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.

118
119
119
120
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
120
121

• 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.

121
122
E-Test Examples
122
123
123
124
124
125
Beta Lactamase Testing
125
126

• 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

126
127
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.
127
128
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.
128
129

• Antimicrobial Resistant Mutants
• Microorganisms have the ability to mutate and
  become resistant to antibiotics. Mutations allow
  for bacteria to circumvent antimicrobial effects
  of drugs.

129
130
Staphylococcus is resistant to m ost antibiotics
130
131

• 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.

131
132

• 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.

132
133

• 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.

133
134

• 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.

134
135
Streptomycin Resistant Mutants
135
136

• 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

136
137

• 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

137
138

• 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

138
139
END UNIT 3
139