Seabolt Bio 209 Exam 3

Front 1

Characteristics of streptococci

Back 1

Gram positive cocci in chains
Catalase negative
Fastidious and require enriched media
Responsible for more infections than any other group of organisms

Front 2

Classification of streptococci

Back 2

Hemolytic activity
Alpha (incomplete) – Streptococcus viridans, S. pneumoniae, S. oralis
Beta (complete) – S. pyogenes, S. agalactiae, S. equisimilis
Non-hemolytic or Gamma (few are pathogenic)

Serological Basis

Group A, B, C, or D
(C not seen in class)

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Streptococcal Group A

Back 3

Isolated from man
S. pyogenes (beta) – cause of numerous diseases including pharyngitis (strep throat), impetigo (skin infection), scarlet fever (erythrogenic toxin production).
Post streptococcal diseases (i.e. complications with immune mechanisms) – rheumatic fever, glomerulonephritis

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Streptococcal Group B

Back 4

Isolated from cattle, man.
S. agalactiae (beta) – In genital tract of 15-30% of all women (causes UTIs). Causes diseases of newborns (septicemia, meningitis, respiratory distress syndrome – 10,000 cases per year with 15-20% mortality

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Streptococcal Group C

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Isolated from lower animals
S. equisimilis (beta) and others.
Pathogens of animals
Occasionally causes pharyngitis, sinusitis, bacteremia, endocarditis in man.

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Streptococcal Group D

Back 6

Intestinal tract of man and animals
Enterococci. Enterococcus faecalis (alpha or non-hemolytic) and S. bovis (alpha or non-hemolytic).
Causes endocarditis, UTIs, and wound infections.

Front 7

Virulence Factors of Streptococci

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A. Hemolysins – dissolve red blood cells
B. Leucocidins – destroy leukocytes
C. Erythrogenic toxin (Group A) – Scarlet Fever.
D. Hyaluronidase – dissolves hyaluronic acid (the cement of connective tissue).
E. Streptokinase – dissolves blood clots.
F. Nucleases – depolymerize DNA.

Front 8

Tests used to differentiate streptococci

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A. Hemolysis
B. Bacitracin
C. Camp (Christi, Atkins, and Munch-Peterson, 1944) and Sodium Hippurate
D. Bile Esculin Test
E. 6.5% NaCl Broth or SF medium

Front 9

Camp
Christi, Atkins, and Munch-Peterson, 1944

Back 9

Group B, S. agalactiae, produces peptide which acts synergistically with β-hemolysin of S. aureus to produce an enhanced zone of hemolysis

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Soduim Hippurate

Back 10

S. agalactiae produces an enzyme called hippuricase, which other beta-hemolytic streptococci lack.
Hippuricase hydrolyzes sodium hippurate into two products, sodium benzote and glycine.
Ninhydrin reagent is used to identify glycine product.

Front 11

Bacitracin

Back 11

place disc (0.04 units) on area of inoculation

Group A – inhibition of growth

Group B and C – growth around disc

Front 12

Hemolysis

Back 12

Stab inoculate blood agar to demonstrate ‘O’ hemolysin

Front 13

Bile Esculin Test

Back 13

Group D Alpha/Gamma Streptococci

Group D hydrolyze esculin (glycoside) to 6,7 dehydroxycoumarin (esculetin), which reacts with iron salts in the medium to produce a black color.

Front 14

6.5% NaCl Broth or SF medium

Back 14

Group D enterococci grow, but Groups A,B, and C (non-enterococci) do not grow

Front 15

Streptococcus pneumoniae

Back 15

Infectious – causes lobar pneumonia, bacteremia, otitis media, and meningitis.

Characteristics:

Gram positive diplococci (cocyloid) that is tapered or lancet-shaped.
Fastidious, and lyse spontaneously with age
Grown best on blood (alpha-hemolytic) and chocolate agar
Form polysaccharide capsules, associated with virulence resistant to phagocytosis
Serotypes (83) based on capsule composition.

Front 16

Tests to differentiate S. pneumoniae

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1. Optochin Test
2. Quellung (Neufield) reactions
3. Bile solubility test
4. Inulin fermentation test
5. Mouse virulence test

Front 17

Optochin Test

Back 17

S. pneumoniae is inhibited by the optochin or “P” disc (ethylhydrocupriene hydrochloride) at 5mg; other alpha hemolytic streptococci are not.

Front 18

Quellung (Neufield) reactions

Back 18

Capsular swelling reaction – sensitive method of detecting S. pneumoniae in sputum.
Pneumococci with capsules (specific polysaccharide) are mixed with capsular antiserum (of the same type). Capsule appears to swell around S. pneumoniae

Front 19

Staphylococci Characteristics

Back 19

Gram positive cocci in clusters
Catalase positive
20 species

Front 20

Tests used to identify staphylococcal organisms.

Back 20

A. Mannitol Salt
B. DNA + Methyl Green
C. Novobiocin Susceptibility
D. Coagulase

Front 21

Mannitol Salt

Back 21

7.5% NaCl, Phenol Red, Mannitol

Front 22

DNA + Methyl Green

Back 22

pH 7.5
Stable Complex of polymerized DNA and methyl green

DNase Nucleotides + Phosphate
DNA Methyl green released, fades at
hydrolysis pH 7.5 (clear zone–DNase produced)

Front 23

Novobiocin Susceptibility

Back 23

5μg disc.
Mueller Hinton Agar.

Front 24

Coagulase

Back 24

Mix plasma and bacteria on slide

Clumping indicates organism is positive for coagulase.

Front 25

Three species most frequently encountered in medical microbiology

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Staphylococcus aureus
Staphylococcus epidermidis
Staphylococcus saprophyticus

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Staphylococcus aureus

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Coagulase positive.

DNase positive

Mannitol positive

Novobiocin sensitive.

Front 27

Staphylococcus epidermidis

Back 27

Coagulase negative

DNase negative

Mannitol negative

Novobiocin sensitive

Front 28

Staphylococcus saprophyticus

Back 28

Implicated in UTIs in sexually active young females.

S. saprophyticus

Coagulase negative

DNase negative

Mannitol positive/negative

Novobiocin resistant.

Front 29

Staphylococcus aureus
Additional Info

Back 29

Causal organism of acne, boils, carbuncles, impetigo (skin infection), pneumonia, osteomyelitis (bone inflammation), endocarditis (inflammation of heart lining), pyelonephritis (kidney inflammation), food poisoning (staphylococcus enterotoxin is resistant to boiling for 20 minutes).

Front 30

Virulence Factors of Staphylococcus aureus

Back 30

Leukocidins – lyse leukocytes (WBC).

Hemolysins – lyse erythrocytes (RBC).

Coagulase – clots plasma (walls off organisms).

Enterotoxin – (staphylococcal enteritis) ingestion of toxin in foods includes bakery, meat, and dairy products

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Kidneys (2, each housing an adrenal gland). Two routes of infection

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Descending (hematogenous) M. tuberculosis, S. aureus, Salmonella spp.
Ascending (via anterior urethra to bladder to ureters to kidney

Front 32

Urethra

Back 32

Urinary tract is nearly always invaded from exterior, via urethra (first 2-3 cm of anterior urethra well colonized with bacteria)

UT infections often begin by colonization of mucosa around the urethra.

Bacteria are usually removed by flushing action of urination. Barriers (stones, enlarged prostate gland, tumors, neurogenic diseases) to free flow contribute to UTI’s.

Front 33

Causal organisms of UTIs

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1. Escherichia coli (80%)
2. Staphylococcus saprophyticus (5-15%)
3. Klebsiella and Proteus mirabilis (occasionally)

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Diagnosis of UTI

Back 34

a urine colony count can be used to establish diagnosis.
Colony count standards:

10^5/mL – significant bacteriuria – UTI confirmed

10^4/mL – with pyuria – suspicious for UTI

10^2-10^3/mL – absence of symptoms – typical contamination of UT.

Front 35

Normal Microbial flora of the Mouth

Back 35

The mouth is colonized with lactobacilli, micrococci, streptococci, yeast, coliforms, viruses, protozoa, and corynebacteria

Staphylococci and pneumococci (air)
Bacteroides, Fusobacterium (feeding and contact with others).

Front 36

Dental Caries (Tooth Decay)

Back 36

Caused by interaction with cariogenic microorganisms.

Organism’s nutrition comes from host’s diet
Main cause is sucrose (table sugar).
Broken down by dextransucrase to glucose and fructose
Forms sticky polymers that allow organisms to bind and form colonies.
Fructose fermentation forms lactic acid, which causes decalcification and softening of dental enamel.

Front 37

Causal Organisms of Tooth Decay

Back 37

1.Streptococcus mutans (most important species)
2.Lactobacillus acidophilus
3.Actinomyces odontolyticus

Front 38

Snyder Test

Back 38

Collect saliva and use to inoculate molten Snyder Agar Deep
Snyder Agar
pH 4.7
Contains glucose and brom cresol green
At pH 4.4 (level at which dental caries form) medium turns yellow
Yellow color indicates acid production by microorganisms
Cultures that turn yellow within 24-48 hours suggest the host is extremely susceptible to tooth decay.

Front 39

Normal Flora of the Throat and Skin

Back 39

Human body contains 10^14 cells, 90 % of which are bacteria.

Front 40

Skin Normal Flora

Back 40

Staphylococci (S. epidermidis), Streptococci, diphtheroids, bacilli, yeast, and mold.

Salt tolerant.

Front 41

Eye conjunctiva

Back 41

Staphylococci, diptheroids, Neisseria

Front 42

Upper Respiratory Tract

Back 42

Mucous membrane of and pharynx are sterile at birth, but colonization with Streptococcus occurs within 4-12 hours.
Aerobic and anaerobic Staphylococci, diptheroids, and members of Neisseria, Branhamella, Haemophilus.

Front 43

Mouth and Teeth

Back 43

Anaerobic organisms including spirochetes, vibrios, and staphylococci

Front 44

Intestinal Tract

Back 44

Sterile at birth
Upper intestine – lactobacilli and enterococci.
Lower intestine and colon
100 distinct types (1011/gm of contents) in sigmoid colon and rectum
96 – 99% anaerobes: Bacteriodes, Clostridium, Lactobacillus, Streptococcus.
1-4% aerobic: coliforms, Proteus, Pseudomonas, yeast.

Front 45

Urinary Tract

Back 45

Usually kidneys and bladder are sterile.
Anterior urethra colonized with bacteria.

Front 46

Genital Tract

Back 46

Normal flora in females mostly lactobacilli and usually acidic due to glycogen metabolism.
Normal flora includes streptococci, anaerobic organisms (clostridia and bacteriodes), gram negative bacilli.

Front 47

Sabouraud’s Dextrose Agar

Back 47

pH 5.6

Selective for yeast and fungi

Throat – yeast (Candida spp.)

Front 48

Mannitol Salt

Back 48

7.4 % NaCl, selects for Staphylococci

Throat – S. aureus.

Skin – S. epidermidis

Front 49

Chocolate Agar

Back 49

Identify Neisseria

Oxidase test

Front 50

Immunology
Definition

Back 50

ability of an individual to resist infection by a particular microorganism due to natural (non-specific) or acquired (specific) defense mechanisms.

Front 51

Natural/Nonspecific defenses of the host

Back 51

Defenses that protect from any pathogen regardless of the species.
Mechanical barriers – skin and mucous membranes are primary defenses
Biochemical factors – sebum, sweat glands (produce perspiration, contains lysozyme, gastric juice in stomach (pH 1.2-3)).
Phagocytosis – ingestion of microorganism or any particulate matter by white blood cells

Front 52

Acquired/Specific Defense of the Host

Back 52

Acquired when individual comes in contact with a microorganism or other foreign substance that is antigenic (has the ability to cause production of proteins know as antibodies).

Specific immunity responds in two ways:

a.Cell-mediated (T lymphocytes)

b. Humoral – production of antibodies by plasma cells (B cells) in response to a specific antigen
Following exposure to specific antigen, individuals produce antibodies called immunoglobulin (eg. IgA, IgG)

Front 53

Diagnostic Immunology

Back 53

Involves the use of antigen-antibody interactions in the direct or indirect detection of antigens/disease

Most often, involving the use of antibodies to detect antigens or diagnose disease, inferring their presence by indirect methods.

Front 54

Agglutination

Back 54

clumping of an antigen

Antigen is mixed, in vitro, with its homologous antibody. Large (macroscopic) 3D lattice aggregates of antigen-antibody form.
Antibodies that combine with specific antigens are called agglutinins.
Performed on a slide or in a test tube

Front 55

Direct Agglutination

Back 55

detect Ab to cellular antigens; or, use Ab-decorated beads to detect cellular Ag

Front 56

Indirect Agglutination

Back 56

first, Ab reacts with antigen attached to latex beads; then, the particles agglutinate

Front 57

Hemagglutination (HA)

Back 57

antigen or antibody mediated clumping of red blood cells

Front 58

Complement Fixation Reactions

Back 58

designed to accurately measure amount of antigen or titer of antibody in a test reaction

Principle: For any normal immunological reaction, complement binds to any formed Ag-Ab complex and is depleted from the reaction (i.e. complement is “fixed”). Any leftover complement (i.e. in excess of antigen-antibody) remaining in a reaction can be measured by adding an “indicator,” e.g., sheep red blood cells plus anti-sheep red blood cell antibody. If complement is present, it can react with and lyse the indicator sheep red blood cells; on the other hand, if all the complement is fixed (indicating presence of antigen and antibody in the original test reaction), the indicator cells remain intact.

Front 59

Neutralization Reactions

Back 59

the harmful effects of a bacterial exotoxin or viral gene product are blocked by an antibody

Antitoxins: neutralizing antibody; antitoxins produced in animals can be injected into humans to provide passive immunity

Schick test: if a test serum has neutralizing antibodies against a virus, they will block viral-mediated hemagglutination

Front 60

Immunofluorescence

Back 60

identification of m/o, antigens using fluorescently-labeled antibodies

Fluorescent-antibody (FA) Techniques:

Direct: detect antigens, epitopes on or within organism with labeled antibody.

Indirect: detect presence/absence of antibodies:

Antigens + “test” serum (± antibodies); to visualize, add antibody conjugate

Front 61

Fluorescence-activated Cell Sorter (FACS)

Back 61

A type of flow cytometry in which different cells within a suspension are detected/separated based on the specific type of fluorescent antibody tag.

Front 62

Enzyme-linked Immunosorbent Assay (ELISA)

Back 62

Similar to fluorescence antibody technique except that the “tag” is an enzyme (that catalyzes the formation of a visible color change in solution) instead of fluorescence; the “matrix” is a microtiter plate.

Front 63

Direct ELISA: detection of antigens

Back 63

In a simple test: primary antibody-enzyme conjugate recognizes antigen

In an “antibody sandwich” test: secondary antibody-enzyme conjugate recognizes the antigen

Front 64

Indirect ELISA: detection of antibodies

Back 64

Antigen bound to the microtiter plate is recognized by any primary IgG antibodies present in the test antiserum; then, a secondary antibody-enzyme conjugate recognizes the primary IgG antibodies; the Ag-Ab complex is visualized by the enzymatic conversion of its substrate to a colored product.

HIV experiment**

Front 65

What does TITER means?

Back 65

the reason for the serial dilution is to determine the titer (relative amount) of antibody (if present); the last dilution in the series that still has some color is considered to be the endpoint titer: a semi-quantitative estimate of amount of antibody. High titers (for example, strong color even in the serum sample diluted 64-fold) correlates with recent infection and/or extremely virulent virus; low titers correlate with a much older infection and/or weak viral pathogen.

Front 66

Bacterial Control

Back 66

Bactericidal (or microbiocidal) – bacteria killing

Bacteriostatic (or microbiostatic) – bacterial growth inhibited

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Where control agents are used

Back 67

Antiseptics – used on (not in) living tissue to control bacterial growth

Disinfectants – used on inanimate objects to inhibit growth of vegetative cells (does not kill spores).

Chemotherapeutic agents – chemicals that destroy bacteria or inhibit growth inside living tissue (Example: Antibiotics).

Front 68

Antibiotics

Back 68

substances derived from living organisms. Usually bacteria, actinomycetes, or fungi (Eg. Penicillin).

Can be bactericidal or bacteriostatic

Classified by range of activity (i.e. how many kinds of microbes they affect.)

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Range of antibiotics

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Narrow range – an example – active against gram positive and a few gram negative bacteria.
Broad range – an example – active against many gram negative and gram positive bacteria.

Front 70

Requirements for Antimicrobial Efficacy

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Selective toxicity harms bacteria not patient.

Does not produce allergic reaction in patient.

Soluble in body fluids

Front 71

Method to determine antimicrobial effectiveness – Kirby Bauer Procedure

Back 71

Inoculate plate with bacteria, apply treatment and incubate.

Following incubation, measure size of the zone free of bacterial growth.

Compare with standard values to determine if bacteria are susceptible to the drug.

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Synergistic versus additive effects

Back 72

Synergistic – when two drugs are more effective than when alone

Additive – when the combined effect of using two drugs together is no better than using the drugs separately

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Advantages of using synergistic drug combinations

Back 73

Reduced incidence of bacterial resistance

Reduced toxicity (smaller dosages can be used).

Increase effectiveness.

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Experiment to determining synergistic effect  Two drug combinations

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Sulfisoxazole and trimethoprim – both affect folic acid synthesis, expect synergistic effect.

Trimethoprim and tetracycline – affect different bacterial processes, expect additive effect.

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How does resistance develop?

Back 75

Point mutation – one or more amino acid substitutions occur during translation. Protein may be inactive, altered or entirely different.

Spontaneous mutations occur at a frequency of 1 x 10-7. (In 10 million bacteria, one cell will possess a different genotype.)

Drug resistance genes can be transferred through transformation, transduction, and conjugation.

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Mutation mechanisms

Back 76

Enzymatic alteration in chemical structure of antibiotic (Example: Beta-lactamases)
Change in selective permeability of cell membranes. (Example: Aminoglycosides cannot cross cytoplasmic membrane.)
Decrease in sensitivity of bacterial enzymes to inhibiting mechanisms. (Example: a bacterium becomes less sensitive to Streptomycin, which interferes with the bacteria’s translation process at the ribosome)
Overproduction of a natural substrate

Front 77

E-Tests

Back 77

type of antimicrobial diffusion test that allows one to detect the minimal inhibitory concentration (MIC) of an antibiotic against a particular organism

Front 78

Beta Lactamase Testing

Back 78

Pencillins and cephalosporins are
known as beta-lactamase antibiotics due to presence of a beta lactam ring
1. Work by preventing cell wall synthesis
2. Bacterial cells become sensitive to osmotic changes and cell lysis
3. Organisms producing beta lactamase are resistant to penicillin and cephalosporin (Most common example of antibiotic resistance)
4. Many bacterial isolates are tested. eg. Neisseria gonorrhoeae and Haemophilus influenzae

Front 79

Beta Lactamase Summary

Back 79

The beta-lactamase test is used to identify those bacteria that produce the enzyme beta-lactamase, which hydrolyzes the beta-lactam ring in penicillin and cepalosporin, rendering the antibiotics ineffective.

Front 80

Factors that affect antiseptic / disinfectant effectiveness

Back 80

- Concentration
- Length of exposure
- Type of microbial population
- Environmental conditions: temp, pH
- Type of material on which microbes exist
- blood, pus, tissue fluids can interact with antimicrobial agent and reduce effectiveness

Front 81

Phenol Coefficent

Back 81

Standard test for antimicrobial effectiveness
Antimicrobial agent is “normalized” against phenol

PC = [concentration of antimicrobial that kills in 10', but not 5']
------------------------------------------------------------------------
[concentration of phenol that kills in 10', but not 5']