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Antimicrobial Drugs

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Title: Antimicrobial Drugs


1
Antimicrobial Drugs
  • Chemotherapy The use of drugs to treat a
    disease
  • Antimicrobial drugs Interfere with the growth of
    microbes within a host
  • Antibiotic Substance produced by a microbe
    that, in small amounts, inhibits another
    microbe
  • Selective toxicity A drug that kills harmful
    microbes without damaging the host

2
  • 1928 Fleming discovered penicillin, produced
    by Penicillium.
  • 1940 Howard Florey and Ernst Chain performed
    first clinical trials of penicillin.

Figure 20.1
3
Table 20.1
4
  • ANTIMICROBIAL SENSITIVITY TESTS
  • _at_ Used to select effective drugs for treatment .
  • _at_ Not performed on commensals or contaminants
  • _at_ This misleads physician and patient to receive
    unnecessary therapy
  • _at_ Such therapy leads to side-effects resistance
    of pathogens.
  • _at_ Used to identify organism if it has a
    characteristic sensitivity pattern.

5
  • LIMITATIONS
  • Measure in vitro not in vivo drug activity.
  • Selection of best drug depends on
  • a) Patient clinical condition
  • b) Type and site of infection.
  • c) History of drug hypersensitivity.
  • 3.Drug activity absorption, diffusion in
    tissues, metabolism, excretion, toxicity, effect
    on patient normal flora,
  • are not known by sensitivity.

6
Table 20.2
7
The Action of Antimicrobial Drugs
  • Broad-spectrum
  • Superinfection
  • Bactericidal
  • Bacteriostatic

8
The Action of Antimicrobial Drugs
Figure 20.2
9
The Action of Antimicrobial Drugs
Figure 20.4
10
Antibacterial Antibiotics Inhibitors of Cell
Wall Synthesis
  • Penicillin
  • Natural penicillins
  • Semisynthetic penicillins

11
Penicillins
Figure 20.6
12
Antibacterial Antibiotics Inhibitors of Cell
Wall Synthesis
  • Penicillin
  • Penicilinase-resistant penicillins
  • Extended-spectrum penicillins
  • Penicillins ?-lactamase inhibitors
  • Carbapenems
  • Monobactam

13
Antibacterial Antibiotics Inhibitors of Cell
Wall Synthesis
Figure 20.8
14
Antibacterial Antibiotics Inhibitors of Cell
Wall Synthesis
  • Cephalosporins
  • 2nd, 3rd, and 4th generations more effective
    against gram-negatives

Figure 20.9
15
Antibacterial Antibiotics Inhibitors of Cell
Wall Synthesis
  • Polypeptide antibiotics
  • Bacitracin
  • Topical application
  • Against gram-positives
  • Vancomycin
  • Glycopeptide
  • Important "last line" against antibiotic
    resistant S. aureus

16
Antibacterial Antibiotics Inhibitors of Cell
Wall Synthesis
  • Antimycobacterium antibiotics
  • Isoniazid (INH)
  • Inhibits mycolic acid synthesis
  • Ethambutol
  • Inhibits incorporation of mycolic acid

17
Antibacterial Antibiotics Inhibitors of Protein
Synthesis
  • Chloramphenicol
  • Broad spectrum
  • Binds 50S subunit, inhibits peptide bond
    formation
  • Aminoglycosides
  • Streptomycin, neomycin, gentamycin
  • Broad spectrum
  • Changes shape of 30S subunit

18
Antibacterial Antibiotics Inhibitors of Protein
Synthesis
  • Tetracyclines
  • Broad spectrum
  • Interferes with tRNA attachment
  • Macrolides
  • Gram-positives
  • Binds 50S, prevents translocation
  • Erythromycin
  • Gram-positives
  • Binds 50S, prevents translocation

19
Antibacterial Antibiotics Inhibitors of Protein
Synthesis
  • Streptogramins
  • Gram-positives
  • Binds 50S subunit, inhibits translation
  • Synercid
  • Gram-positives
  • Binds 50S subunit, inhibits translation
  • Oxazolidinones
  • Linezolid
  • Gram-positives
  • Binds 50S subunit, prevents formation of 70S
    ribosome

20
Antibacterial Antibiotics Injury to the Plasma
Membrane
  • Polymyxin B
  • Topical
  • Combined with bacitracin and neomycin in
    over-the-counter preparation

21
Antibacterial Antibiotics Inhibitors of Nucleic
Acid Synthesis
  • Rifamycin
  • Inhibits RNA synthesis
  • Antituberculosis
  • Quinolones and fluoroquinolones
  • Ciprofloxacin
  • Inhibits DNA gyrase
  • Urinary tract infections

22
Antibacterial Antibiotics Competitive Inhibitors
  • Sulfonamides (Sulfa drugs)
  • Inhibit folic acid synthesis
  • Broad spectrum

Figure 5.7
23
Figure 20.13
24
Antifungal DrugsInhibition of Ergosterol
Synthesis
  • Polyenes
  • Amphotericin B
  • Azoles
  • Miconazole
  • Triazoles
  • Allylamines

Figure 20.15
25
Antifungal DrugsInhibition of Cell Wall Synthesis
  • Echinocandins
  • Inhibit synthesis of ?-glucan
  • Cancidas is used against Candida and Pneumocystis

26
Antifungal DrugsInhibition of Nucleic Acids
  • Flucytocine
  • Cytosine analog interferes with RNA synthesis
  • Pentamidine isethionate
  • Anti-Pneumocystis may bind DNA

27
Antifungal DrugsInhibition of Microtubules
(Mitosis)
  • Griseofulvin
  • Used for superficial mycoses
  • Tolnaftate
  • Used for athlete's foot action unknown

28
Antiviral DrugsNucleoside and Nucleotide Analogs
Figure 20.16a
29
Antiviral DrugsNucleoside and Nucleotide Analogs
Figure 20.16b, c
30
Antiviral DrugsEnzyme Inhibitors
  • Protease inhibitors
  • Indinavir
  • HIV
  • Inhibit attachment
  • Zanamivir
  • Influenza
  • Inhibit uncoating
  • Amantadine
  • Influenza
  • Interferons prevent spread of viruses to new
    cells
  • Viral hepatitis

31
Antiprotozoan Drugs
  • Chloroquine
  • Inhibits DNA synthesis
  • Malaria
  • Diiodohydroxyquin
  • Unknown
  • Amoeba
  • Metronidazole
  • Damages DNA
  • Entamoeba, Trichomonas

32
Antihelminthic Drugs
  • Niclosamide
  • Prevents ATP generation
  • Tapeworms
  • Praziquantel
  • Alters membrane permeability
  • Flatworms
  • Pyantel pamoate
  • Neuromuscular block
  • Intestinal roundworms

33
Antihelminthic Drugs
  • Mebendazole
  • Inhibits nutrient absorption
  • Intestinal roundworms
  • Ivermectin
  • Paralyzes worm
  • Intestinal roundworms

34
Disk-Diffusion Test
Figure 20.17
35
E Test
Figure 20.18
36
  • MIC Minimal inhibitory concentration
  • MBC Minimal bactericidal concentration

37
Broth Dilution Test
Figure 20.19
38
Figure 20.20
39
Antibiotic Resistance
  • A variety of mutations can lead to antibiotic
    resistance.
  • Mechanisms of antibiotic resistance
  • 1. Enzymatic destruction of drug
  • 2. Prevention of penetration of drug
  • 3. Alteration of drug's target site
  • 4. Rapid ejection of the drug
  • Resistance genes are often on plasmids or
    transposons that can be transferred between
    bacteria.

40
Antibiotic Resistance
  • Misuse of antibiotics selects for resistance
    mutants. Misuse includes
  • Using outdated, weakened antibiotics
  • Using antibiotics for the common cold and other
    inappropriate conditions
  • Use of antibiotics in animal feed
  • Failure to complete the prescribed regimen
  • Using someone else's leftover prescription

41
Effects of Combinations of Drugs
  • Synergism occurs when the effect of two drugs
    together is greater than the effect of either
    alone.
  • Antagonism occurs when the effect of two drugs
    together is less than the effect of either alone.

42
Effects of Combinations of Drugs
Figure 20.22
43
The Future of Chemotherapeutic Agents
  • Antimicrobial peptides
  • Broad spectrum antibiotics from plants and
    animals
  • Squalamine (sharks)
  • Protegrin (pigs)
  • Magainin (frogs)
  • Antisense agents
  • Complementary DNA or peptide nucleic acids that
    binds to a pathogen's virulence gene(s) and
    prevents transcription

44
  • TECHNIQUES
  • Mainly two
  • Diffusion technique.
  • Dilution technique.

45
  • DIFFUSION SENSITIVITY TECHNIQUE
  • _at_ Used in routine sensitivity testing.
  • _at_ A disc of filter paper is impregnated with a
    known volume concentration of a drug placed
    on an agar medium inoculated with a test
    organism.

46
Control organisms are inoculated _at_ On same
plate (Stokes technique). _at_ On a separate plate
(Kirby-Bauer technique). Drug diffuses into
medium.
47
  • _at_ After an overnight incubation, culture is
    examined for areas of no growth (inhibition
    zones) around discs
  • Sensitive bacteria are inhibited at a distance
    from disc.
  • Resistant bacteria grow up to the edge of disc.

48
  • _at_ In Stokes technique
  • inhibition zone is compared directly with that
    of control .
  • _at_ In Kirby-Bauer technique
  • zone is measured compared against a previously
    prepared scale that correlates zone size with MIC.

49
_at_ MIC is the minimum drug concentration required
to inhibit bacterial multiplication under
standard conditions. _at_ It is measured by the
dilution sensitivity technique. _at_ Inhibition
zone increases when MIC decreases.
50
  • Inhibition zones vary in size due to
  • Difference in molecular structures of drugs
    (larger zones are obtained when drugs diffuse
    rapidly in medium).
  • When bacterial growth is heavy (zones are
    smaller, vice versa) .
  • Factors affecting the medium
  • (volume, moisture, pH, constituents).
  • 4.Factors affecting the disc (drug
    concentration, storage, application).

51
  • DILUTION SENSITIVITY TECHNIQUE
  • _at_ Performed under conditions
  • Patient not responding to therapy
  • Patient is immunosuppressed.
  • _at_ Measures MIC.
  • _at_ Measures the minimum bacterial
  • concentration (MBC)
  • (The minimum concentration of drug required to
    kill bacteria).

52
Technique _at_ Dilutions of drug are added to a
medium. _at_ A standard inoculum of organism is
added. _at_ After overnight incubation, MIC is
reported .
53
_at_ Clinical response is assessed by comparing MIC
obtained with already known concentrations of the
drug _at_ MBC may be determined by subculturing
last tube in the dilution series to show visible
growth _at_ Other tubes should detect no growth on
subculture.
54
_at_ Dilution techniques require good
standardization good control of inoculum,
medium, drugs , incubation time, diluting
techniques, reading of results. _at_ MIC may be
determined by automated machines.
55
DRUG ASSAYS _at_ Performed to check enough
drug concentration in a body fluid to
give adequate therapy (e.g.
treatment of endocarditis) make sure
that concentrations of toxic
aminoglycosides remain below their toxic
levels in pt. serum.
56
_at_ Specimens submitted for drug assays are
accompanied by drug dose, time of
administration, time of collecting the specimen,
and if patient is receiving other treatments.
57
  • STOKES DISC DIFFUSION SENSITIVITY TESTING
  • _at_ Has the following advantages
  • Both test control strains are inoculated on
    same plate.
  • Inoculum gives semi-confluent growth
  • (neither too heavy nor too light).

58
3. The activity of each disc is controlled. 4.
Inhibition zone of test organism is compared
directly with that of control. 5. Errors due to
too heavy or too light inocula are easily
detected. 6. Different media may be used.
59
REQUIREMENTS 1) SENSITIVITY TESTING AGAR _at_
Best is Mueller - Hinton agar. _at_ Depth
of medium is 4mm (25 ml) _at_ Pour plates on a
level surface. _at_ Too thin too thick
media give false inhibition zones.
_at_ patient not used antibiotic before 3 days. _at_
Plates are stored in plastic bags at 2 -
8C for up to 2 weeks.
60
_at_ Before use dry plates with lids slightly open
for ½ hr at 37C. _at_ 5 blood is added to M-H
agar to test for fastidious organisms
(Neisseria, Haemophilus, Streptococcus).
61
  • Factors affecting antimicrobial sensitivity
  • Media containing substances inhibiting action of
    aminoglycosides, tetracyclines, trimethoprim,
    e.g the substance thymidine.
  • pH of media False large zones are formed if
    medium is acidic (tetracycline), or false small
    zones if medium is alkaline (aminoglycosides).
  • Fermentable sugars are not added to medium to
    avoid production of acid and change of pH.

62
2) ANTIMICROBIAL DISCS _at_ To select drugs for
sensitivity, consult clinicians. _at_ Drug list
must be limited reviewed at regular
intervals. _at_ If resistance developed, one member
of each drug group is selected.
63
Other factors include Manufacturer
instructions regarding discs store temp., expiry
date, etc. are followed Bring discs to
room-temp. one hour before use. Do not expose
discs to sunlight. Quality control of discs
essential. Avoid dryness heat that decrease
control zone size.
64
  • 3) CONTROL STRAINS
  • _at_ Selected according to
  • Site of infection in patient.
  • Drug concentration at this site.
  • Strain must respond to treatment with normal
    doses .
  • Strain must grow at same rate as test organism.

65
  • Recommended control strains
  • S.aureus Oxford strain (NCTC 6571) Used for
    all except polymyxins, for pathogens of all
    specimens except urine.
  • E.coli NCTC 10418 . Used for all drugs against
    pathogens from urine.
  • Ps.aeruginosa NCTC 10662 Used for
    controlling all drugs against Ps.

66
  • Precautions for control strains are
  • Strain is cultivated on N.A. slopes.
  • Strains are stored in dark at room
    temp.(20-28C).
  • Subculture is made every 3 6 months
  • Each week, a nutrient broth or agar culture is
    made stored at 2 8C. from this culture,
    suspensions are
  • prepared for daily use.

67
4) TURBIDITY (OPACITY) STANDARD _at_ It is a
standard of barium chloride for matching
turbidity of test control strains inocula. _at_
Turbidity of standard is equivalent to an
overnight broth culture. _at_ After matching ,
dont incubate test or control strains for two
hrs.
68
  • Preparation of Turbidity Standard
  • Add I ml of H2SO4 to 99 ml water to make 1
    H 2SO4.
  • Dissolve 2.35g barium chloride in 200 ml water .

69
3. Mix 0.5 ml barium chloride solution to 99.5 ml
H2SO4 solution 4. Transfer turbid solution to
screw-cap bottle of same type as that used to
prepare test control strain suspension 5.
Store turbidity standard sealed in dark at room
temp. for 6 months.
70
INDIRECT SENSITIVITY TESTING _at_ Indirect
(Secondary) test Inoculum is a pure culture. _at_
Direct (primary) test Inoculum is a
specimen.
71
  • METHODS OF INDIRECT TESTING
  • _at_ Apply Stokes technique as follows
  • Emulsify colonies of organism in Muller-Hinton
    broth.
  • 2. Match turbidity developed against standard
    turbidity .
  • To match, view against a printed card
  • No incubation.

72
3. Using a sterile a 4mm loop , apply the
organism suspension to center of sensitivity
plate. _at_ Using a sterile swab, spread inoculum
across the center third of the plate.
73
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74
_at_ Do not use same swab for both application and
spreading. _at_ Judge your turbidity with turbidity
standard, not by naked eye.
75
4. Similarly, inoculate the broth culture of
control strain across the upper and lower thirds
of plate, leaving 5 mm. on each side of test
organism. _at_ Control suspension must be
standardized against the standard. 5. Allow the
inocula to dry for few minutes, with the Petri
dish closed.
76
6. Place antibiotic discs (after warming to room
temp.) between test and control inocula. Press
disc down a little, and do not move once it is
placed. 7. After ½ hr. incubate at 37C . _at_ For
methicillin , incubate at 35C.
77
8. Read test when _at_ Growth of both test
and control strains is not too heavy or too
light. _at_ Control inhibition zones measure 8-15
mm. radius. Measurement is from edge of
disc to edge of zone.
78
_at_ Using an electric rotary inoculator, test
strain may be inoculated in a ring around the
control strain. _at_ If growth of test control
strains is not semi confluent, sensitivity test
is repeated.
79
INTERPRETATION OF RESULTS Test is reported
sensitive, intermediate, resistant a)
Sensitive Test zone is _at_ wider than
control zone _at_ or equal to control zone
_at_ or not than 3 mm smaller of
control zone.
80
b)IntermediateTest zone is _at_ more than 3
mm smaller of control zone
but not less than 3 mm in diameter. c)
Resistant Test zone is _at_ 2 mm or less.
_at_ No zone of inhibition
81
  1. Intermediate zone drugs must be prescribed in
    high doses to cure infection, or when drug is
    concentrated at site of infection, e.g. UTI.
  2. With sulphonamides trimethoprim, slight growth
    may occur within inhibition zone. This is due to
    presence of inhibitors (thymidine), and it must
    be ignored.

82
3. Strains are considered resistant if _at_
Growth is heaped-up at zone edge
without gradual fading up towards disc
(penicillin-resistant Staph.) _at_ Large
colonies are seen growing within
inhibition zone (Staph.aureus) 4. To test for
sensitivity of co- trimoxazole , test for
sensitivity of sulphamoxazole trimethoprim
separated, using individual discs each.
83
5. Colistin polymyxin give smaller zones of
inhibition because of their large molecular size.
Hence control zone must be at least 3-4mm.. 6.
If Proteus swarms across its inhibition zone, no
problem, the zone is clear. 7. Check your discs
daily for any decrease in zone size resulting
from drug deterioration.
84
  • DIRECT SENSITIVITY TESTING
  • _at_ Performed when
  • Gram smear stain showed large
  • number of one type of organism.
  • 2. To obtain a presumptive result for
  • serious cases
  • 3. For urine, pus, pos. blood cultures,
  • 4. To isolate identify a pathogen or
  • to detect a resistant strain.

85
_at_ Sensitivity plate must not replace routine
culture plate. _at_ Blood is added to Muller
Hinton agar to be used for direct sensitivity _at_
Procedure for direct sensitivity is same as for
indirect sensitivity . _at_ Result of direct
sensitivity must be confirmed by indirect
sensitivity .
86
_at_ Do not report direct sensitivity result if
Growth of bacteria is too heavy or
too light. Zone size is smaller than
that of the control.
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