Gram Positive Bacilli and Gram Negative Cocci (trans 7)

Question 1

GRAM POSITIVE BACILLI

Answer 1

 Includes Bacillus anthracis, Bacillus cereus, Bacillus thuringiensis, Corynebacterium diphteriae, Listeria monocytogenes, Propionibacterium acnes, Erysipelothrix rhusopathiae, Lactobacillus
 Used for production of: Antibiotic (ex Polymixin, Bactiracin [source: soil bacillus]), Alcohol, Solvents (enzymes)

Question 2

GRAM POSITIVE SPORE-FORMING BACILLI
 Gram (+) rods in chains
 Includes Bacillus and Clostridium species. Bacillus = aerobes, Clostridium = anaerobes
 Spore for Bacillus is for survival, spore for fungi is for reproduction
 Spores can be seen in dead animal tissue or in soil because of depletion of nutrition, water content, temperature, pH, etc (requirement for growth is not present)

Answer 2

1. Bacillus anthracis
2. Bacillus cereus
3. Bacillus thuringiensis

Question 3

GRAM POSITIVE SPORE-FORMING BACILLI
Sporulation and germination process of spore-former bacilli. Under favorable condition, there is a chain of vegetative cell. It will keep on multiplying and multiplying until the infective dose is enough to produce disease. But if the vegetative cell is in the soil or dead animals under unfavorable condition, it produces spore. Sporulation will take place, and the vegetative form lyses.

Answer 3

The vegetative cell is the one that will keep on multiplying. Sometimes, you can see the spore outside the cell. This is because the vegetative cell has already lysed. If nutrient is present, they are arranged in rods in short chains in tissue of living animal or humans. They undergo germination.

Question 4

GRAM POSITIVE SPORE-FORMING BACILLI:
BACILLUS ANTHRASIS - Characteristics
 Anthrax, a disease of sheep, cattle and sometimes horses, hogs and goats (herbivores)
 Was first isolated by Robert Koch
 Has a vaccine (Anthrax, Diptheria, Rabies) for active immunization (heat attenuated culture) developed by Louis Pasteur
 Anthrax attack: this organism was used for Bioterrorism back in Sept 18, 2001. This was possible because anthrax can be grown in large quantities and spores are resistant to destruction.

Answer 4

 A pathogen of animals (herbivores); humans are just accidental host.
 Humans may acquire it in agricultural settings or industrial settings (in processing of hides or animal hair via inhalation of spores)
 Source: contaminated blood and tissues of dead animals

Question 5

GRAM POSITIVE SPORE-FORMING BACILLI: 
BACILLUS ANTHRASIS - Characteristics
Morphology:
o Large bacilli with spore
o Straight rods, with right angle end
o Non-motile
o May occur in singles, pairs or chains 
o Capsulated (made of poly-D glutamatic acid) in infected animals/human

Answer 5

Spore
o Oval, does not distend the cell; centrally located
o Dormant in culture, soil, dead animals
o Extremely resistant to chemical and physical env’t
o Viable for years in contaminated pasture
o Inactivated at 120oC for 15 minutes using autoclave

Question 6

GRAM POSITIVE SPORE-FORMING BACILLI:

BACILLUS ANTHRASIS - Mode of Transmission

Answer 6

 Bacillus anthracis is primarily transmitted by direct contact with B. anthracis-infected animals or their carcasses or with contaminated products from infected animals, including meat, hides, wool, or items made with those products, such as drums or wool clothing.
 Anthrax in humans is not generally considered to be contagious; person-to-person transmission of cutaneou anthrax has rarely been reported.

Question 7

GRAM POSITIVE SPORE-FORMING BACILLI:
BACILLUS ANTHRASIS - Mode of Transmission
Primary Host: Herbivores
1. Heat resistant spores are found in soil
2. Spores contaminate spiny vegetations that are ingested by herbivores OR Spores are inhaled when they contaminate the hides/furs of animals
3. DEATH

Answer 7

Accidental Host: man
3 Modes of Transmission to Man
1. SKIN CONTACT (cutaneous anthrax)
 “Malignant pustule” via skin contact with contaminated animal products such as hides, wool and hair. Abrasion in the skin increases susceptibility.
2. INGESTION (gastrointestinal anthrax)
 Gastroenteritis via ingestion of contaminated food products such as meat from infected animals.
3. INHALATION (pulmonary anthrax)
 “Woolsorter’s disease” via inhalation of contaminated dust or fur.

Question 8

GRAM POSITIVE SPORE-FORMING BACILLI:
BACILLUS ANTHRASIS - Virulence Factors
**Capsule (antigenic)

Answer 8

 Composed of D-glutamic acid
 Plasmid-encoded (pXO2) must be present to produce capsule (if not present, then the organism is unencapsulated)
 Function: anti-phagocytic

Question 9

GRAM POSITIVE SPORE-FORMING BACILLI: 
BACILLUS ANTHRASIS - Virulence Factors 
**Exotoxin (antigenic)
 Plasmid encoded (pXO1)
 Heat labile
 There are 3 types of exotoxins:

Answer 9

a. Protective Antigen (PA) - for adherence
b. Edema Factor (EF)
o Calmodulin-dependent adenylate cyclase
o Generates cAMP within the host cell and inhibits neutrophil phagocytosis
o PA + EF = Edema Toxin – inhibits neutrophil function and elevation of intracellular cAMP, resulting in impaired maintenance of water homeostasis.
c. Lethal Factor (LF)
o Induce the death of macrophages via activation of IL1, TNF, and O2 radicals
o PA + LF = Lethal Toxin – major virulence factor; causes apoptosis, tissue necrosis, and death
* PA + EF = Gross local edema
* PA + LF = Death
* PA + EF + LF = Edema & Death

Question 10

GRAM POSITIVE SPORE-FORMING BACILLI:

BACILLUS ANTHRASIS - Pathogenesis

Answer 10

After transmission, capsule protects the bacilli from phagocytes.
1. Anthrax bacteria flood in to the blood stream following infection
2. Bacteria produces 3 exotoxins (PA,LF,EF)
3. PA attaches to the receptor of the cell membrane and penetrates, allowing LF and EF to enter
o Release of Edema Factor = Increase cAMP leads to production of fluid = leading to edema and inhibition of neutrophil phagocytosis
4. Once inside, lethal toxin kills the macrophage via activation of IL1, TNF, and Oxygen radicals

Question 11

GRAM POSITIVE SPORE-FORMING BACILLI:
BACILLUS ANTHRASIS - CLINICAL SIGNIFICANCE
Anthrax infection provides potent immunomodulating effects. In human, approx 95% = cutaneous anthrax; 5% = pulmonary anthrax; rare = GIT anthrax

Answer 11

Types of Anthrax infection:

a) Cutaneous Anthrax
b) Inhalational (Pulmonary) Anthrax/Woolsorter’s Disease
c) Gastrointestinal Anthrax

Question 12

GRAM POSITIVE SPORE-FORMING BACILLI:

BACILLUS ANTHRASIS - Types of Anthrax infection

Answer 12

Cutaneous Anthrax
 Spores from the soil or infected animals gain access to wound and/or to subcutaneous tissues, releasing exotoxin and form a painless dark pustule (called malignant pustule or eschar)
 The organisms may invade regional lymph nodes (lymphadenopathy) and then the general circulation which can lead to fatal septicemia, meningitis, and death.
 Lesion is painless and is found in the hands, forearms, face, or neck.
 Begins 1-7 days after infection (resembles an insect bite) as a papule that rapidly changes into a vesicle, and a necrotic ulcer develops. Lesion is typically 1-3 cm and have a characteristic central black eschar
 There is edema, fever, malaise, and headache.
 After 7-10 days, eschar is fully developed.

Question 13

GRAM POSITIVE SPORE-FORMING BACILLI:

BACILLUS ANTHRASIS - Types of Anthrax infection

Answer 13

Inhalational (Pulmonary) Anthrax/Woolsorter’s Disease
 There is evident “Massive enlarged mediastinal lymph nodes” in chest x-rays. Mediastinum has widened itself.
 Due to inhalation of spores by people who handle raw wool, hides or horsehair => phagocytosed in the lungs => transported by the lymphatic drainage to the mediastinal lymph nodes => germination occurs => toxin production
 Fever, malaise and cough with bloody pleural effusion, which progress to a severe infection with hemorrhagic necrosis leading to respiratory distress and cyanosis.
 Inflammation of the lymph nodes
 Death within 24 hours, if left untreated. 100% mortality

Question 14

GRAM POSITIVE SPORE-FORMING BACILLI:

BACILLUS ANTHRASIS - Types of Anthrax infection

Answer 14

Gastrointestinal Anthrax
 Rare form of anthrax in humans
 Due to ingestion of meat contaminated with spores
 Abdominal pain, vomiting, and bloody diarrheathrax

Question 15

GRAM POSITIVE SPORE-FORMING BACILLI:
BACILLUS ANTHRASIS - Diagnostic Laboratory Tests
 The typical cells measures 1x3-4μm; have square ends and are arranged in long chains; spores are located in the center of the non-motile bacilli.
 Stained smears will show Gram positive blunt-ended bacilli that occur singly, in pairs, or frequently in long chains [Jawetz]
 Can be identified in dried smears by immunofluorescence staining techniques

Answer 15

SPECIMEN: Malignant pustule, sputum, blood
AGAR WITH BACTERIOPHAGE
(+) lysis = B. anthracis
(-) lysis = other Bacillus species
MOUSE INOCULATION
 Isolated organism => Mouse inoculation => Mouse death in 2-5 days
SEROLOGY: ELISA
 Antibodies to the organism can be demonstrated

Question 16

GRAM POSITIVE SPORE-FORMING BACILLI:

BACILLUS ANTHRASIS - TREATMENT

Answer 16

 Ciprofloxacin or Doxycycline WITH one or two antibiotics (Rifampin, Vancomycin, Penicillin, Imepenem)

Question 17

GRAM POSITIVE SPORE-FORMING BACILLI:

BACILLUS ANTHRASIS - PREVENTION

Answer 17

 Dead animals infected with B. anthracis should be buried deep enough to prevent spread of spores to new pastures.
 Animal products (hides, wools) coming from places where there is anthrax should be sterilized using gas sterilizer (autoclave sterilizer is not enough)
 Anthrax vaccine: Live spore of Stearne strain of B. anthracis (active immunization for animals); killed spore vaccine for humans to control outbreak.
 No effective vaccine against anthrax for humans but a cell-free vaccine is available for workers in high-risk occupations.

Question 18

GRAM POSITIVE SPORE-FORMING BACILLI:

BACILLUS CEREUS - characteristics

Answer 18

 “Chinese Restaurant Syndrome”
 Cause of food-borne illness
o Spores are heat-resistant thus can survive cooking
o Can cause food poisoning
o Common food affected: FRIED RICE, pasta, contaminated sauce (due to long exposure time before eating)
 B. cereus colonies DO NOT have Medussa Head characteristic of that of B. anthracis
 Usually motile; Penicillin and cephalosporin resistant

Question 19

GRAM POSITIVE SPORE-FORMING BACILLI:

BACILLUS CEREUS - Virulence Factor

Answer 19

1. Enterotoxin
   o Causes diarrhea
2. Glycoprotein S-layer
   o for adherence and promotion of interactions with polymorphonuclear cells

Question 20

GRAM POSITIVE SPORE-FORMING BACILLI:
BACILLUS CEREUS - CLINICAL MANIFESTATIONS
The diseases produced are more of intoxication due to toxins produced by the organism rather than a food-borne infection. The enterotoxin may be pre-formed in the food or intestine.
B. cereus may be found in the normal stool so it is not diagnostic. Concentration of 100,000 or more bacteria per gram of food is diagnostic.
Food poisoning caused by B. cereus has 2 distinct forms:

Answer 20

1. Emetic Type
   o Heat STABLE enterotoxin
   o Associated with fried rice/pasta
   o Staphylococcus-like enterotoxin
   o Incubation period: 1-6 hrs (shorter)
   o Vomiting > Diarrhea
   o There is nausea, vomiting, abdominal cramps, and occasionally diarrhea. Self-limiting, recovery occurs in 24 hours.
2. Diarrheal Type
   o Heat LABILE enterotoxin
   o Associated with meat dishes and sauces
   o Cholera-like enterotoxin (watery diarrhea)
   o Incubation period: 6-24 hrs (longer)
   o Profuse diarrhea with abdominal pain and cramps. Fever and vomiting are uncommon.
   o Associated with uncooked meat dishes, vegetables (salads), and sauces

Question 21

GRAM POSITIVE SPORE-FORMING BACILLI:

BACILLUS CEREUS - Other Diseases

Answer 21

o Ophthalmitis (inflammation of the eye) can occur after traumatic penetrating eye injuries with soilcontaminated object
o Keratitis, endophthalmitis, panophthalmitis (whole eye) due to organisms introduced by foreign bodies through eye trauma.
o Medical device or in IV drug use may predispose to endocarditis, meningitis, osteomyelitis, and pneumonia.
o Treatment: Clindamycin, Vancomycin, Gentamicin with or without an Aminoglycoside

Question 22

GRAM POSITIVE SPORE-FORMING BACILLI:

BACILLUS THURINGIENSIS

Answer 22

 Used as a larvicide because it produces a protein which is lethal to some insect larvae (e.g. flies and mosquito)
 Genes from Bacillus thuringiensis coding for insecticidal compounds have been inserted into the genetic material of some commercial plants.
 It can also cause food poisoning and opportunistic infections in immunocompromised patients.

Question 23

Bacillus A-C-T 05

Answer 23

o Composed of group of international scientists that collaborated to share their findings about ACT (anthracis, cereus, thuringiensis) (05 because it was formed in 2005) regarding their genomes, gene regulation, physiology, pathogenesis, etc.
o Recently, they found out that B. cereus can produce zwittermicin A which can be used as a broad-spectrum antibiotic

Question 24

GRAM POSITIVE NON-SPORE FORMING BACILLI

Answer 24

1. Corynebacterium diphtheriae
2. Listeria monocytogenes
3. Erysipelothrix rhusiopathiae

Question 25

GRAM POSITIVE NON-SPORE FORMING BACILLI: CORYNEBACTERIUM DIPHTHERIAE
 Man is the only reservoir
 Causative agent of Diphtheria History
 Loeffler – 1st isolated the bacteria
 Yersin – the one who discovered the exotoxin
 Man is the only known reservoir, therefore it can be eradicated through vaccination

Answer 25

3 Biotypes (Produces the same amount of toxins but differs in the size of the colony)
 Gravis – most severe type, largest
 Intermedius
 Mitis – smallest

Question 26

GRAM POSITIVE NON-SPORE FORMING BACILLI:

CORYNEBACTERIUM DIPHTHERIAE - Characteristics

Answer 26

 Small, slender, pleomorphic, gram-positive rods that are non-motile, unencapsulated, anaerobic and do not form spores [Jawetz]
 “Chinese letter arrangement”
 X,V, or Y was formed during snapping division
o Club-shaped swelling at one or both ends because of irregular swellings at one end
o Metachromatic or Volutin or Babes-Earnts granules – pockets of inorganic phosphate that causes the swelling of the bacteria

Question 27

GRAM POSITIVE NON-SPORE FORMING BACILLI:
CORYNEBACTERIUM DIPHTHERIAE - Virulence Factor
Exotoxin (lysogenic property)
o Encoded by beta-prophage that carries the toxigenic gene
- Absence of beta-prophage means it is nontoxigenic
o Inhibits protein synthesis and Elongation Factor 2
o It has 2 subunits:
A – has ADP- ribosylation activity; pathogenic
B – binds to cell surface
o All toxigenic strains produce immunologically identical toxin
o Low iron levels inside the host cell will allow the release of toxin.

Answer 27

*Lysogenic property - it stays inside the bacterial cell but will not destroy the bacteria. It only carries the gene for the toxin. This only means that diphtheria is non-toxigenic inside the bacteria pod, but it is the toxin that makes it pathogenic.

Question 28

GRAM POSITIVE NON-SPORE FORMING BACILLI:

CORYNEBACTERIUM DIPHTHERIAE - Mode of Transmission

Answer 28

 Via respiratory droplets from throat through coughing and sneezing
 Direct contact with infected person
 Human is the only reservoir

Question 29

GRAM POSITIVE NON-SPORE FORMING BACILLI:

CORYNEBACTERIUM DIPHTHERIAE - Pathogenesis

Answer 29

1. Subunit B binds to host membranes
2. It enters thru toxin receptor by endocytosis
3. Subunit A leaves the lysosome where cleavage occurs
4. Subunit A interrupts the transfer of the polypeptide chain of ribosome
5. EF2 ceases and halts protein synthesis
6. Cell death

Question 30

GRAM POSITIVE NON-SPORE FORMING BACILLI:
CORYNEBACTERIUM DIPHTHERIAE - Clinical Manifestations
1. Cutaneous (via punctured wound)
 Can be caused by both toxigenic and nontoxigenic strains
 Deep erosive non-healing ulcer with gray-brown membrane

Answer 30

2. Respiratory
    Colonized tonsils, nasal cavity, and throat
    Pseudomembrane – results from death of mucosal epithelial cell (greenish to gray film)
    “Bull neck” appearance due to pronounced swelling of lymph node because of mumps virus
    Any attempt to remove the pseudomembrane exposes and tears the capillaries and thus results in bleeding
   **Complications
    If it hardens from inflammation, it can lead to airway obstruction
    Myocarditis
    Cranial nerve and muscle paralysis

Question 31

GRAM POSITIVE NON-SPORE FORMING BACILLI:
CORYNEBACTERIUM DIPHTHERIAE - Laboratory Diagnosis
1. Nasopharynx or throat swab
o Blood agar plate (gray to white colonies)
o Loeffler’s slant (enhance the formation of metachromatic granules; (+) growth is the presence of cream-yellow colonies; then when stained with methylene blue and viewed under the microscope, the granules will be seen)
o Potassium Tellurite Plate (real culture media for diphtheria; presence of grayish-black colonies)
o All three subtypes of Corynebacterium diptheriae (gravis, intermedius, mitis) produce the same toxins. They only differ in the size of their colonies. Mitis has the smallest size of colonies, biggest is gravis.

Answer 31

2. Elek’sToxigenicity Test
   o Used to test if organism is toxigenic or not
   o Precipitation of (+) control culture meet with the precipitation of test organism
   o An antitoxin disc is placed on an agar plate and the culture to be tested is inoculated 9mm away from the disc.
   o After 48hours, the toxin will be precipitated by the antitoxin from the disc as a whitish material between the disc and growth
   **Elek’s Toxigenicity Test. Filter paper contains antitoxin, (+) control produces toxin, (-) control does not produce toxin. Antitoxin from the filter paper and the toxin from the test organism migrate in the agar and produces precipitate in the area where they are specific.

Question 32

GRAM POSITIVE NON-SPORE FORMING BACILLI:

CORYNEBACTERIUM DIPHTHERIAE - Treatment

Answer 32

 Antitoxin – to prevent attachment of fragment B

 Antibiotic (Penicillin, Erythromycin) – adjunct to antitoxin; arrests toxin production

Question 33

GRAM POSITIVE NON-SPORE FORMING BACILLI:
CORYNEBACTERIUM DIPHTHERIAE - Prevention
 Toxoid vaccine
 DTaP vaccine
Prophylaxis
 Should be given for those who are exposed to patient with respiratory diphtheria and be placed under close surveillance

Answer 33

Mnemonic for Diphtheria: ABCDEFG
A – ADP-ribosylation
B – Beta prophage
C – Corynebacterium
D – Diphtheriae
E – Elongation Factor 2
G – Granules (Monochromatic)

Question 34

GRAM POSITIVE NON-SPORE FORMING BACILLI:
LISTERIA MONOCYTOGENES
 The organism has a very serious effect on pregnant, new born, elderly, and immunocopromised individuals.
 It can be found in the soil, water, animals, meat products, sea food, raw milk, plants
 Short, motile, gram-positive, non-spore-forming rod
 Facultative intracellularly located, facultative anaerobe
 Endotoxin (+) – only gram (+) that has endotoxin
 Catalase (+), narrow zone of beta hemolysis

Answer 34

 Can tolerate cold (2-4°C) under conditions of low pH and high Na+, can survive in refrigerated food (can multiply even if placed in the refrigerator)
 Has a tumbling motility brought about by actin filaments at 22-28°C but not at 37°C
 Tumbling motility- differentiates it from diptheroids that are members of the normal flora of the skin
 Only gram-positive that has endotoxin
 Habitat: GIT & female genito-urinary tract (GUT)

Question 35

GRAM POSITIVE NON-SPORE FORMING BACILLI:
LISTERIA MONOCYTOGENES - Virulence factors
For adhesion:
 D-galactose residue adheres to D-galactose receptor of the host cell
**Iron is an important virulence factor. Listeriae produce siderophores and are able to obtain iron from transferrin.
**Immunity is primarily cell mediated. It can be transferred by sensitized lymphocytes but not by antibodies.

Answer 35

For invasion:
 Internalin (protein) – mediates invasion and attach to macrophages (Inl A, Inl B)
 Listeriolysin O (LLO) – acts as hemolysin to escape from phagosome
 Actin A – promotes polymerization of actin which is a component of host cell
 Phospholipase substance (PlcA, PlcB)
 Catalase, superoxide dismutase – neutralizes the effect of phagocytic oxidative outburst

Question 36

GRAM POSITIVE NON-SPORE FORMING BACILLI:

LISTERIA MONOCYTOGENES - Life Cycle

Answer 36

 Enters body through ingestion of contaminated food
 Listeria monocytogenes has a cell wall surface protein called internalin that interacts with E-cadherin, a receptor on epithelial cells, promoting phagocytosis into the epithelial cells
 After phagocytosis, the bacterium is enclosed in a phagolysosome, where the low pH activates the bacterium to produce listeriolysin O (LLO)
o LLO lyses membrane of the phagolysosome -> listeria escapes to cytoplasm
 Organisms proliferate and Actin A (ActA), another listerial surface protein, induces host cell actin polymerization, which propels them to the cell membrane
 Pushing against the host cell membrane, they cause formation of elongated protrusions called filopods/Listeriapods
 Listeriapods are ingested by adjacent cells,macrophages, and hepatocytes and the listeriae are released causing the life cycle to begin again

Question 37

GRAM POSITIVE NON-SPORE FORMING BACILLI:
LISTERIA MONOCYTOGENES - Clinical Manifestation
**Pregnant

Answer 37

 Septicemia (due to endotoxin)
 Placental abscess
 Spontaneous abortion

Question 38

GRAM POSITIVE NON-SPORE FORMING BACILLI:
LISTERIA MONOCYTOGENES - Clinical Manifestation
**Neonates (Early Granulomatous infantiseptica)

Answer 38

 Early onset syndrome (granulomatous infanseptica) - (1-3 days after delivery) is the result of infection in utero and is a disseminated form of the disease characterized by neonatal sepsis, pustular lesions and granulomas containing L. monocytogenes in multiple organs. Death may occur before or after delivery.
 Late onset syndrome (meningitis) (5 days) - causes the development of meningitis between birth and the third week of life; it is often caused by serotype IVb and has a significant mortality rate.

Question 39

GRAM POSITIVE NON-SPORE FORMING BACILLI:
LISTERIA MONOCYTOGENES - Clinical Manifestation
**Adults

Answer 39

 Can develop listeria meningoencephalitis, bacteremia, and (rarely) focal infections
 Meningoencephalitis and bacteremia occur most commonly in immunocompromised patients

Question 40

GRAM POSITIVE NON-SPORE FORMING BACILLI:

LISTERIA MONOCYTOGENES - Laboratory Diagnostics

Answer 40

 Tumbling motility – differentiates it from the diphtheroids that are members of the normal flora of the skin
 B hemolysis on blood agar (specimen: blood and spinal fluid)
 (+) catalase, (+) esculin hydrolysis
 CSF (high PMN, low glucose, normal protein)

Question 41

GRAM POSITIVE NON-SPORE FORMING BACILLI:

LISTERIA MONOCYTOGENES - Treatment

Answer 41

 Ampicillin
 Vancomycin
 Ciprofloxacin
 Trimethoprim-sulfamethoxazole – drug of choice for CNS infections for patients allergic to Penicillin

Question 42

GRAM POSITIVE NON-SPORE FORMING BACILLI:

LISTERIA MONOCYTOGENES - Prevention

Answer 42

 Pregnant women, immunocompromised patients should not ingest unpasteurized milk products or raw vegetables

Question 43

GRAM POSITIVE NON-SPORE FORMING BACILLI:
ERYSIPELOTHRIX RHUSIOPATHIAE
 Gram-positive rods in short chain, non-motile, nonencapsulated
 Animal pathogen: sea and land animals (most common in swines)
 Commonly affected: fisherman, butcher, veterinarian
 Catalase, oxidase, and indole negative [Jawetz]
 Resistant to: Smoking, pickling process, dessication
 Subcutaneous inoculation from animals or animal products
 Isolated from meat of animals and fishes

Answer 43

Culture Characteristics
 Produces small, transparent glistening colonies
 Alpha hemolytic on blood agar
 Gram stains: sometimes looks gram negative because it decolorizes easily. May appear singly, in short chains, randomly, or in long non-branching filaments.
 Triple sugar iron (TSI) agar: Hydrogen sulfide is produced turning the TSI butt black
 The colony morphology and gram stain appearance vary depending on the growth medium, incubation temperature, and pH

Question 44

GRAM POSITIVE NON-SPORE FORMING BACILLI:

ERYSIPELOTHRIX RHUSIOPATHIAE - Virulence factor

Answer 44

 Hyaluronidase (Spreading Factor) - hydrolyses hyaluronic acid in connective tissues
 Neuraminidase - cleaves with N-acetyl neuramic acid.
 Increases ability of the organism to attach to host receptors.

Question 45

GRAM POSITIVE NON-SPORE FORMING BACILLI:

ERYSIPELOTHRIX RHUSIOPATHIAE - Clinical Manifestation

Answer 45

 Distributed in land and sea animals worldwide, including variety of vertebrates and invertebrates.
 Transmission to man: direct contact with infected meat
 The most important impact is in swine, in which it causes erysipelas. In humans, erysipelas is called erysepeloid and also caused by group B B-hemolytic streptococci.

Question 46

GRAM POSITIVE NON-SPORE FORMING BACILLI:
ERYSIPELOTHRIX RHUSIOPATHIAE - Clinical Manifestation
**Erysipeloid

Answer 46

o A nodular type of cellulitis is the most common E rhusiopathiae infection in humans
o Infection is via direct inoculation of a wound with animal products. Manifested by inflammation or swelling of traumatized skin.
o Occurs on the fingers by direct inoculation on the site of a cut (“seal finger ” or “whale finger”).
o After 2-7 days incubation, severe pain and swelling occur. The lesion is raised and violaceous in color. Pus is usually not present at the infection site, which helps differentiate it from staphylococcal and streptococcal skin infections.
o Resolves in 3-4weeks, or more rapidly with antibiotic treatment.
o May also cause a diffuse cutaneous disease and bacteremia with endocarditis

Question 47

GRAM POSITIVE NON-SPORE FORMING BACILLI:

ERYSIPELOTHRIX RHUSIOPATHIAE - Laboratory Diagnostics

Answer 47

 Specimen - aspirates or biopsy from the margin of lesion

 Culture on BAP- uses 5-10% CO2 and will result to alpha hemolysis

Question 48

GRAM POSITIVE NON-SPORE FORMING BACILLI:

ERYSIPELOTHRIX RHUSIOPATHIAE - Treatment

Answer 48

 Highly susceptible to penicillin G, the drug of choice for severe infections
 Intrinsically resistant to Vancomycin

Question 49

GRAM POSITIVE NON-SPORE FORMING BACILLI:

ERYSIPELOTHRIX RHUSIOPATHIAE - Prevention

Answer 49

 Live attenuated vaccine - for animals
 Protection from direct contact with infected animal secretion.
 Proper handling of animal products

Question 50

GRAM NEGATIVE COCCI

Answer 50

1. Neisseria meningitidis
2. Neisseria gonorrhea
3. Moraxella catarrhalis

Question 51

GRAM NEGATIVE COCCI - NEISSERIA SPECIES

Answer 51

 Gram-negative cocci in pairs (diplococcus); nonmotile
 Flattened on one side
 Cellular structures:
o Lipooligosaccharide (LOS) – antigenic (Ag)
o Pore (porin proteins)
o Outer membrane (antigenic)
o Peptidoglycan
o Pilus (antigenic)
 Different stereotypes are antigenically heterogenous (capable of changing surface antigen)
 Importance: antigen produced previously cannot attack the new one, because it has switched from one antigen form to another

Question 52

GRAM NEGATIVE COCCI - NEISSERIA SPECIES
**Characteristics
 Sensitive to drying, chilling and sunlight
 N. gonorrhoeae organism can live up to 15 minutes in contaminated objects (toilet seat, towel, etc)
 No need for infective dose, one bacterium is enough to cause disease.
 Utilized carbohydrates as energy source
o N. meningitides: maltose and glucose (MG)
o N. gonorrhoeae: glucose only (G)
 Oxidase test (+)
o Purple color
o Reagent: tetramethyl-p-phenyl enediamene
o Oxidase will destroy NO in the macrophage
 Catalase test (+)
o Culture (catalase) + 3% H2O2 = H2O + O2

Answer 52

 Iron is required for growth
o Siderophores: if iron is not enough in the bacterial cell, siderophore will go out of the cell to find a source of iron, goes back bringing the iron with it.
 Culture medium is blood agar plate, when heated becomes chocolate agar
o To isolate Neisseria alone: THAYER-MARTIN or MARTIN-LEWIS MEDIUM:
- Addition of vancomycin and lincomycin (against gram positives), colistin (against gram negatives), and nystatin (against yeasts)
- Results to dry and white colonies of Neisseria
 Atmospheric requirement: 3-10% CO2
o Neisseria can’t grow in an atmosphere that contains oxygen alone, it also needs CO2
o Put CO2 tablet in culture, place inside Ziplock bag (to increase moisture which activates CO2 production) to have an enriched atmosphere for the bacteria

Question 53

GRAM NEGATIVE COCCI:

MORAXELLA (BRAMHAMELLA) CATARRHALIS

Answer 53

 Old name: Neisseria catarrhalis
 Gram negative diplococci
 Part of the normal flora of the respiratory and genital tracts
 Can cause infection of the middle ear, eyes, CNS, joints
 Can be cultured on BAP and chocolate agar
 Non-motile, non-fermentative (doesn’t ferment CHO), and oxidase positive

Question 54

GRAM NEGATIVE COCCI:

NEISSERIA MENINGITIDIS - Gram staining characteristics

Answer 54

 Gram (-) diplococci, intracellularly located (located inside PMN). But for chronic gonorrhea infection, sometimes the bacteria can be seen outside the PMNa.

Question 55

GRAM NEGATIVE COCCI:

NEISSERIA MENINGITIDIS - Structures

Answer 55

 Pili – for attachment, transfer of genes
 Capsule – (in yellow) enclosing an (orange-staining) diplococci; used for vaccine production, stereotyping and can only be found in fresh specimen

Question 56

GRAM NEGATIVE COCCI:

NEISSERIA MENINGITIDIS - Classification System

Answer 56

 13 serotypes based on capsular polysaccharide: A, B C, D, X, Y, Z, E, W-135, H, I, K, L
 A, B, C, Y, W-135 – pathogenic; most common cause of disease in humans (the other serotypes may be the ones found in our body as normal flora)

Question 57

GRAM NEGATIVE COCCI:

NEISSERIA MENINGITIDIS - Virulence factors

Answer 57

 Polysaccharide capsule – antigenic
 Opacity associated protein (Opa) – for adhesion
 Pili (Type IV) – antigenic
 IgA protease – helps in intracellular location
**These four are used when escaping the host immune system
 Endotoxin (LOS) – most important virulent factor. The active endotoxin interacts with IL-1, IL-6, TNF, causing toxic effects – meningococcemia, necrosis of the host cell, and fever.

Question 58

GRAM NEGATIVE COCCI:
NEISSERIA MENINGITIDIS - Pathogenesis
**Humans are the only natural hosts for whom meningococci are pathogenic
1. Meningococci, from an asymptomatic carrier, enters the nasopharynx or conjunctiva of humans.
2. At the upper respiratory tract, pili (type IV) binds to the human cell surface protein CD46. They may form part of the transient flora without producing symptoms.
3. In less than a week, they go to the bloodstream, producing bacteremia.

Answer 58

4. In the blood stream,
   o Meningococci are engulfed by macrophages
   o This causes release of endotoxin, activation of cytokines (IL-1, IL-6, TNF-a), and activation of free radicals (H2O2, NO)
5. TNF-a is released in the vital organs which causes:
   o Vasodilation and decreased plasma volume => septic shock => death
   o Causes blockage of small blood vessels that supply the vital organs => multi-organ failure (Waterhouse-Friedrichsen Syndrome), disseminated intravascular coagulation

Question 59

GRAM NEGATIVE COCCI:
NEISSERIA MENINGITIDIS - Clinical Findings
3 syndromes:
a. Meningitis
b. Meningitis and meningococcemia
c. Meningococcemia w/o meningitis
**If patient survives, sequelae: CN VIII deafness, CNS damage
 Risk Factors:
1. Close contact w/ patient w/ primary invasive disease
2. Recent viral respiratory illness (flu)
3. Smoking or exposed to secondary smoke
4. Household overcrowding

Answer 59

a. Meningitis
o Most common complication of N. meningitidis infection
o Usually begins with sudden intense headache, vomiting, stiff neck, and progresses to coma within few hours
o Meninges are acutely inflamed, with thrombosis of blood vessels and exudation of PMN leukocytes, so that the surface of the brain is covered with a thick purulent exudate
b. Meningitis and meningococcemia Meningococcemia:
o There is thrombosis of many small blood vessels in organs, with perivascular infiltration and petechial hemorrhages
o There may be interstitial myocarditis, arthritis, and skin lesions
c. Meningococcemia w/o meningitis
o Vasculitis purpura – hallmark of fulminant meningococcemia

Question 60

GRAM NEGATIVE COCCI: 
NEISSERIA MENINGITIDIS - Diagnostic Laboratory Tests
 Specimens:
o CSF – meningitis
o Blood (buffy coat, contains WBC) – meningococcemia
o Nasopharyngela swab – carrier surveys
 Serology:
o Counter current immunoelectrophoresis
o Latex agglutination

Answer 60

 Culture media
o Thayer Martin Medium – favors growth on Neisseria sp., inhibits many organisms
- Vancomyin (+)
- Colistin (-)
- Nystatin (yeast)
o Martin William Medium
- Lincomycin (+)
- Colistin
- Nystatin
 Biochemical Tests
o Catalase (+)
o Oxidase (+)
o Glucose (+)
o Maltose (+)

Question 61

GRAM NEGATIVE COCCI:
NEISSERIA MENINGITIDIS - Management
Prompt action is important

Answer 61

Treatment:
o Penicillin G– drug of choice
o 3rd generation of cephalosporins (cefotaxime, ceftriaxone) – for those allergic to penicillin
 Prophylaxis
o for carriers or exposed person
o 5-30% of normal population harbors meningococcus
o During epidemic, the carrier rate goes up to 70-80%
*Rifampin (600mg daily for 2 days) – eliminates the bacteria in the nasopharynx
*Ciprofloxacin (500g single dose) – eradicates carriers

Question 62

GRAM NEGATIVE COCCI:
NEISSERIA MENINGITIDIS - Susceptibility
Predisposing factors to Meningococcal infection:
o People with immune complex disease (Systemic Lupus Erythematosus, Rheumatoid arthritis, Severe liver disease, Nephrotic syndrome)
o People with C5-C9 deficiencies
 Cannot form Membrane Attack Complex (MAC)
 MAC has lytic activity

Answer 62

Vaccine:
Monovalent or polyvalent vaccine (A, C, W135, Y)
o single dose of 50 μg
o used only for outbreak or epidemic
o not recommended routinely for children
o recommended for:
 travelers to highly endemic areas
 persons with complement deficiency (C6-C9)
 closed population (military camp, dormitory)

Question 63

GRAM NEGATIVE COCCI:

NEISSERIA GONORRHOEAE

Answer 63

 Diplococcus, unencapsulated, kidney shaped
 Ferments only glucose
 Differ antigenically from other neisseriae

Question 64

GRAM NEGATIVE COCCI:

NEISSERIA GONORRHOEAE - Antigenic Structures

Answer 64

 N. gonorrhoeae is antigenically heterogenous capable of changing its surface structures to avoid host defenses

Question 65

GRAM NEGATIVE COCCI:
NEISSERIA GONORRHOEAE - Antigenic Structures
**Pili
 Hair-like appendages extending from gonococcal surface
 Enhance attachment to host cells and resistance to phagocytosis
 Made up of pilin proteins
o Amino terminal- hydrophobic
o Amino acid near mid portion- serves in attachment to host cells; less prominent in immune response
o Carboxyl terminal- prominent in immune response

Answer 65

 For adhesion
 It confers resistance to phagocytosis
 Antigenic variation
o By shuffling and recombining chromosomal regions of genes
o A single strain can at different times synthesize multiple pilins that have different amino acid sequence
o A single gonococcal cell has the genetic capacity to produce at least 8 antigenic variant pili
 PILIN (antigen) helical peptide
o All genes are not expressed at one time
o Ability to switch genes (variability of infection)

Question 66

GRAM NEGATIVE COCCI:
NEISSERIA GONORRHOEAE - Antigenic Structures
**Outer Membrane Protein (OMP)

Answer 66

 OMP I – used for serotyping, ELISA
 OMP II – mediates attachment to host cell
 OMP III – complexed with OMP I to form porins

Question 67

GRAM NEGATIVE COCCI:
NEISSERIA GONORRHOEAE - Antigenic Structures
**Lipooligosaccharide (LOS)

Answer 67

 major binding sites for IgG
 important role in immunity
 Unlike gram (-), gonococcal LPS does not have Oantigen side chains
 Endotoxic
 Structurally resemble cell membrane glycosphingolipids
o Thus, also reacts with monoclonal antibodies
o Evades immune recognition

Question 68

GRAM NEGATIVE COCCI:
NEISSERIA GONORRHOEAE - Antigenic Structures
**Peptidoglycan

Answer 68

 Possess intrinsic toxicity for human fallopian tube

 Damages the mucosa resulting to sloughing of ciliated cells

Question 69

GRAM NEGATIVE COCCI:
NEISSERIA GONORRHOEAE - Antigenic Structures
**IgA protease

Answer 69

 All gonococci produce protease to a Subclass IgA-1

Question 70

GRAM NEGATIVE COCCI:
NEISSERIA GONORRHOEAE - Antigenic Structures
**Penicillinase

Answer 70

 Also known as beta-lactamase
 PPNG (penicillinase-producing N.gonorrhea)
 Penicillinase (Beta-lactamase) Producing N. gonorrhea strain destroy the B-lactam ring of penicillin
o In gram (-) they are located in the middle
o In gram (+) on surface

Question 71

GRAM NEGATIVE COCCI:

NEISSERIA GONORRHOEAE - Susceptibility

Answer 71

 persons with C6-C9 deficiency => cannot form MAC

Question 72

GRAM NEGATIVE COCCI:
NEISSERIA GONORRHOEAE - Pathogenesis
 Neisseria attaches to mucus secreting epithelial cells
 If the microorganism gets inside T helper cells => T helper cells tell interferon that there is a foreign body
 Neutrophils, macrophage, dendritic cells will send cytokines
 Antigen presenting cells activate IL-6, IL-23, TNF, IL-18
 T-helper cells activate IL-6, TNF, Interferon
 Once they are inside, after 14 days incubation period, signs and symptoms of inflammatory reaction occurring in sub-epithelial connective tissue can be observed.
 There will be dense infiltration of PMNs

Answer 72

Common organs and tissues involved
o Cervix
o Urethra (periurethral ducts)
o Rectum
o Pharynx
o Conjunctiva
 Complications: obstruction of ducts and glands by exudate

Question 73

GRAM NEGATIVE COCCI:
NEISSERIA GONORRHOEAE - Clinical Findings
In males: urethritis
o 10% asymptomatic
o Yellow, creamy pus/discharge (w/in a week)
o Painful urination
o May extend up to epididymis
o As suppuration subsides, fibrosis occurs
o Complications :
 Epididymitis (small mass in scrotum, unilateral testicular pain and swelling of testicles)
 Prostatitis

Answer 73

In females: Cervicitis, urethritis
o primary infection in the endocervix extending to urethra and vagina
o Asymptomatic (20-80%)
o Dyspareunia (pain during intercourse)
o Yellow purulent vaginal discharge (pus-forming cocci)
o Burning sensation or vaginal itching
o Mild lower abdominal pain
o Progress to uterine tubes salphingitis, fibrosis, obliteration of tubes
o Infertility occurs in 20% with gonococcal salphingitis

Question 74

GRAM NEGATIVE COCCI:
NEISSERIA GONORRHOEAE - Clinical Findings
**Syndromes in Females

Answer 74

1. Accessory glands infection – Skene’s glands and Bartholin’s glands
2. PID (Pelvic Inflammatory Disease)
   o results to fibrosis of the fallopian tube.
   o IF fibrosis is in the cervix, it may go up to the fallopian tube and cause scarring or blockage of the tube and can result to ectopic pregnancy or sterility for females due to blocking of the passage of the ova.
3. Fitz-Hugh-Curtis syndrome
   o Also called Perihepatitis
   o inflammation of the peritoneum and tissues surrounding the liver
   o Characterized as “violin string” adhesions in the liver capsule
   o Occurs almost exclusively in women

Question 75

GRAM NEGATIVE COCCI:
NEISSERIA GONORRHOEAE - Clinical Findings
**Syndromes in Males and Females
1. Pharyngitis
o For those who practice fellatio (oral sex)
o Show a purulent pharyngeal exudate
o Viral fever-like symptoms
2. Anorectal
o Constipation and purulent discharge for those who practice anal sex
o Rectal colonization
o Painful defecation

Answer 75

3. Ophthalmia neonatorum
   o occurs in newborns who are delivered through the birth canal of infected mothers
   o Initial conjunctivitis rapidly progresses and, if left untreated, may result to blindness
   o May be prevented by tetracycline, erythromycin ointment or 1% silver nitrate
4. Disseminated Gonococcal Infection (DGI)
   o Leads to skin lesions (hemorrhagic papules and pustules) on the hands, forearms, feet and legs
   o Also leads to tenosyvitis and suppurative arthritis usually of the knees, wrists and ankles.
   o Septic arthritis: fever, chills, skin lesion

Question 76

GRAM NEGATIVE COCCI:
NEISSERIA GONORRHOEAE - Diagnostic Laboratory Tests
 Specimen
o Pus and secretions from urethra, cervix, rectum, conjunctiva, throat, or synovial fluid for culture and smear.
o Blood or synovial fluid if disseminated infection
 Smears
o Gram-stained smears of urethral or endocervical exudates will reveal diplococci within pus cells
 Culture
o Culture at 37oC with 5% CO2
1. Thayer Martin medium (vancomycin, colistin, nystatin)
2. Modified Thayer-Martin medium (V,C,N) + Trimethoprim (proteus)
3. Stuart’s medium (transport medium)
4. Chocolate agar

Answer 76

Biochemical tests:
**N. Meningitidis = (+) maltose, glucose and oxidase
**N. Gonorrhea = (+) glucose oxidase
 Nucleic Acid Amplification (PCR)
o Preferred tests for genitourinary specimens
 Serology
o Detects antibodies in the serum (ELISA)
o RIA
o Immunofluorescence

Question 77

GRAM NEGATIVE COCCI: 
NEISSERIA GONORRHOEAE - Treatment
**For uncomplicated urogenital, anorectal, pharyngea
1. Ceftriaxone 125 mg, IM, Once
2. Ciprofloxacin 500 mg, Orally, Once
3. Ofloxacin 400 mg, Orally, Once

Answer 77

If chlamydial infection is not ruled out, plus:
1. Azithromycin 1 g, Orally, Once
2. Doxycyclin 100 mg, Orally, 2x a day for 7 days
 All sexual partners should be treated
 Abstain from sex until all organisms have been cleared

Question 78

GRAM NEGATIVE COCCI:

NEISSERIA GONORRHOEAE - Prevention and Control

Answer 78

 Avoid having multiple sexual partners
 Rapid eradication of the bacteria from infected individuals by early treatment and diagnosis
 Condoms
 Gonococcal ophthalmia neonatorum prevented by local application of 0.5% erythromycin ophthalmic ointment or 1% tetracycline ointment to conjunctiva of newborns