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Streptococcus

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Title: Streptococcus


1
UNIT III
Streptococcus Staphylococcus Immunology Chemic
al control of microorganisms Antibiotic
susceptibility testing
1
2
  • Streptococcal Pathogens
  • Characteristics of streptococci
  • Gram positive cocci in chains (divide in one
    plane).

2
3
  • Catalase negative (differentiates from
    staphylococci and micrococci).
  • Fastidious and require enriched media (blood,
    chocolate).
  • Responsible for more infections than any other
    group of organisms.

3
4
  • Classification of streptococci
  • 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)

4
5
Alpha Hemolysis
5
6
Beta Hemolysis
6
7
7
8
  • Serological Basis (Lancefield Groups)
  • Lancefield Groups divided by serological
    testing of extractable carbohydrate (C-substance)
    in the cell wall with antiserum produced in
    rabbits.
  • Groups are used in medical field to describe
    isolates and diseases.
  • A, B, C, D are most frequently isolated
  • Groups correlate with source

8
9
  • Streptococcal Groups
  • Group A
  • 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.

9
10
Group A
Streptococcus (group A) Rheumatic Fever 
Streptococcus pyogenes
10
11
  • Group B
  • 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.)

11
12
Group B
Streptococcus agalactiae
Streptococcus agalactiae
12
13
  • Group C
  • Isolated from lower animals
  • S. equisimilis (beta) and others.
  • Pathogens of animals
  • Occasionally causes pharyngitis, sinusitis,
    bacteremia, endocarditis in man.

13
14
Group C
14
15
Group D
Enterococcus faecalis
15
16
  • Group D
  • Intestinal tract of man and animals
  • Enterococci. Enterococcus faecalis (gamma or
    non-hemolytic) and S. bovis (gamma or
    non-hemolytic).
  • Causes endocarditis, UTIs, and wound infections.

16
17
  • Virulence Factors of Streptococci
  • Hemolysins dissolve red blood cells
  • Leucocidins destroy leukocytes
  • Erythrogenic toxin (Group A) Scarlet Fever.
  • Hyaluronidase dissolves hyaluronic acid (the
    cement of connective tissue).

17
18
  • Streptokinase dissolves blood clots.
  • Nucleases depolymerize DNA.

18
19
  • Tests used to differentiate streptococci
  • Hemolysis Stab inoculate blood agar to
    demonstrate O hemolysin
  • Bacitracin place disc (0.04 units) on area of
    inoculation.
  • Group A inhibition of growth
  • Group B and C growth around disc

19
20
Bacitracin
20
21
  • Camp (Christi, Atkins, and Munch-Peterson, 1944)
    and Sodium Hippurate
  • Camp Group B, S. agalactiae, produces peptide
    which acts synergistically with ß-hemolysin of S.
    aureus to produce an enhanced zone of hemolysis.
  • Soduim Hippurate 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.

21
22
CAMP Test
22
23
  • Bile Esculin Test
  • 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.
  • 6.5 NaCl Broth or SF medium
  • Group D enterococci grow, but Groups A,B, and C
    (non-enterococci) do not grow

23
24
  • Streptococcus pneumoniae
  • 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.

24
25
25
26
  • Tests to differentiate S. pneumoniae
  • Optochin Test S. pneumoniae is inhibited by the
    optochin or P disc (ethylhydrocupriene
    hydrochloride) at 5mg other alpha hemolytic
    streptococci are not.

P
26
27
Optochin Test
27
28
  • Classical tests for identification
  • A. Quellung (Neufield) reactions
  • 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.
  • Bile solubility test
  • Inulin fermentation test
  • Mouse virulence test

28
29
  • Staphylococcal Pathogens
  • Staphylococci Characteristics
  • Gram positive cocci in clusters (divide in 2 or
    more planes).

29
30
  • Catalase positive (Streptococci negative)
  • 20 species

30
31
  • Tests used to identify staphylococcal organisms.
  • Mannitol Salt
  • 7.5 NaCl, Phenol Red, Mannitol.

31
32
  • DNA Methyl Green
  • 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 zoneDNase
    produced)

32
33
  • Novobiocin Susceptibility
  • 5µg disc.
  • Mueller Hinton Agar.

33
34
  • Coagulase
  • Mix plasma and bacteria on slide
  • Clumping indicates organism is positive for
    coagulase.
  • Coagulase
  • Plasma Fibrin Clot
  • (fibrinogen) or clumps

34
35
35
36
  • Three species most frequently encountered in
    medical microbiology.
  • Staphylococcus aureus
  • 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).

36
37
  • Virulence Factors
  • 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

37
38
  • Non-toxic factors
  • DNase depolymerization of DNA.
  • Lipases gelatinase, staphylodinase (dissolves
    clots).

38
39
  • S. aureus
  • Coagulase positive.
  • DNase positive
  • Mannitol positive
  • Novobiocin sensitive.

39
40
Staphylococcus aureus
40
41
Staphylococcus aureus
41
42
  • Staphylococcus epidermidis
  • Predominant organism on skin and mucosal
    surfaces.
  • Normally non-pathogenic, but produces diseases
    when penetrates skin.
  • Grows well on biosynthetic material (prosthetic
    devices joints and heart valves) producing
    endocarditis and skin lesions.

42
43
  • S. epidermidis
  • Coagulase negative
  • DNase negative
  • Mannitol negative
  • Novobiocin sensitive

43
44
Staphylococcus epidermidis
44
45
  • Staphylococcus saprophyticus
  • Implicated in UTIs in sexually active young
    females.
  • S. saprophyticus
  • Coagulase negative
  • DNase negative
  • Mannitol positive/negative
  • Novobiocin resistant.

45
46
Staphylococcus saprophyticus
46
47
DIRECT AGGLUTINATION FOR THE IDENTIFICATION OF
STAPHYLOCOCCUS AUREUS Although the coagulase
test has long been recognized as a principal tool
in the identification of S. aureus, it is a slow
test that can take as long as 24 hours to become
positive. S. aureus can be differentiated by a
rapid slide agglutination procedure using latex
particles coated with antibody specific for the
protein A component of the cell wall that is
unique to S. aureus. When S. aureus is mixed with
the Staphylococcus Reagent, agglutination
(clumping of particles) will be visible to the
naked eye.
47
48
Test Method If fewer than six test are
performed, the test card may be cut with scissors
and the unused portion saved for later use. 1.
Mix the latex reagent by shaking expel any latex
from the dropper for complete mixing. 2.
Dispense 1 drop of Test Latex onto one of the
circles on the reaction card and 1 drop of
Control Latex onto another circle.
48
49
3. Using an applicator stick to pick up and
smear 5 suspect colonies onto the Test Latex
containing circle and mix into the Test Latex
reagent. Spread to cover the circle. 4. Repeat
step 3 for the Control Latex 5. Pick up and
hand rock the card for up to 20 sec and observe
for agglutination under normal lighting
conditions. Read macroscopically do not use a
magnifying glass.
49
50
6. Dispose of the reaction card in an
appropriate biohazard container. 7. Re-cap the
bottles.
50
51
  • NORMAL MICROBIOTA DISCUSSION
  • UTIs account for more than 7 million visits to
    physicians offices and complicate well over 1
    million hospital admissions annually in the US.

51
52
  1. Urinary Tract System

52
53
  • Kidneys (2, each housing an adrenal gland). Two
    routes of infection
  • Descending (hematogenous) M. tuberculosis, S.
    aureus, Salmonella spp.
  • Ascending (via anterior urethra to bladder to
    ureters to kidney
  • Ureters (connect kidneys to bladder)
  • Bladder

53
54
54
55
  • Urethra
  • 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 UTIs.

55
56
Urethra
56
57
  • Facts about UTIs
  • UTIs are 14 times more common in females than
    males
  • Causal organisms
  • 1. Escherichia coli (80)
  • 2. Staphylococcus saprophyticus (5-15)
  • 3. Klebsiella and Proteus mirabilis
    (occasionally)

57
58
  • Symptoms
  • Suprapubic pain and tenderness
  • Increased urinary frequency and urgency
  • Dysuria (painful urination)
  • Urine shows presence of leukocytes (pyuria)
  • Inflammation of the bladder (cystitis)
  • Inflammation of the kidney and renal pelvis
    (pylelonephritis)

58
59
  • Diagnosis
  • Urine becomes heavily contaminated during
    passage therefore, a urine colony count can be
    used to establish diagnosis.
  • Colony count standards
  • 105/mL significant bacteriuria UTI confirmed
  • 104/mL with pyuria suspicious for UTI
  • 102-103/mL absence of symptoms typical
    contamination of UT.

59
60
Urinary Tract Infection
Positive plate for UTI
60
61
  • Normal Microbial flora of the Mouth
    Susceptibility to dental carries.
  • 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).

61
62
  • Saliva
  • Contains millions of bacteria
  • pH range is 5.7 7.0 (average is 6.7)

62
63
Normal Mouth Flora
S. epidermidis
S. aureus
Streptococcus mutans
63
64
  • Dental Caries (Tooth Decay)
  • Caused by interaction with cariogenic
    microorganisms.
  • Organisms nutrition comes from hosts 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.

64
65
Dental Carries
65
66
  • Causal Organisms
  • Streptococcus mutans (most important species)
  • 2. Lactobacillus acidophilus
  • 3. Actinomyces odontolyticus

66
67
  • Determining host susceptibility to dental caries
    and identifying organisms of the mouth.
  • Procedure (Snyder Test)
  • 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.

67
68
Synder Agar
Tube 1  Uninoculated Synder tube Tube 2  No
color change indicates little or no
susceptibility to forming dental caries Tube 3 
Sight color change indicates mild susceptibility
to forming dental caries Tube 4  Significant
color change indicates moderate susceptibility to
forming dental cariesTube 5  Complete color
change indicates high susceptibility to forming
dental caries.
68
69
  • Normal Flora of the Throat and Skin
  • Human body contains 1014 cells, 90 of which are
    bacteria.
  • Skin
  • Staphylococci (S. epidermidis), Streptococci,
    diphtheroids, bacilli, yeast, and mold.
  • Salt tolerant.

69
70
  • Eye conjunctiva
  • Staphylococci, diphtheroids, Neisseria
  • Upper Respiratory Tract
  • 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.

70
71
  • Mouth and Teeth
  • Anaerobic organisms including spirochetes,
    vibrios, and staphylococci.
  • Intestinal Tract
  • 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.

71
72
  • Urinary Tract
  • Usually kidneys and bladder are sterile.
  • Anterior urethra colonized with bacteria.
  • Genital Tract
  • 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.

72
73
  • Procedures for isolating normal flora of the
    throat and skin
  • Obtain sample by swabbing.
  • Culture on various media.
  • Blood Agar
  • Identifies alpha and beta hemolysis
  • Streptococci and Staphylococci

73
74
  • Sabourauds Dextrose Agar
  • pH 5.6
  • Selective for yeast and fungi
  • Throat yeast (Candida spp.)

Candida susceptibility test
74
75
  • Mannitol Salt
  • 7.4 NaCl, selects for Staphylococci
  • Throat S. aureus.
  • Skin S. epidermidis

Selective for Staphylococcus. Differentiates
between S. aureus and S. epidermidis. S. aureus
is able to ferment Mannitol.
75
76
  • Chocolate Agar (enriched medium)
  • Identify Neisseria
  • Oxidase test

76
77
  • Immunology
  • Definition ability of an individual to resist
    infection by a particular microorganism due to
    natural (non-specific) or acquired (specific)
    defense mechanisms.

77
78
  • Natural/Nonspecific defenses of the host
  • 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

78
79
  • Acquired/Specific Defense of the Host
  • 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
  • Cell-mediated (T lymphocytes)
  • 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)

79
80
  • Diagnostic Immunology
  • 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.

80
81
  • Definition detection and study of
    antigen-antibody reactions in vitro.
  • Used in diagnosis of clinical diseases.
  • Examples of serological tests include
    agglutination, precipitation, complement
    fixation, ELISA, etc

81
82
  • Agglutination (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

82
83
Agglutination
83
84
  • Agglutination reactions involve reaction of
    antibodies to particulate or soluble antigens
    bound to particles or beads or to cellular
    antigens detection by clumping of Ag Ab complex
  • Direct detect Ab to cellular antigens or, use
    Ab-decorated beads to detect cellular Ag
  • Indirect first, Ab reacts with antigen attached
    to latex beads then, the particles agglutinate
  • Hemagglutination (HA) antigen or antibody
    mediated clumping of red blood cells

84
85
Direct Agglutination
85
86
  • Immunofluorescence 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

86
87
Direct Fluorescent-antibody
87
88
Indirect Fluorescent-antibody
88
89
89
90
  • Fluorescence-activated Cell Sorter (FACS)
  • A type of flow cytometry in which different cells
    within a suspension are detected/separated based
    on the specific type of fluorescent antibody tag.

90
91
Fluorescence-activated Cell Sorter
91
92
  1. Enzyme-linked Immunosorbent Assay (ELISA)
    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.

92
93
  • Direct detection of antigens
  • In a simple test primary antibody-enzyme
    conjugate recognizes antigen
  • In an antibody sandwich test secondary
    antibody-enzyme conjugate recognizes the antigen

93
94
Direct ELISA
94
95
  • Indirect detection of antibodies
  • 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.

95
96
Indirect ELISA
96
97
Indirect ELISA test for the identification of HIV
Antibodies Introduction The indirect ELISA test
(enzyme-linked immunosorbent assay) is a
screening test that is currently used to detect
the presence of antibodies to HIV (it is not a
direct test for the presence of the virus). The
principle steps involved are outlined next
97
98
I. Reaction of antigen with putative (suspected)
antibodies in a test (patient) serum 1. Binding
of antigen to the wells of a microtiter plate.
HIV specific antigens are chemically adsorbed to
the plastic wells of a microtiter plate
(typically, this is a plastic plate that has 96
wells arranged in a 8 horizontal row by 12
vertical column configuration). The wells of
the microtiter plate that are used in this
exercise have already been coated.
98
99
2. Addition of Antiserum to the wells of a
microtiter plate. An example of a basic clinical
method is to place a drop of blood on a piece of
clean filter paper. The sample is placed in a
microtiter well that has previously been coated
with HIV antigens and allowed to incubate. Any
HIV primary antibodies present in the blood
sample will then bind to the antigens on the
surface of the well. Alternatively, fresh
antisera can be directly added to the wells
thats what the assay in the lab calls for. In
either case, the well is then usually rinsed to
wash away any unbound antibodies.
99
100
NOTE In this exercise, you are to add two
different sera to appropriately lettered rows
and, you make a serial dilution of the sera. You
will not wash out the unbound antibodies
100
101
Heres how the serial dilution is made 1. Add
three drops of serum to well 1 for each serum
sample used the row that matches the letters on
the patient serum assigned to you 2. Add three
drops of water to wells 2-7 for each of the
serum samples 3. Add three drops of serum to
well 2, mix (makes a 12 dilution) withdraw
three drops of mixed sample transfer to well 3.
101
102
4. Mix the transferred drops with the water
already in well 3 (makes a 14 dilution)
withdraw 3 drops transfer to well 4. 5.
Repeat step 4 until you have serially diluted the
sample serum to well 7 discard the last three
drops after youve completed the mixing step in
well 7 (making a 164 dilution).
102
103
NOTE 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.
103
104
II. Visualization of the Antigen-Antibody
Complex A. Addition of Secondary
Antibody-Enzyme Conjugate In order to
visualize antigen-antibody reaction, the human
antibody (from the serum) is recognized by a
second set of antibodies (secondary antibodies
these secondary antibodies are raised in closely
related but non-human sources (e.g., goat sheep,
mouse).
104
105
These anti-human IgG antibodies will attach to
the HIV antibodies that are already bound to the
HIV antigens on the well surface, creating a
sandwich with the HIV antibodies in the middle
(HIV antigen HIV antibody conjugated
antibody). The well is then usually rinsed to
wash away any unbound secondary antibody enzyme
conjugate If there are no HIV antibodies in
the patients sample, the conjugated antibodies
will be washed away. This results in the lack of
color development.
105
106
NOTE In this exercise, you will add the 1
drops of conjugate to the same wells to which you
have already added the patient sera once again,
you will not wash out the unbound antibody.
106
107
B. Addition of Substrate chromogen. The last
step is to add 1 drop substrate chromogen to
the wells. This substrate will undergo a chemical
reaction when it comes in contact with its
enzyme, and will change color. If the patient has
HIV antibodies, the HIVantigen-HIVantibody
complex will be detected when this substrate is
added a dark orange or red color is positive for
HIV antibodies a light yellow or clear color is
a negative test result.
107
108
  • I. Bacterial Control
  • Chemical classification
  • Bactericidal (or microbiocidal) bacteria
    killing
  • Bacteriostatic (or microbiostatic) bacterial
    growth inhibited.

108
109
  • Where control agents are used
  • 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).

109
110
  • II. Antimicrobial Agents
  • A. Antibiotics 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.)

110
111
  • 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.

111
112
  • B. Synthetic Antimicrobial Agents substances
    created (synthesized) in the laboratory.
  • Requirements for Antimicrobial Efficacy
  • Selective toxicity harms bacteria not patient.
  • Does not produce allergic reaction in patient.
  • Soluble in body fluids

112
113
D. Modes of action of antimicrobial agents
Mode of Action Example
Inhibits cell wall synthesis Penicillin, bacitracin, vancomycin
Alters cytoplasmic membrane Polymyxins
Inhibits protein synthesis Tetracycline, chloramphenicol, streptomycin, gentamicin
Competitive inhibition Sulfonamides- bind to active folate synthetase site where para-aminobenzoic acid should bind (prevents folic acid synthesis) Trimethoprim also inhibits the same pathway
113
114
  • Method to determine antimicrobial effectiveness
    Kirby Bauer Procedure
  • 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.

114
115
115
116
  • Synergistic Effect of Drug Combinations
  • Synergistic versus additive effects
  • 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.

116
117
  • Advantages of using synergistic drug
    combinations
  • Reduced incidence of bacterial resistance
  • Reduced toxicity (smaller dosages can be used).
  • Increase effectiveness.

117
118
  • Experiment to determining synergistic effect ?
    Two drug combinations
  • 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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MIC/MBC
MIC (Minimum Inhibitory Concentration) The
lowest concentration of an antibiotic needed to
inhibit the growth of bacteria MBC (Minimum
Bactericidal Concentration) The lowest
concentration of an antibiotic needed to kill
bacteria Video of MIC/MBC test http//www.medsch
ool.lsuhsc.edu/Microbiology/Flash/MICMBC.htm
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  • E-Tests
  • Designed and produced by a Swedish company,
    E-Tests are a type of antimicrobial diffusion
    test that allows one to detect the minimal
    inhibitory concentration (MIC) of an antibiotic
    against a particular organism. This is a much
    better indication of the in vivo effect of an
    antibiotic.
  • MIC is determined by detecting where the growth
    of organism intercepts the numbered strip. For
    example, if the growth intercepted the number
    256, then the MIC will be reported as 256
    micrograms of the particular antibiotic, which
    would be needed to inhibit the organism in vivo.

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E-Test Examples
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Beta Lactamase Testing
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  • Principle
  • A. 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

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II. Application
A. Beta lactamase test determines if
bacterial isolate is resistant to B-
lactam antibiotics B. Substrate is
nitrocefin-turns pink when it is hydrolyzed.
Impregnated disk is coated with the
bacterium. Other methods.
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III. Summary
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 cephalosporin,
rendering the antibiotics ineffective.
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  • Antimicrobial Resistant Mutants
  • Microorganisms have the ability to mutate and
    become resistant to antibiotics. Mutations allow
    for bacteria to circumvent antimicrobial effects
    of drugs.

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Staphylococcus is resistant to m ost antibiotics
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  • How does resistance develop?
  • 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
  • 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 bacterias translation
    process at the ribosome)
  • Overproduction of a natural substrate.

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  • Practical application of this lab
  • In clinical settings, diseases (Tuberculosis, for
    example) are usually treated with two or more
    drugs.
  • The probability that one M. tuberculosis cell
    will mutate is about 1106. The probability that
    the organism is resistant to two drugs is
    1 x 10-10.

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  • A patient instructed to take antibiotics for 10
    days will frequently discontinue medication
    before that period is complete.
  • Why is that bad? The drug first kills off
    susceptible bacteria. By not continuing the
    treatment, the patient selects for mutants which
    may be resistant to antibiotics. These mutants
    can cause subsequent, and potentially more
    severe, infections.

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Streptomycin Resistant Mutants
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  • Mutationchange in base sequence of a gene one
    source of genetic variability
  • Change enables cell to survive in adverse
    conditions e.g.. antibiotic resistance
  • Major clinical importance
  • Increased number of bacterial resistance strains
    due to overuse and misuse
  • Select for drug resistant strains by their cidal
    effects on several cell types
  • These agents select for resistant mutants and do
    not act as inducers for the mutation

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  • Ways in which drug resistant organisms circumvent
    the cidal effects of a drug
  • Production of an enzyme that alters the chemical
    structure of the antibiotic, e.g.. penicillin
    resistance
  • Change in selective permeability of the cell
    membrane, e.g.. streptomycin resistance
  • Decrease in the sensitivity of the organisms
    enzymes to inhibiting mechanisms such as strep
    resistance which interferes with translation at
    the ribosomes
  • Overproduction of a natural substrate to compete
    effectively with the drug, e.g.. resistance to
    sulfas

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  • Isolation of a streptomycin resistant mutant from
    a wild type E. coli by gradient plate technique
  • Requires preparation of the double layered agar
    plate
  • The lower slanted medium lacks streptomycin
  • Molten agar containing streptomycin is poured
    over the initial layer
  • The E. coli is inoculated onto the final surface
  • Colonies of a region of high streptomycin
    concentration is indicative of streptomycin
    resistant mutants

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END UNIT 3
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