This document discusses antibiotic sensitivity and resistance. It covers the historical development of antibiotics, classifications of antibiotics based on chemical structure and mechanism of action, and mechanisms of antibiotic resistance. Antibiotic resistance can occur via genetic or non-genetic means, including production of enzymes that break down antibiotics, modification of antibiotic targets, and changes to cell permeability. Testing methods like disc diffusion and dilution tests are used to determine antibiotic sensitivity.

1. ANTIBIOTIC SENSITIVITY
AND RESISTANCE
Presented by: Dr. Mitali Thamke
(I MDS)

2. INDEX
 INTRODUCTION
 HISTORICAL ASPECT
 DEFINITION
 CLASSIFICATION
 MECHANISMS OF ACTION OF ANTIMICROBIAL
DRUGS
 RESISTANCE TO ANTIMICROBIAL DRUGS
o PRODUCTION OF ENZYMES
o PRODUCTION OF ALTERED ENZYMES
o SYNTHESIS OF MODIFIED TARGETS
o ALTERATION OF PERMEABILITY OF CELL WALL
o ALTERATION OF METABOLIC PATHWAY

3.  BASIS OF RESISTANCE
o NONGENETIC BASIS
o GENETIC BASIS
 CHROMOSOME- MEDIATED RESISTANCE
 PLASMID- MEDIATED RESISTANCE
 TRANSPOSONS- MEDIATED RESISTANCE
 ANTIBIOTIC SENSITIVY TESTING
 DISC DIFFUSION TESTS
 DILUTION TESTS
 Mechanism of biofilm resistance against antimicrobials
 Factors promoting resistance
 Prevention of drug resistance
 Conclusion

4. INTRODUCTION
 Antibiotics have not only saved patients lives, they
have played a pivotal role in achieving major
advances in medicine and surgery.
 They have successfully prevented or treated infections
that can occur in patients who are receiving
chemotherapy treatments; who have chronic diseases
such as diabetes, end-stage renal disease, or
rheumatoid arthritis; or who have had complex
surgeries such as organ transplants, joint
replacements, or cardiac surgery.

5.  Antibiotics have also helped to extend expected life
spans by changing the outcome of bacterial infections.
 In 1920, people in the U.S. were expected to live to be
only 56.4 years old; now, however, the average U.S. life
span is nearly 80 years.
 Antibiotics have had similar beneficial effects
worldwide. In developing countries where sanitation is
still poor, antibiotics decrease the morbidity and
mortality caused by food-borne and other poverty-
related infections.

6. HISTORICAL ASPECT
 Paul Ehrlich coined the term chemotherapy in 1913 to
mean “injury of invading organism without injury to
the host”.
Ehrlich’s Magic Bullets

7. Gerhard Domagk - Prontosil
 The modern era of chemotherapy of infection started
by Domagk in 1935 with the demonstration of
therapeutic effects of prontosil, a sulfonamide dye, in
pyogenic infection.

8. Fleming and Penicillin
 In 1928 Alexander Fleming observed that a
contaminating mould on a staphylococcal culture plate
caused the adjacent bacterial colonies to undergo lysis.
Fleming named antibacterial principle penicillin.

9.  Chain and Florey followed up this observation in 1939
which culminated in the clinical use of penicillin in
1941.
 It was not extensively used until the 2nd World War
when it was used to treat war wounds

10. Selman Waksman
 In 1940’s Waksman and his colleagues undertook a
systematic search of Actinomycetes as source of
antibiotics and discovered streptomycin in 1944.

11. DEFINITIONS
 ANTIBIOTICS:
• Antibiotics are the substances produced by
microorganism, which selectively suppress the growth of
or kill other microorganisms at very low concentrations.
 CHEMOTHEARPY:
• Treatment of systemic infections with specific drugs that
selectively suppress the infecting micro-organisms
without significantly affecting the host.

12.  Due to analogy between the malignant cells and the
pathologic microbes, treatment of neoplastic diseases
with drugs is also called chemotherapy
• It would be more meaningful to use the term
antimicrobial agent to designate synthetic as
well as naturally obtained drugs that
attenuate microorganisms.

13. CLASSIFICATION OF
ANTIBIOTICS
A. WITH RESPECT TO THEIR CHEMICAL STRUCTURE
B. ACCORDING TO SPECTRUM OF ACTIVITY
C. TYPE OF ACTION
D. ACCORDING TO MECHANISM OF ACTION

14. A. With respect to their
chemical structure
 SULPHONAMIDE AND RELATED STRUCTURES
 Sulfadiazine and others
 Sulfones – Dapsone (DDS), Paraaminosalicylic acid
 DI-AMINO-PYRIMIDINES
 Trimethoprim
 pyrimethamine

15.  QUINOLONES
 Nalidixic acid
 Norofloxacin
 Ciprofloxacin etc.
 TETRACYCLINE
 Oxytetracycline
 Doxycycline
 NITROBENZENE DERIVATIVE
 CHLORAMPHENICOL
 LACTAM ANTIBIOTICS
 Penicillin
 Cephalosporin
 Monobactum
 Cabapenems

16.  AMINOGLYCOSIDES
 Streptomycin
 Gentamycin
 MACROLIDE ANTIBIOTICS
 Erythromycin
 Roxithromycin
 Azithromycin
 POLYPEPTIDE ANTIBIOTIC
 Polymyxin-B
 Colistin
 Bacitracin

17.  Glycopeptides
Vancomycin
Teicoplanin
Oxazolidinone
Linezolid
Nitrofuran derivatives
 Nitrofurantoin
 Furazolidone
 Nitroimidozoles
 Metronidozole
 Tinidazole

18. Nicotinic acid derivatives
 Isoniazid
 Pyrazinamide
 Ethionamide
Polyene antibiotics
 Nystatin
 Amphotericin-B
Azole derivatives
 Miconazole
 Clotrimazole
 Ketoconazole
Others
 Rifampin
 Lincomycin
 Clindamycin

19.  According to mechanism of action
1) Inhibit cell wall synthesis
• Penicillins
• Cephalosporin
• Vancomycin
• Bacitracin
2) Damage to the cytoplasmic membrane:
 Polypeptides
 Polyene
3) Inhibit DNA gyrase
• Fluoroquinolones
• Ciprofloxacin
4) Interfere with DNA function
 Rifampicin
 Metronidazole

20. Classification
D) Spectrum of
activity:
 Narrow spectrum:
 Penicillin G
 Streptomycin
 Erythromycin
Broad spectrum:
Tetracyclines
Chloramphenicol
Intermediate spectrum
Aminopenicillin
Cephalosporins
Fluoroquinolones

21. Classification
E) Type of action:
1. Primarily
bacteriostatic
• Sulfonamides
• Tetracyclines
• Chloramphenicol
• Macrolide
• Ethambutol
2. Primarily bactericidal
• Penicillins
• Aminoglycosides
• Rifampin
• Cotrimoxazole
• Cephalosporins
• Vancomycin
• Quinolones

22. Mechanism of action of
antimicrobial drug
 The antibiotics act against bacteria by following
mechanism:
1) Inhibition of cell wall synthesis
2) Inhibition of protein synthesis
3) Inhibition of nucleic acid synthesis
4) Alteration of cell membrane function

23.  Inhibition by cell wall synthesis:
 Penicillin , cephalosporin, vancomycin and caspofungin
acts against bacteria by interfering cell wall synthesis.
 Penicillin and cephalosporins are called 𝛽 -lactum
antibiotics because they posses an intact 𝛽-lactam
ring, essential for antimicrobial activity.

24. Antibiotic Mechanism of action
Penicillin inhibiting penicillin –binding
proteins(PBPs)also known as
transpeptidases that link the cross-
bridges between NAMs, thereby, greatly
weakening the cell wall meshwork
Cephalosporin Similar to penicillin
Vancomycin Similar to penicillin
Caspofungin It kills bacteria by preventing synthesis of
𝛽-glucan, a polysaccharide component of
bacterial cell wall.

25.  Inhibition of protein synthesis :
 Bacteria have 30S and 50S ribosomal
units, whereas mammalian cell have 80S
ribosomes.
 The subunit of each type of ribosome,
their chemical composition, and their
functional speficities are sufficiently
different, which explain why these
antimicrobial drugs can inhibit protein
synthesis in bacterial ribosomes without
having major effect on ribosomal
ribosomes

26.  Aminoglycosides and tetracyclines act at the level of
30S ribosomal subunits, whereas
erythromycins,chloramphenical and clindamycin act at
the level of 50S ribosomal subunits.

27. Antibiotic Mechanism of action
Aminoglycosides binding to 30S subunit ribosome which
block the initiation complex, leading to no
formation of peptide bonds or polysomes
Tetacycline acts by inhibiting protein synthesis of
bacteria by blocking the binding of
aminoacyl t-RNA to 30S ribosomal
subunits
Macrolides inhibiting protein synthesis of bacteria by
blocking the release of t-RNA after it has
transferred its amino acid to the growing
peptide.
Chloramphenocol binds to 50S subunit of the ribosome and
block peptidyl transferase, the enzyme
that delivers the amino acid to growing
polypeptide, resulting in the inhibition of
bacterial protein synthesis
Clindamycin It inhibits bacterial protein synthesis by
blocking the release of t-RNA

28.  Inhibition of nucleic acid synthesis:
 Sulphonamide ,trimethoprim,quinolones and
rifampicin are examples of drugs that act by inhibition
of nucleic aid synthesis.

29. Antibiotic Mechanism of action
Sulphonamide antibiotic inhibit the synthesis of
tetrahydrofolic acid, the main donor of
the methyl groups that are essential to
synthesis adenine, guanine and cytosine
Trimethoprim enzyme reduce dihydrofolic to
tetrahydrofolic acid, leading to decreased
synthesis of purines and ultimately DNA
Quinolones act by inhibiting bacterial DNA synthesis
by blocking DNA gyrase.
Rifampicin Rifampicin inhibits bacterial growth by
binding strongly to the DNA-dependent
RNA polymerase of bacteria

30. Alteration of cell membrane
function
 The cytoplasm of all living cells is surrounded by the
cytoplasmic membrane, which serves as a selective
permeability barrier.
 The cytoplasmic membrane carries out active transport
functions, and thus controls the internal composition of cell.
 If the functional integrity of cytoplasmic membrane is
disrupted, macromolecules and ions escape from cell, and
cell damage or death ensues.

31.  Antifungal drugs act by altering the cell membrane
function of fungi.
 They show selective toxicity because cell membrane of
fungi contain ergosterol whereas human cell
membrane has cholesterol.
 Bacteria with exception of mycoplasm do not have
sterol in their cell membranes, hence resistant to
action of these drugs

32.  Polymix b which is an antibacterial binds to
lipopolysaccharide in outer membrane of Gram
negative bacteria and disrupt both outer and inner
membrane

33.  Amphotericin B and azoles are frequently used
antifungal drugs.
 Amphotericin B acts against the fungi by disrupting
the cell membrane by binding at the site of ergosterol
in the membrane.
 Azoles such as ketoconazole inhibits synthesis of
ergosterol, hence are toxic to fungi.

34. Resistance to antimicrobial
drugs
 As early as 1945, Sir Alexander Fleming raised the
alarm regarding antibiotic overuse when he warned
that the “public will demand [the drug and] ... then will
begin an era ... of abuses.
 The overuse of antibiotics clearly drives the evolution
of resistance.
 Epidemiological studies have demonstrated a direct
relationship between antibiotic consumption and the
emergence and dissemination of resistant bacteria
strains.

35. Mechanism of antibiotic
resistance
1. Production of enzymes
2. Production of altered enzymes
3. Synthesis of modified targets
4. Alteration of permeability of cell wall
5. Alteration of metabolic pathway

36. Basis of resistance
 The resistance by bacteria against antibiotic may be
classified as:
1. Nongenetic basis
2. Genetic basis

37. Nongenetic basis
 Nongenetic basis of resistance plays less important
role in development of drug resistance:
1. Certain bacteria under ordinary circumstances are
usually killed by penicillin. But these bacteria , if they
loss their cell wall and become protoplast, become
nonsusceptible to the action of cell wall-acting drug
such as penicillins.

38. 2. In certain condition as in abscess cavity, bacteria can be
walled off which prevent drug to penetrate effectively into
bacteria. Surgical drainage of the pus, however, makes
these bacteria again susceptible to action of antibiotics

39. 3. The presence of foreign bodies such as surgical
implants and catheters and penetration injury caused by
splinters and sharpeners make successful antibiotic
treatment more difficult.

40. 4. Nonreplicating bacteria in their resting stage are less
sensitive to the action of cell wall inhibitors such as
penicillin and cephalosporin.
This is particularly true for certain bacteria such as
Mycobacterium tuberculosis that remains in resting stage
in tissues for many years, during which it is insensitive to
drugs.
However when these bacteria begin to multiply, they
become susceptible to antibiotic

41. Genetic basis of Drug
Resistance
 The genetic basis of drug resistance , mediated by
genetic change in the bacteria, is most important in
development of drug resistance in bacteria. This is of 3
types as follows:
1. Chromosomes-mediated resistance.
2. Plasmid –mediated resistance.
3. Transposons-mediated resistance.

42. Chromosome-mediated
resistance
 Chromosome-mediated resistance occurs as a result of
spontaneous mutation.
 This is cause by mutation in gene that codes for either
the target of drugs or the transport system in the
membrane of cell wall that controls the entry of drugs
into cells
 The frequency of chromosomal mutation is much less
than the plasmid-mediated resistance

43. Plasmid-mediated resistance
 Plasmid-mediated drug resistance in bacteria occurs
by transfer of plasmid and genetic materials.
 It is mediated by resistance plasmid, otherwise known
as R-factor

44. R-factors
 They are circular, double stranded DNA molecules that
carry the genes responsible for resistance against
variety of antibiotics.
 These factors may carry one or even two or more
resistant genes.
 These genes encode for a variety of enzymes that
destroy the antibiotic by degrading antibiotics or
modify membrane transport.

45. Plasmid-mediated antibiotic
resistance
Antibiotic Mechanism of resistance
𝛽 −lactams 𝛽-lactamases break down the 𝛽 −lactam ring to an
inactive form
Aminoglycosides Aminoglycosides modifying enzymes:
acetyltransferases, phosphotransferases and
nucleotidyltransferases
Erythromycin and
clindamycin
Induced enzymatic activity due to methylating
ribosomal RNA
Chloramphenicol Acetylation of the antibiotic to an inactive form
Tetracycline Alteration of cell membrane, decreases permeability to
antibiotic

46.  Plasmid mediated resistance plays a very important
role in antibiotics usage in clinical practice. This is
because
a. A high rate of transfer of plasmids from one
bacterium to another bacterium takes place by
conjugation
b. Plasmid mediated resistance to multiple antibiotics
c. Plasmid mediated resistance occurs mostly in Gram-
negative bacteria.

47. Transposons-mediated drug
resistance
 Drug resistance is also mediated by transposons that
often carry the drug resistance genes
 Transposons are small pieces of DNA that move from
one site of bacterial chromosome to another and from
bacterial chromosome to plasmid DNA.
 Many R factors carry one or more transposons.

48. Difference between mutational and
transferable drug resistance
Mutational drug resistance Transferable drug resistance
Chromosome mediated Plasmid mediated
Resistance to one drug Resistance to multiple drug
Resistance is nontransferable Resistance is transferable
Virulence of organism lowered Virulence of organism not lowered
Low-degree of resistance High-degree of resistance
Due to decreased permeability
,development of alternative metabolic
pathway or inactivation of drug
Due to production of many degrading
enzymes.

49. Specific mechanism of
resistance
 Penicillins
Resistance to penicillin is mainly mediated by 3
mechanism:
1. Production of penicillin-destroying enzymes(𝛽-
lactamases)
2. Mutation in genes coding for PBP
3. Reduced permeability to drug

50. 1. Production of penicillin destroying
enzymes(𝛽lactamases):
 Resistance to penicillins may be determined by
organism’s production of penicillin-destroying
enzymes(𝛽-lactamases).
 𝛽 -lactamases such as penicillinases and
cephalosporinases open the 𝛽-lactam ring of penicillin
and cephalosporin and abolish their antimicrobial
activity.
 𝛽-lactamases have been described for many species of
Gram-positive and Gram-negative bacteria.

51.  Some 𝛽-lactamases are plasmid-
mediated(eg,penicillinase of S.aureus),while other are
chromosomally mediated(eg,many species of Gram-
negative bacteria such as Enterobacter spp.,Citrobacter
spp.,Pseudomonas spp.,etc)
 There is one group of 𝛽-lactamases that is occasionally
found in certain species of Gram-negative bacilli,
usually Klebsiella pneumoniae and E.coli

52.  These enzymes are termed extended-spectrum 𝛽-
lactamases because they confer upon the bacteria the
additional ability to hydrolyze the 𝛽-lactam rings of
cefotaxime,ceftazidime,or azetreonam.

53. 2. Mutation in genes coding for PBP:
 This form of resistance occur due to absence of some
penicillin receptors(PBP) and occurs as a result of
chromosomal mutation
 The mechanism is responsible for both low-level and
high-level resistance seen in S.pneumonia to penicillin
G and in S.aureus to nafcillin.

54. 3.Reduced permeability to drug:
 Low-level resistance of Neisseria gonorrhoeae to
penicillin is caused by poor permeability of drug.
 However, high-level resistance is mediated by plasmid
coding for penicillinase.

55. Vancomycin
 Resistance to vancomycin is mediated by change in D-
ALA-D-ALA part of peptide in the peptidoglycan to D-
ALA-D-lactate.
 This result in the inability of vancomycin to bind to a
bacteria.
 Vancomycin resistance in Enterococcus is been
increasingly documented in different clinical
conditions.

56. AMINOGLYCOSIDES
 Resistance to aminoglycoside is mediated by three
important mechanism as follows:
1. Plasmid-dependent resistance to aminoglycosides
enzymes is most important mechanism. It depends on
production of plasmid-mediated phosphorylating, and
acetylating enzymes that destroy the drug

57. 2. Chromosomal resistance of microbes to
aminoglycosides in second mechanism.
The chromosomal mutation in the genes results in lack of
a specific protein receptor on the 30S subunit of
ribosome, essential for binding of the drug.

58. 3. A ‘permeability defect’ is the third mechanism.
This lead to an outer membrane change that reduces
active transport of aminoglycosides into cell so that the
drug cannot reach the ribosome.
Often this is plasmid-mediated

59. Tetracycline
 Resistance to tetracycline occurs by 3 mechanisms:
a. Efflux
b. Ribosomal protection, and
c. Chemical modification
 The first two are the most important

60.  Efflux pumps, located in bacterial cell cytoplasmic
membrane, are responsible for pumping the drug out
of the cell.
 Tet gene products are responsible for protecting the
ribosome, possibly through mechanism that induce
conformational changes.
 These conformational changes either prevent binding
of the teracyclines or causes their dissociation from the
ribosome.
 This is often plasmid-controlled.

61. Antibiotic sensitivity testing
 Antibiotic sensitivity testing is carried out to determine
the appropriate antibiotic agents to be used for a
particular bacterial strain isolated from clinical
specimen
Antibiotic sensitivity
Disc difution
test
kirby -
bauer
Stokes test
Dilution test
AGAR
DILUTION
TUBE
DILUTION
disc diffusion
&dilution
E -TEST
qualitative quantitative

62. Disc diffusion tests
 Most commonly used
 In this method as name suggests, disc is impregnated
with known concentrations of antibiotics are placed on
agar plate that has been inoculated with a culture of
bacterium to be tested.
 The plate is incubated at 37℃ for 18- 24 hours

63.  After diffusion the concentration of antibiotic usually
remain higher near the site of antibiotic disc but
decreases with distance.
 Susceptibility to the particular antibiotic is determined
by measuring the zone of inhibition of bacterial
growth around the disc.
Zone of inhibition

64. Selection of media
 Media that support both test and control strain is
selected for carrying out AST of the bacteria.
 For e.g., Mueller Hinton agar is used for testing Gram-
negative bacilli and Staphylococcus spp;
 Blood agar for Streptococcus spp. And Enterococcus
spp
 Chocolate agar for Haemophilus influenza
 Wellcotest medium for sulphonamide and
cotrimoxazole .

65.  Medium is prepared by pouring onto the flat
horizontal surface of petri dishes of 100mm to a depth
of 4mm.
 pH is maintained at 7.2-7.4
 More alkaline pH increases activity of tetracyclines
,novobiocin and fusidic acid
 Acidic pH reduced the activity of aminoglycosides and
macrolides such as erythromycin.
 Plates are stored at 4℃ for upto 1 week.

66. Type of disc diffusion tests
 Disc diffusion tests are of following types:
1. Kirby-Bauer disc diffusion method
2. Strokes disc diffusion method
3. Primary disc diffusion test

67. Kirby-Bauer disc diffusion
method
 Most common method used
 Both test strain and control strain are tested in
separate plates.
 Test organism is inoculated in suitable broth solution,
followed by incubation at 37℃ for 2-4 hours.
 0.1 ml of broth is inoculated on surface of agar
medium by streaking with a sterile swab

68. THE TEST BACTERIA IS
ISOLATED FROM ITS
CULTURE PLATE
A LIQUID CULTURE OF
THE TEST BACTERIUM
IN A SUITABLE BROTH
IS PREPARED IN A TEST
TUBE

69. THIS IS THEN POURED ON TO
A SUITABLE SOLID AGAR
MEDIUM(NUTRIENT/MUELLE
R-HINTON) IN A PETRI DISH
WHICH IS TILTED TO ENSURE
UNIFORM SPREADING
EXCESS BROTH IS PIPETTED
OFF.

70. Alternately,a sterilized cotton swab may be
dipped in the liquid bacterial suspension and
streaked across the solid agar medium in
different angle to ensure uniformity.
The plate is dried at 37 degree Celsius for 30
minutes
The antibiotic filter paper(4/5 per 10cm
dish)are placed with sterile forceps and
incubated overnight
A ’lawn’culture develops with zone of in
hibition

71.  If the bacterium is susceptible to the drug growth is
inhibited,but if it is resistant no zone of inhibition will be seen.

73. Stroke’s method
 Similar to Kirby-bauer method,however
control organism is used.
 The control organism used are
 Staph.Aureus
 E.coli
 Ps.aeruginosa
 A standard sesitive control organism is
inoculated on one side of the plate.
 The test bacterium is inoculated on the
other side of the plate
 Antibiotic disc are placed at junction of two
layers.
 Comparison of zone of inhibition indicates
the susceptibility with respect to
Std.bacterium.

74.  In modified Strokes method, control strain is inoculated
in the central part but test strains are inoculated on the
upper and lower third of plate.
 Reporting of result is carried out by comparing the zone
of inhibition of test and control bacteria.

75.  The zone is measured from the edge of the disc to
edge of zone. It is interpreted as follows:
1. Sensitive (S): the zone of test bacterium is equal to or
more than that of control strain. The difference
between the zone sizes of control and test strain
should not be more than 3mm if the zone size of test
bacterium is smaller than that of control.
2. Intermediate sensitive(I): The zone size of the test
bacterium should be at least 2 mm, and the differences
between the zone of test.
3. Resistant (R): The zone size of test bacterium is 2mm or
less.
Antibiotic disc Inner zone: resistant strain
Black zone:
intermediate
susceptibility
Outer zone: suceptible strain

77. Interpretation of disc
diffusion tests:
 Results of disc diffusion tests such as Kirby-Bauer and Strokes
method are interpreted as follows:
 Sensitive(S): infection treatable by the normal dosage of the
antibiotic
 Intermediate(I): Infection may respond to higher dosage
 Resistant(R): Unlikely to respond to usual dosage of the
antibiotics

78. Primary disc diffusion test:
 It is carried out directly on clinical specimens unlike
Kirby-Bauer or Strokes diffusion method which are
performed on pure cultures of bacteria isolates from
clinical specimens.
 In this method the clinical specimen is inoculated
uniformly on surfaces of the agar to which antibiotic
are applied directly.
 Plate is incubated overnight at 37℃ for demonstration
of zone of inhibition.

79.  Method is used to know antibiotic sensitivity result
urgently but these results should always be confirmed
by testing isolates subsequently by Kirby-Bauer or
Strokes diffusion method.

80. Dilution tests
 Dilution test is performed to determine the minimum
inhibitory concentration of (MIC)antimicrobial agent.
 MIC is defined as the lowest concentration of the
antimicrobial agent that inhibits the growth of
organisms.

81.  Estimation of the MIC is useful to:
a. Regulate the therapeutic dose of antibiotic accurately
b. Test antimicrobial sensitivity patterns of slow growing
bacteria such as M.tuberculosis.

82. Following method are carried out to
determine the MIC:
1. Broth dilution method
2. Agar dilution method
3. Epsilometer test

83. Broth dilution method
 Quantitative method for determining MIC.
 The antimicrobial agent is serially diluted in Mueller-
Hinton broth by doubling dilution in tubes, and then a
standard suspension of broth culture of test organism
is added to each of the antibiotic dilution and control
tube.
 This is mixed gently and incubated for 16-18 hours at
37℃

84.  An organism of known susceptibility is included as
control.
 MIC is recorded by noting the lowest concentration of
the drug at which there is no visible growth as
demonstrated by the lack of turbidity in tube.
 Main advantage is that this is a simple procedure for
testing a small number of isolates.

85.  Added advantage is that by using same tube ,the
minimum bactericidal concentration (MBC) of bacteria
an be determined.
 MBC is determined by subculture from each tube,
showing no growth on a nutrient agar without
antibiotics

86.  The plates are examined for the growth, if any, after
incubation overnight at 37℃
 The tube containing the lowest concentration of drug
that fail to show any growth on subculture plate is
considered as the MBC of the antibiotic for that strain.

87. Agar dilution method
 It is quantitative method for determining the MIC of
antimicrobial agent against test organism.
 It is useful:
A. To test organism from serious infection like bacterial
endocarditis
B. To verify equivocal results of disc diffusion test

88.  Mueller-Hinton agar is used
 Serial dilution of antibiotic are made in agar and
poured into petri dishes.
 Dilution are made in distilled water and added to the
agar which has been melted and cooled to more that
60℃
 One control plate without antibiotic
 Organism to test is inoculated and incubated
overnight at 37℃.

89.  Plates examined for presence or absence of bacteria.
 The concentration at which bacterial growth is
completely inhibited is considered as the MIC of
antibiotic.
 Organisms is reported sensitive, intermediate, or
resistance by comparing test MIC value
 Main advantage is that number of organisms can be
tested simultaneously on each plate containing an
antibiotic solution.

90. Epsilometer test
 Combines the principle of disc
diffusion and agar dilution
 Similar to Kriby-Baurer method
but uses a thin inert plastic strip
impregnated with a known
gradient of varying drug
concentration along its length.
 Here, a symmetrical inhibition
ellipse is produced.
 The intersection of inhibitory zone
of edge and the calibrated carrier
strip indicates the MIC value.
 E test is a very useful test for easy
interpretation of the MIC of an
antibiotic.
MIC

91. Mechanisms of Biofilm
Resistance
Against Antimicrobials

92. Mechanisms responsible for a
resistance in biofilms are as
following
1) Biofilm Impermeability to Antimicrobial Agents
Antimicrobial molecules must reach their target in order to
inactivate the enmeshed bacteria. The biofilm glycocalyx
protects infecting cells from humoral and cellular host
defence systems as well as the diffusion of the antimicrobial
molecules to the target, acting as a barrier by influencing the
rate of transport of molecules into the biofilm interior.

93.  Physical (coaggregation and coadhesion),
metabolic, and physiological (gene expression and
cell–cell signalling) interactions yield a positive
cooperation among different species within the
biofilm.
 A key role is played by F. nucleatum, able to form the
needed “bridge” between early and late colonizers.
 The presence of F. nucleatum, enables anaerobes to
grow, even in the aerated environment of the oral
cavity.
 In the absence of F. nucleatum, P. gingivalis cannot
aggregate with the microbiota

94.  Because subgingival bacteria are organized in biofilms,
in principle, they are less susceptible to antimicrobials.
 Therefore, The oral plaque biofilm needs to be
mechanically debrided or disturbed in order for
antimicrobials to be effective

95. 2) Altered Growth Rates in Biofilm Organisms
 the growth rate of the organisms is significantly slower than
the growth of planktonic (biofilm free) cells; therefore, the
uptake of the antimicrobial molecules is diminished.
3) The Biofilm Microenvironment Antagonizing The
Antimicrobial Activity
 Relatively large amounts of antibiotic-inactivating enzymes
such as b-lactamases which accumulate within the
glycocalyx produce concentration gradients that can protect
underlying cells.

96.  The most prominent b-lactamase-producing
organisms belonged to the anaerobic genus
Prevotella. Other enzyme-producing anaerobic
strains were F. nucleatum, and Peptostreptococcus
sp.

97. 4) The Role of Horizontal Dissemination
in the Biofilm
Horizontal transmission of genetic information between
bacteria can occur by three gene transfer mechanisms:
 conjugation,
 transduction, and
 transformation

98.  Conjugation requires that the donor cell have a
conjugative element, usually a plasmid or a transposon,
and that physical contact be made between donor and
recipient cells to initiate transfer of the DNA molecule.
 Transduction process involves transducing
bacteriophage particles that harbour the foreign DNA.
 Gene transfer by transformation, does not require a
living donor cell, since free DNA released during cell
death and lysis is the principal source of the donor DNA.

99. These all above mentioned 3 mechanisms of horizontal gene
transfer require basic components i.e. plasmids, conjugative
transposons (CTn), and bacteriophages.
 Plasmids
 Usually exist as independently replicating units.
 Plasmids are common in both gram-positive and gram-
negative organisms isolated from the oral cavity. Among
the most important plasmids in mediating broad host-
range gene transfer are those of the IncP group.

100.  Conjugative Transposons
 The gram-negative oral bacterium A.
actinomycetemcomitans and E.corrodens has been
implicated as a causative agent of several forms of
periodontal diseases. The conjugative tetracycline
resistance transposon Tn916 was transduced to their
recipients as a unit.

101. o Bacteriophage
 Bacteriophage can contribute to horizontal gene
transfer by transduction or lysogenic conversion.
 A range of bacteriophages specific for species of
Veillonella spp, Actinomyces spp, S. mutans ,
Enterococcus faecalis , and A. actinomycetemcomitans
have been described in dental plaque samples or in
saliva.

102. Communications Systems
(Quorum Sensing)
 Gram-positive bacteria generally communicate via small
diffusible peptides, while many gram-negative bacteria
secrete acyl homoserine lactones (AHLs).
 AHLs are involved in quorum sensing whereby cells are
able to modulate gene expression in response to
increases in cell density.
 Several strains of P. intermedia, F. nucleatum, and P.
gingivalis were found to produce such activities.

103.  van Winkelhoff et al and Slots revealed that systemically
administered antibiotics provided a clear clinical benefit in
terms of mean periodontal attachment level “gain” post
therapy when compared with groups not receiving these
agents.
 Meta-analyses performed by Herrera et al and Haffajee
et al indicated that adjunctive systemically administered
antibiotics can provide a clinical benefit in the treatment
of periodontal infections.

104.  The tetracyclines, metronidazole, and b-lactams are
among the most widely used agents for treating
periodontal conditions.
 Mechanisms of bacterial resistance to these antibiotics
have been extensively described and attributed to
resistance genes.
 Many genes for bacterial resistance to tetracycline
have been identified and characterized. These include
tet A, B, C, D, E, G, H, I, K, L, M, O, Q, and X
associated with gram-negative bacteria, and tet K,
L, M, O, P, Q, S, Otr A, B, and C (oxytetracycline
resistance determinants) associated with gram-
positive bacteria.

105.  Kuriyama et al. found that bacteria of the genus
Porphyromonas and of the genus Fusobacterium
showed susceptibility to cephalosporins
 Fosse et al. found resistance to tetracycline frequently
associated with β-lactamase production, with 50% of
Gram-negative oral anaerobes isolated resistant both
to tetracycline and to penicillins.
 Andres et al.were able to link erythromycin and
tetracycline resistance and β-lactamase production in
Gram-negative anaerobes, concluding that they were
associated with conjugative elements in oral Prevotella
species. The co-transfer of resistance determinants to
these three antibiotic classes occurs frequently within
this genus

107. Antibiotics promote
resistance
 If a patient taking a course of antibiotic treatment does
not complete it
 Or forgets to take the doses regularly,
 Then resistant strains get a chance to build up
 The antibiotics also kill innocent bystanders bacteria
which are non-pathogens
© 2008 Paul Billiet ODWS

108.  This reduces the competition for the resistant
pathogens
 The use of antibiotics also promotes antibiotic
resistance in non-pathogens too
 These non-pathogens may later pass their resistance
genes on to pathogens

109. Resistance gets around
 When antibiotics are used on a person, the numbers of antibiotic
resistant bacteria increase in other members of the family
 In places where antibiotics are used extensively
e.g. hospitals and farms
antibiotic resistant strains increase in numbers
© 2008 Paul Billiet ODWS

110. Prevention of drug
resistance
 It is utmost clinical importance to curb development of
drug resistance. Measures are:
1. No indiscriminate and inadequate or unduly
prolonged use of antimicrobial agents(AMA’s) should
be made. This would minimize the selection pressure
and resistance strain will get less chance to
preferentially propagate.

111. 2. Prefer rapidly acting and selective(narrow spectrum)
AMA’s whenever possible; broad-spectrum drug should
be used only when a specific one cannot be determined
or is not suitable.

112. 3. Use combination of AMA’s whenever prolonged
therapy is undertaken,eg, Tuberculosis ,SABE
4. Infection by organism notorious for developing
resistance e.g., staph. Aureus,
E.coli,M.tberculosis,Proteus,etc must be treated
intensively.

113.  For acute localized infections in otherwise healthy
patient ,symptom determined shorter courses of
AMA’s are being advocated now

114. Conclusion
 Antibiotic resistance is rising to dangerously high
levels in all parts of the world.
 New resistance mechanisms are emerging and
spreading globally, threatening our ability to treat
common infectious diseases.
 A growing list of infections – such as pneumonia,
tuberculosis, blood poisoning and gonorrhea – are
becoming harder, and sometimes impossible, to treat
as antibiotics become less effective.

115.  Where antibiotics can be bought for human or animal
use without a prescription, the emergence and spread
of resistance is made worse.
 Similarly, in countries without standard treatment
guidelines, antibiotics are often over-prescribed by
health workers and veterinarians and over-used by the
public.

116.  Without urgent action, we are heading for a post-
antibiotic era, in which common infections and minor
injuries can once again kill.

117. References
1. Essentials of medical pharmacology,6th edition ,K.D
Tripathi
2. Textbook of Microbiology and Immunology ;Subhash
Chandra Parija
3. Carranza’s Clinical periodontology,11th edition
4. Rajiv Saini Biofilm: A dental microbial infection. J Natural
science,biology and medicine 2011;2: 71-75
5. Basics & clinical pharmacology,9th edition,Bretram G.
Katzung
6. L.C .Sweeny et.al. Antibiotic resistance in general dental
practice J.Antimicrob.chemotherapy 2004;53: 567-76

118. THANK
YOU