. coli is negative.
Human Disease of the Enterobacteriaceae
1) Abscesses

2) Pneumonia

3) Meningitis

4) Septicemia Wounds

5) UTIs

6) Intestinal Infections

7) Endotoxin Shock: Causes fever, leucopenia, capillary hemorrhage, hypotension, circulatory collapse. Assayed by the limulus lysate assay, which is a reagent prepared from the amebocytes of the horseshoe crab (Limulus polyphemus).
Virulence of the Enterobacteriaceae
80% of all gram negatives recovered, 50% of all clinically significant isolates and 70% of all UTI’s.

1) Shigella / Salmonella: Diarrheal dysenteric illness.
2) K. pneumoniae: Pneumonia with ”brick red” sputum. Friedlander’s Bacillus.
Endotoxin Produced by the Enterobacteriaceae
Pharmacologically active lipopolysaccharide that is part of the Gram negative cell wall. Consists of 3 layers: an outer variable carbohydrate portion that determines O-antigen specificity, middle core polysaccharide that is structurally similar among species and central highly conserved lipid moiety (lipid A, toxic moiety). No treatment against this endotoxin.
Antigenic Characterization
Way to differentiate the Enterobacteriaceae.

1) Flagellar Antigens (H antigens): Heat labile. Generate antibody response.

2) Capsular Antigens (K antigens): K antigens in Salmonella are “Vi” for virulence. K88 antigen in E.coli is a pili antigen. Heat labile (affected). Can be protein or polysaccharide.

3) Somatic Antigens (O antigens): Polysaccharide antigens that compose the outer region of LPS. Heat stable. IgM antibody response.
Media for the Detection of Carbohydrate Utilization
Kligler Iron Agar (KIA) and Triple Sugar Iron Agar (TSI): Top is aerobic, butt is anaerobic.

1) KIA: Lactose and Glucose. Glucose and lactose evenly distributed throughout the medium. Lactose is present 10:1 compared to glucose. Ferrous sulfate is there to detect H2S production.

2) TSI: Dextrose (glucose), lactose, sucrose. Sucrose to Glucose ratio is 10:1. Lactose to glucose is also 10:1.
Primary Isolation Media
Nonselective: Use Blood agar.

Selective or Differential: Use MacConkey or Eosin Methylene Blue Agar.
MacConkey Agar
Differential plating for selection and recovery of Enterobacteriaceae. Bile salts and crystal violet inhibit gram-positive bacteria. Lactose is the sole carbohydrate present.

1) Strong lactose fermenters produce red colonies (Escherichia, Klebsiella, Enterobacter).

2) Weak lactose fermenters produce colorless or slightly pink colonies (Citrobacter, Providencia, Serratia and Hafnia).

3) Species of Proteus, Edwardsiella, Salmonella, Shigella appear colorless.
Eosin Methylene Blue Agar
Differentiate between lactose fermenters and non lactose fermenters.

1) Strong lactose fermenters form green black colonies with a metallic sheen (E. coli).

2) Weak fermenters give purple colonies (Serratia, Klebsiella).

3) Sucrose fermenters and non-lactose fermenters produce purple to black colonies (Shigella, Shigella-salmonella and citrobacteer).
5
5
Hektoen Agar
Highly selective for Enterobacteriaceae. Good for enhanced recovery of Salmonella and Shigella.

1) Rapid Lactose Fermenters: Bright orange and pink (E. coli).

2) Salmonella: Blue green black centers.

3) Shigella: More green colonies.
XLD Agar
Xylose Lysine Deoxycholate Agar. Less inhibitory to growth than HE agar and is designed to detect shigella in feces.
Enrichment Broths for Recovery of Enterobacteriaceae
1) Selenite Broth: Used for the recovery of Salmonella and Shigella from feces or sewage. This medium will maintain the predominant E. coli in lag phase and allow the Salmonella and Shigella to grow out. Sodium selenite inhibits E. coli.
Tribe Escherichieae
Genera in this tribe are:

1) Escherichia coli (Most common isolate in the clinical lab)

2) E. blatte

3) E. fergusonii

4) E. hermannii

5) E. vulneris

6) Shigella (16S related, but not biochemically).
Mixed Acid Fermentation of Escherichieae
Positive. Glucose converted into products.

Acid Neutral
4 1
CO2 H2
1 1
Virulence Factors of E. coli
Capsules, pili, siderophores, endotoxin (most potent factor), toxins, enterotoxins, colonization factors antigens.
Important Structures of E. coli
1) Fimbriae (pili): Type 1 bind to urinary slime (Hemagglutination activity). Allow for adherence to the intestinal wall

2) LPS “O” Antigen: 170 varieties

3) Flagellar "H" Antigen: 50 varieties

4) Capsular "K" Antigen: 90 varieties
Serotypes of E. coli
The genes in the different E. coli strains are carried on mobile genetic elements.

1) Plasmids carry ETEC toxin genes.

2) Transposons carry ETEC heat stable toxin ST.

3) Bacteriophages carry EHEC Shiga toxin.
Virotypes of E. coli
Virulence-associated classification scheme. There are over 1000 different antigenic types of E. coli.

1) E. coli K1: Causes neonatal meningitis.

2) E. coli O55 and O111: Cause neonatal diarrhea.

3) E coli MNEC: Spread from meninges due to sepsis. Gain access to CNS via membrane-bound vacuoles in endothelial cells.
Intestinal Tract Infection by E. coli
2 steps: Attachment by pili and then enterotoxin production.

1) Pili allow the cells to attach to the cells of the jejunum and ileum.

2) Enterotoxins (Plasmid Encoded): Act on the cells of the jejunum and ileum and not the cells of the colon because they lack receptors.
Enterotoxins of E. coli
LT (heat labile) and ST (heat stable).
LT Enterotoxin
High molecular weight. Heat-labile toxin acts by catalyzing the addition of ADP-ribose to the coupling protein that stimulates the cyclase. The resultant increase in intracellular cAMP concentration causes an outpouring of fluid (K and Cl). Test is 30 minutes at 100C. Impacts synthesis and release of prostaglandin. GMI.
A) LT1: Shares 75% amino acid with cholera toxin. 5 ‘B’ subunits, 1 A subunit. Binds same receptor as cholera toxin (GM1). B subunit of LT binds to host GM1. B subunit gets internalized. Inside the cell, A subunit binds to a protein that controls cAMP production in the intestinal cells, the rise in cAMP causes Cl ion secretion to increase, uptake of NaCl is inhibited. GS is a protein that controls cAMP production. Inactivation of GS causes an increase in cAMP. Activates a chloride channel (CFTR) and chloride secretion is increased. Uptake of NaCl is inhibited. Result is loss of water flow control by the intestinal cells (diarrhea).
ST Enterotoxin
ST: Low molecular weight, heat stable toxin, which stimulates guanylate cyclase. Family of small peptide toxins. 2 groups:

1) Methanol Soluble (ST): Receptor for STA is host-cell guanolate cyclase (regulates GMP) and leads to same fluid loss. STB: Mild and watery diarrhea.

2) Methanol Insoluble
Systemic Infections by E. coli
Two structural components play a role here:

1) Capsule: Interferes with phagocytosis. Implied with infant meningitis.

2) Endotoxin: Results in fever, hypotension, and disseminated intravascular coagulation.
Colicins
Coliform bacteria produce a cell-free substance that inhibits other bacteria. The producing strain is immune to its own colicin, but sensitive to other colicins (such as bacteriocin). Their modes of actions include: Membrane depolarization, DNA endonuclease, and ribosome inactivation.

Colicin E1 arrests macromolecular synthesis and is made by various strains of E. coli. Many are encoded on plasmids. Some plasmid sequences were utilized for vector development with rDNA. E2 inhibits DNA synthesis.
Gastroenteritis Caused by E.coli
1) Enterotoxigenic E. coli (ETEC)
2) Enteropathogenic E. coli (EPEC)
3) Enteroinvasive E. coli (EIEC)
4) Enterohemorrhagic E. coli (EHEC)
5) Enteroaggregative E. coli (EAEC)
6) Diffusely adherent E. coli (DAEC)
ETEC
Traveler’s Diarrhea. LT and ST do not damage mucosal epithelium. Strains adhere to the small intestine mucosa, but do not invade. Instead produce toxins.

Contaminated food and water. Disease symptoms: Profuse watery diarrhea, mild abdominal cramps, vomiting. Seen in all ages. Transmission by the fecal-oral route, require 106-109 organisms. Note surface attachment of the cells to the intestinal epithelial cells is required for toxin production. E. coli strains, 06, 08, 015, 020. In infants disease is called “cholera infantum”. Virulence factor is the enterotoxin. No histologic change in host cells. Uncommon in the US. Adhesins pili type 1&2.
EPEC
Infantile diarrhea. Bacterial cells attached to the epithelial cells in localized micro-colonies causes attaching and effacing lesions. Nosocomially acquired.

Disease symptoms, in infants, are a low-grade fever, malaise, vomiting, diarrhea, large amount of mucous with no gross blood. Occurs in infants. Transmission via nurse’s hands. E. coli serogroups 055, 086, 0111, 0119. When they adhere, dramatic alterations in the structure of host mucosal cells. Microvilli are elongated (attaching and affixing). No exotoxins. Adhere to mammalian cells. Bacteria attach to host cell by bundle forming pili. This attachment causes the host cell tyrosine kinase to be activated, Ca+ levels increase.
EIEC
Disease is dysentery-like diarrhea. Bacteria invade epithelial cells. Usually from contaminated food and water. Disease symptoms include dysentery, fever and colitis. Urgency and painful straining at stools, Blood mucous and WBC’s in the stool.

Affects all ages Most common strains are E. coli 028ac, 029, 0112ac, 0124. Virulence mechanism is invasiveness.
EHEC
Hemolytic-Uremic Syndrome or Hetero Hemolytic E coli. Can also be called STEC (sero toxigenic E. coli) or VTEC (Viro toxigenic E. coli). Elaboration of cytotoxins (Stx 1). Stx 1 is a verocytotoxin, similar to the S. dysenteriae serotype 1 toxin (Shiga toxin).

Contaminated food and water. Disease symptoms are bloody diarrhea without WBC’s and no fever. Abdominal pain is common. May progress to HUS (red cell lysis, kidney failure, death). All ages affected. E. coli 0157:H7 cause Hemorrhagic colitis, abdominal cramps, watery diarrhea, hemorrhagic discharge resembling G.I. tract bleeding. This strain expresses the 157th (O) antigen and the 7th (H) antigen.
Virulence mechanism is the cytotoxin. Transmission by undercooked hamburger and lettuce. Adhesions are similar to the EPEC strains. Toxin is the Shiga toxin (Stx). Stx 1 and 2 (associated with pus) receptors for this toxin are found on kidney cells as well as intestinal cells. Thus, involvement in acute kidney failure. The gene (Stx) is encoded on a temperate bacteriophage (lysogenic). STx1 is like Shuga toxin.
EAEC
Bacterial cells adhere to epithelial cells in a pattern resembling a pile of stacked bricks and produce an ST-like toxin. Non-invasive. No histological change in intestinal cells. Don’t adhere uniformly. Disease symptoms are watery diarrhea, low-grade fever, vomiting, dehydration, and less commonly abdominal pain. Virulence mechanism is adherence. Recovered from children with chronic diarrhea.
DAEC
Cells adhere to epithelial cells in a diffuse pattern and carry a gene encoding a surface fimbria F1845. Patients have watery diarrhea without blood or fecal WBC’s.
E. coli 0157:H7
Lives in the soil. The strain is also observed in pigs, sheep, deer and rabbits. Occurs in water since the microbe contaminates water from feces. 0157:H7 is the prototype serotype with others including: 0111:NM, 0145:NM. NM means non motile. ID50 low.
Detection of 0157:H7
Possible only in the acute phase of the illness. Can't be detected before 5-7 days. Direct plating on sorbitol MacConkey agar (SMAC) or cefixime-SMAC. Confirm with 0157:H7 antisera. E. coli strains are all sorbitol + except for 0157:H7, which is sorbitol negative.
Destruction of E. coli 0157:H7
Thorough cooking of ground beef kills the strain. Cook spinach at 160C for 15 seconds.
Treatment of 0157:H7
Certain antibiotics such as fluoroquinolones and trimethoprim make the disease caused by this strain worse. The listed antibiotics cause DNA damage as a side effect of their primary action. Vaccines to Toxins: Works. No vaccines, no antibiotics.
Recovery from 0157:H7
Diarrhea often bloody and accompanied by abdominal cramps. Fever may be absent or mild. Adults can recover in about 1 week. The young and elderly can develop HUS.
Treatment E. coli Infections
1) UTI: Sulfonamides and ampicillin

2) Sepsis: Gentamicin

3) Neonatal Meningititis: Ampicillin and gentamicin

4) Diarrheal diseases: Antibiotics not given, however trimethoprim-sulfamethoxazole may shorten the term.
Genus Escherichia
1) Straight gram-negative rods.
2) Motile by peritrichious flagella or non-motile.
3) Facultatively anaerobic.
4) Optimal temp 37C (mesophillic).
5) Oxidase negative.
6) Citrate negative.
7) H2S negative.
8) Indole +
9) VP –
10) Urease –
11) Lysine, Ornithine decarb +
12) Gas from glucose +
13) KIA: Acid slant/acid deep.
Other Escherichia Strains
1) E. hermannii: Found in human wounds, sputum, feces, not believed to cause Infection. Yellow pigmented.

2) E. fergusonii: Recovered from blood, gall bladder, urine, feces.
Diarrhea
Higher volume and frequency of bowel movements than normal. It results from insults to the GI tract by 2 mechanisms:

1) Invasion and destruction of mucosal epithelial cells with subsequent loss of re-absorptive function.

2) Production of an exotoxin that mediates fluid secretion from intestinal epithelial cells and thus is called an enterotoxin.
Genus Shigella
They cause classic Bacillary Dysentery, severe cramping, abdominal pain, and diarrhea with blood and mucous. The bacteria invade the mucosal cells of the distal ileum and colon but seldom beyond. Local inflammation and ulceration occurs, but the bacteria rarely penetrate the wall or enter the blood stream. One of the most commonly recognized bacterial diarrhea in the U.S. As few as 200 organisms are able to cause disease.
Identification of Shigella
1) Gram-negative rod
2) No gas from glucose fermentation
3) H2S negative
4) Non-motile
5) Lactose Negative: Colonies colorless on MacConkey’s.
6) TSI: Alkaline slant, acid butt.
Virulence Factors of Shigella
1) The “O” antigens divide the genus into 4 groups, A, B, C, D.

2) Heat labile Exotoxin (Shiga Neurotoxin): Produced by S. dysenteriae. Inhibits protein synthesis, causes paralysis and death when injected into mice.
– Consists of 2 chains. A subunit responsible for biologic activity, and 5 B subunits responsible for binding the toxin to susceptible cells. Subunit A inhibits protein synthesis and alters epithelial cells ability to transport electrolytes, this leads to fluid accumulation in the intestine. Subunit A also mediates neurotoxic activity, which results in convulsions-coma.

3) Extrachromosomal element associated with invasiveness. A series of genes regulates the production/secretion of invasins that localize on the Shigella outer membrane. These mediate inter-intracellular spread of the organism.
Subgroups of Shigella
1) Group A: S. dysenteriae. 15 serotypes
2) Group B: S. flexneri. 8 serotypes
3) Group C: S. boydii. 19 serotypes
4) Group D: S. sonnei. 1 serotype
Shigellosis
A human disease transmitted from person to person. Carriers are asymptomatic.
5 F’s: Fingers, flies, food, fomites and feces. Principle factors in an outbreak. Food-borne out number water borne 2:1.Outbreaks occur, day care centers, hospitals, cruise ships. Incubation period 1-4 days, symptoms fever abdominal cramps followed by diarrhea. Watery first then blood and mucous. Disease varies from mild to severe, depends on the
patient’s age and strain involved.
− S. dysenteriae: Most severe usually seen in the U.S. in travelers from abroad. Toxin production. Usually see severe diarrhea, convulsions, coma, intestinal perforation and hemorrhage.
− S. sonnei: Mild form of the disease isolated from 80 % of all individuals with shigellosis in the U.S.
Prevention and Control of Shigellosis
Isolate and treat active cases and carriers. Emphasize proper hygiene, sewage disposal and treatment, chlorinated water supply and warn travelers to be vigilant. Treat with fluid replacement and ampicillin.
Tribe Edwardsielleae
One genus and three species. Usually found in nature, fresh water and fish. Makes lots of Hydrogen Sulfide. Wound infections from trauma in water.
− E. tarda
− E. hoshinae
− E. ictaluri
Tribe III Salmonelleae
1 genus (Salmonella). Salmonella are confirmed with a polyclonal antisera test. Three antigenic markers provide up to 2,500 serotypes. Can be categorized into 3 species.
3 Antigenic Markers of Salmonelleae
1) “O” antigens of the LPS.

2) “H” antigens that are flagella.

3) “Vi” antigens that are capsular or associated with virulence.
3 Species of Salmonella
S. bongori: Animal Bacteria

S. enterica: Human Bacteria

S. typhi: Human Bacteria. Humans are its only reservoir.
S. typhi
1) Mucate and Acetate negative.

2) Citrate negative

3) H2S positive

4) Non-lactose

5) Motility postivie

6) Urease negative.

7) Indole Negative

8) Methyl Red Positive

9) Vouges Proskauer Negative

10) Citrate Negative
Why S. typhi is so Invasive
Due to the production of the Vi antigen. This is a capsular polysaccharide composed of N-acetylglucosamine uronic acid. S. typhi is able to survive in phagocytes. The strain also appears to wrap its chromosomes to protect it from oxidative damage in the phagolysosome, a DNA supercoiling effect that E.coli strains lack and thus can not survive in the phagocyte.
Isolation of Salmonella
1) MacConkey Agar: Colorless.

2) Hektoen Agar: Black due to H2S production.

3) Enrichment Broths (Selenite or Tetrathionate).
Incidence and Source of Salmonellosis
Human infections caused by ingestion of food, water or milk contaminated by human or animal excreta. Salmonella primary pathogens lower animals. This is usually the source for non-typhoidal salmonellosis in humans.

− Salmonella in Eggs: Contaminate the outer surface of the egg and get inside via hairline cracks.
Pathogenesis of Salmonella
3 types of infections:

1) Enterocolitis
2) Enteric Fevers (Typhoid Fever)
3) Septicemia
Enterocolitis
The bacteria penetrate the mucosal cells, results in inflammation and diarrhea. 100,000 bacteria of Salmonella are required to produce infection. Most frequent salmonella infection. Self-limiting disease that resolves without treatment. Use fluid and electrolyte replacement. Antibiotic administration does not shorten the illness.
Enteric Fevers (Typhoid Fever)
Infection begins in small intestine with few GI symptoms. Cells enter draining lymph nodes where the bacterium multiplies and spreads to the phagocytes of the liver, gallbladder and spleen. This survival in the phagocytes is an unusual feature of the disease. This disease is bimodal. Caused by S. typhi. A “Carrier State” can exist in which patients will shed the microbe in their feces, for up to 1 year. “Rose Spots” can be seen. Treatment, ampicillin, chloramphenicol.

Has 2 phase. 1st is fever and constipation. Blood culture is +, stool is -. 2nd phase is diarhetic phase: Blood is -, stool is +. Serodiagnosis: “Widal Reaction”is used to diagnose typhoid fever. Measures agglutinating host antibodies to “O” and “H” antigens of S.typhi when the strain cannot be isolated.
Septicemia
5-10% of infections. Patient usually has an underlying disease (cancer) or it’s a child with enterocolitis. Blood culture is +. No GI symptoms.
Disease Process of Salmonellosis
Ingestion of >105 bacteria needed to start an infection. Bacteria invade mucosal cells and multiply in the macrophages. Inflammatory response to bacteria in tissue causes pain and leads to the release of prostaglandins. The prostaglandins increase cAMP levels in mucosal cells. Na+ uptake inhibited and Cl- secretion increased. Loss of water by mucosal cells and diarrhea.
Adherence and Invasion seen with Salmonellosis
1) Adherence: S. typhimurium produce a number of adhesions: Type 1 fimbriae: Plasmid encoded fimbriae, long polar fimbriae and thin aggregative fimbriae. The long polar fimbriae mediate attachment to the Peyer’s patches.

2) Membrane ruffling: Trigger mammalian cells to form pseudopods that engulf the bacteria. The bacteria get inside a endocytic vesicle.

3) Pathogenicity Islands: Clustered virulence genes, cause inhibition of phagosome-lysosome fusion.

4) Acid tolerance: S. typhimurium is acid tolerant in the lab down to pH 3.0. The ability to accomplish this is due to a component regulatory system, PhoPQ.
Clinical Types of Salmonella Infection
1) Gastroenteritis: Most frequent manifestation, mild to fulminant diarrhea, low grade fever, nausea-vomiting.

2) Bacteremia or Septicemia: Without major GI symptoms, high spiking fever and + blood cultures.

3) Enteric fever: Mild fever and diarrhea, early phase, fever and constipation, blood cultures +, stool cultures -; second phase (diarrheic), blood culture-, stools+. Bimodel.

4) Carrier state: Seen in patients with a previous infection, excrete the organism for up to one year following remission of symptoms. Organism resides in the gall bladder. Typhoid Mary.
Vaccines for Salmonella
Vi antigen material (capsule) seems to be effective. Cooking thoroughly kills the bacteria.
Phage Therapy for Diarrheal Disease
Phages are good antigens and would stimulate the production of antibodies that would destroy the phages. Therefore really not useful for injection. With diarrheal diseases perhaps the oral route would work? Phage resistance via cell wall receptor modification would occur in a small percentage of the strains thus allowing phage resistant strains to cause the disease to re-appear.
Tribe IV Citrobactereae
1 genus: Citrobacter. Divided into 11 species. This genus is isolated from feces, soil and water samples. Resistant to antimicrobials.
Citrobacter Species Include
1) C. freundii: Possible diarrhea outbreaks.
2) C. koseri: Meningitis in children.
Tribe V Klebsielleae
VP + so a butanediol fermenter. Takes glucose through glycolysis, makes pyruvate and makes 2,3-Butanediol, ethanol, lactate, succinate, acetate, CO2 and H.

Divided into 5 generas:
1) Klebsiella

2) Enterobacter

3) Pantoea

4) Hafina

5) Serratia
Characteristics of Klebsiella
Friedlander’s Bacillus. Found in soil, water and the GI tract of humans and animals. Large, mucoid colonies, that are usually opportunistic and cause nosocomial infections, UTIs and pneumonia.

2 species: K. pneumoniae divided into 3 subsp (Rhinosplaramatus, pneumoniae and zoniae) and K. oxytoca. The indole reaction differentiates these two species, K. pneumoniae is indole negative, the other species is indole positive. Harbor antimicrobial-resistance plasmids that are chromosomally encoded. All clinical strains are resistant to ampicillin and carbenicillin. These isolates appear to make, extended spectrum beta lactamases (ESBL)’s as seen in K. pneumonia.
Pathogenesis of Klebsiella
K. pneumoniae causes pneumonia in patients with debilitating illnesses. The pneumonia is destructive and leads to necrosis and its sputumis brick red. This species can also cause enteritis, meningitis, UTI and septicemia. Nosocomial.
Enterobacter
These are motile rods that are usually MR-, VP+ and H2S-. Found in nature, soil, water, sewage and vegetables. Does not cause diarrhea. 16 species in the genera. Two most common strains: E. aerogenes, E. cloacae.

Serotyping is done by “O”, “H” and “K”. Chromosomal beta lactamase, not expressed.
Differentiate Between Cloacae and Aerogenes
1) Lysine Poitive: Aerogenes
2) Arginine Positive: Cloacae
3) Glycerol Fermentation: Aerogenes
4) Esculin Positive: Aerogenes
Species of Enterobacter
1) E. cloacae: Most frequently isolated species from humans and animals. Found in feces but not an enteric pathogen. Makes SLT toxin and causes pus-like issue in infants.

2) E. sakazakii (Coronobacter sakazakii): An emerging infant formula-borne pathogen that causes meningitis, sepsis and enterocolitis.

3) E. aerogenes: Found in human and animal feces but is not an enteric pathogen. It is an opportunistic pathogen from the respiratory tract, urinary tract and wounds.
Pantoea
Comprised of former members of the E. agglomerans. Saprophytic microbes frequently isolated from plants, flowers, seeds and vegetables.

-P. agglomerans: Biopesticide (extends shelf-life of apples and pears).
Hafnia
-H. alvei: Biochemically similar to the Enterobacter and makes the scent of human feces (skatole). Found in soil and dairy products. Can cause gastroenteritis, virulence genes for adhesion to epithelial cells. Non-motile and lactose negative.
Serratia
This genus produces three hydrolytic enzymes: Lipase (spirit blue agar), Gelatinase (in broth) and DNase. Produces a red pigment (prodigiosin) at 25C and not at 37C. Secondary metabolism. Subway experiments.

-S. marcescens: Associated with human infections, pneumonia and septicemia, resistant to antibiotics.