Antibiotics &

Antibiotic
Resistance

Year 1 2012/2013
Module: Infection and Immunity
FF1244

Professor Dr Nordiah Awang Jalil

Department of Medical
 Antibiotics

Antibacteria derived from:

bacteria or molds
de novo synthesis
 Groups of Antibiotics
-lactams: Penicillins, Cephalosporins
Aminoglycosides: Gentamycin, Amikacin
Glycopeptides: Vancomycin, teicoplanin
Macrolides, Lincomycin & Streptogramin
Tetracyclines: Tetracycline, Doxycycline
Carbepenems: Imipenem, Meropenem
Polypeptides: Bacitracin, Colistin, Polymyxin
B
Quinolones: Ciprofloxacin, Moxifloxacin
Others
 Antibiotics

Mechanisms of Action :
Inhibiting cell wall synthesis

Interfering nucleic acid synthesis :

DNA/RNA
Inhibiting protein synthesis

Interfering with folic acid metabolism

Disrupting cell membrane

 DNA
replication

Isoniazid
RNA synthesis
Ethambuto
l

Oxazolidino
 Activity of Antibiotics
1.Bactericidal: they kill bacteria
Penicillins, cephalosporins, vancomycin,
quinolones carbepenems, aminoglycosides,
polymyxins
2.Bacteriostatic: they slow bacterial growth
tetracyclines, chloramphenicol, Erythromycin,
clarithromycin, azithromycin, clindamycin,
lincomycin, and sulfonamides
However, bactericidal drugs may be
bacteriostatic against certain microorganisms
and vice versa.
Antibiotic Resistance
Ability of a bacterial cell to resist
the bacteriostatic or bactericidal
effect of an antibiotic Rx failure
In vitro, the tested antibiotic does
not inhibit / kill the organism
Mechanisms of
Antimicrobial Resistance
1. Enzymatic Inactivation
2. Altered Target Enzymes
3. Altered Ribosomal Target
4. Altered Permeability
5. Efflux
BARRIER
IMPERMEABILITY
1. Enzyme Inactivation
A. -lactamase
destroy lactam ring of -lactam
antobiotics
Produce by GPC, GNB, Anaerobes
i. Penicillinase
Staph. aureus, N. gonorrhoeae, H. influenzae
R to Penicillin
S to cephalosporins
S to antistaph. Penicillin eg cloxacillin
inhibited by -lactamase inhibitors
eg clavulanic acid, sulbactam,tazobactam
ii. -lactamase in Enterobacteriaceae
TEM-1, TEM-2, SHV-1
TEM = 1st 3 letters of pt. TEM -lactamase
1st isolate
SHV = sulfhydryl variable
R to Penicillin, 1st & 2nd -gen. Cephalosporins
S to 3rd & 4th-generation cephalosporins
inhibited by -lactamase inhibitors
eg clavulanic acid, sulbactam, tazobactam
iii. Extended-spectrum -lactamase
(ESBL)
Mutation of TEM & SHV enzymes
> 60 TEM-type , > 30 SHV-type
ESBLs
R to cephalosporins

iv. Carbepenemases
R to imipenem, meropenem
B. Aminoglycoside-modifying

Enzymes
Phosphotransferases (APH)
Acetyltransferases (AAC)
Nucleotidyltransferases (ANT)
inactivate aminoglycoside before it
reach the ribosomes
major mechanism of R in
aminoglycoside
C. Chloramphenicol
Acetyltransferase
Main mechanism R to
chloramphenicol
Inactivate chloramphenicol by
acetylating OH group
Unable to bind 50S ribosom to
inhibit protein synthesis
D. Erythromycin
Esterase
/
Erythromycin Esterase
Phosphotransferase
Hydrolyze lactone ring of macrocylic
nucleus
Inactivate macrolide
Erythromycin Phosphotransferase
inactivate macrolide by introducing a
phosphate on the 2- OH group
Erythromycin Resistance mainly via
alteration of ribosomal target site
Antibiotic Resistance
Mechanisms

Enzyme Inactivation
-lactamase
Aminoglycoside-
modifying enzymes
Chloramphenicol
acetyl transferase
Erythromycin
Esterase
/ Phosphotransferase
2. Altered Target
Enzymes
Penicillin-binding proteins (PBP) are
peptidoglycan transpeptidase for
synthesis of PG cell wall.
-lactam antibiotics bind with PBP to
inhibit PG synthesis
Alteration of PBPs
Affinity, loss of PBPs, emergence of new
PBPs
R to Penicillin & other -lactam antibiotics
Main mechanism resistance for penicillin-
resistant pneumococci (PRP) , MRSA
Also in enterococci, gonococci, H.influenzae
 Altered Target Enzymes
DNA gyrase coded by gene gyrA, gyrB
Responsible for supercoiling of DNA
Quinolone acts on DNA gyrase
Inhibit supercoiling of DNA
Rapid killing of bacteria
Mutation of gyrA, gyrB altered DNA gyrase
Quinolone resistant

Altered Dehydrofolate reductase

Trimethoprim resistant
3. Altered Ribosomal
Target
Resistant to Macrolides, Lincosamides
& Streptogramins (MLS),
Tetracyclines, Aminoglycosides
Unable to bind the ribosomal target
site
Unable to inhibit protein synthesis &
cell growth
 Antibiotic Resistance
Mechanisms
Altered target site
Penicillin binding
proteins (penicillins)
DNA gyrase
(quinolones)
RNA polymerase
(rifampin)
30S ribosome
(streptomycin)
 4. Altered
Permeability
Cell wall of GNB consists of outer
membrane
GNB R to Penicillin
OMP porin allows penetration of antibiotics
or by active transport
Altered / loss of OMP porin Imipenem R
Pseudomonas aeruginosa
Streptococci, enterococci & anaerobes lack
necessary oxidative pathway for transport
of aminoglycoside resistant
 Microbe Library
American Society for Microbiology
www.microbelibrary.org
5. Efflux
An efflux pump is essentially a channel
that actively exports antimicrobial and
other compounds out of the cell
Antimicrobial enters the bacterium
through porin
Pumped back out of the bacterium by the
efflux pump. By actively pumping out
antimicrobials, the efflux pumps prevent
the intracellular accumulation necessary
to exert their lethal activity inside the cell.
Active
Efflux
Resistant to
Tetracycline
Quinolones
Macrolides
 Antibiotic Resistance
Mechanisms
Active efflux
Tetracycline
Quinolones
Macrolides
Genetic Transfer of
Resistance
Resistance genes can be
transferred between bacteria
by several means, most often
by:
a.Conjugation
b.Transduction
c.Transformation
 Reasons for Bacterial
Resistance
Antibiotic overuse, misuse or abuse
Prolonged use for therapy/prophylaxis
Misuse for viral infection
Abuse in animal husbandry/agriculture
eg Avoparcin (vancomycin) use in livestock:
VRE jumping from animals to humans
Poor hand hygiene and failure of
infection control measures
Spread of resistant bacteria
Counterfeit Drugs
Low efficacy
Impact of Antibiotic
Resistance
morbidity & mortality
Length of hospital stay (LOS)
Cost burden
Less antibiotics in the pipeline
 Campaign to Prevent Antimicrobial Resistance in Healthcare Settings

Antimicrobial Resistance:
Key Prevention
Antimicrobial-Resistant
Susceptible Pathogen
Strategies Pathogen
Pathogen
Prevent Prevent
Transmission Infection

Antimicrobial Infection
Resistance

Effective
Optimize Diagnosis
Use & Treatment
Antimicrobial
Use
 12 Steps to Prevent Antimicrobial Resistance: Hospitalized Adults

Preventions of Antimicrobial
Resistance
Clinicians hold the solution
Take steps NOW to prevent antimicrobial resistance!

12 Break the chain

11 Isolate the pathogen Prevent Transmission
10 Stop treatment when cured
9 Know when to say no to vanco
8 Treat infection, not colonization Use Antimicrobials Wisely
7 Treat infection, not contamination
6 Use local data
5 Practice antimicrobial control
4 Access the experts
3 Target the pathogen Diagnose & Treat Effectively
2 Get the catheters out
1 Vaccinate Prevent Infections
Preventions of
Antimicrobial Resistance
Active Antibiotic Stewardship Program
Judicious use of antibiotics

Education of prescribers regarding

proper antimicrobial usage

Antimicrobial formulary with restricted

prescribing of targeted agents

Prospective audit of antimicrobial

prescribing with feedback to

prescribers.
 Antibiotic Susceptibility
Tests
1. Disk Diffusion Technique
2. Dilution Tests

- minimum inhibitory concentration

(MIC)
- minimum bactericidal concentration
(MBC)
3. Antibiotic Assays
Disk Diffusion
Technique
Easy, cheap, flexible
Qualitative result for the organism
against the tested antibiotics
Sensitive, Intermediate, Resistant
Not useful for organisms
* slow growers eg Mycobacterium TB
* fastidious organism eg Mycoplasma
 Disk Diffusion
Technique
Filter paper disks containing
particular antibiotic concentrations
applied onto sensitivity agar plate
lawn with the tested bacteria
Diameter zone of inhibition measured
linear log2 MIC antibiotic
- compared with the chart
 NCCL
S for Clinical Laboratory
National Committee
Standards

Clinical and Laboratory Standards

Institute
(1st January 2005)

CLSI
 CLSI Method

Muller Hinton
Agar

MHA + 5%
Stoke Method UK
- Test & control organisms on same
plate
Stokes Method
Disks are placed at the interface
Zones of inhibition are compared
The use of a sensitive control
shows that the antibiotic is
active, so that if the test
organism grows up to the disk it
may safely be assumed that the
test organism is resistant to that
drug.
Factors Influencing the
Test
Media
- Thickness of media: 4 mm
- Mg ++, Ca++ contents
- pH
- Type: DST, MHA, MHA+blood,
Haemophilus test media
Antibiotic Concentration, potency,
diffusibility
Organism inoculum density & growth
rate
Enviroment Temp. & incubation time
 MIC
Minimum antibiotic concentration to
inhibit growth
Give quantitative value
Broth & agar plate doubled antibiotic
dilution
After incubation, 1st Tube/plate with
minimum antibiotic concentration shows
NO Growth = MIC value
The more resistant an organism is, then
the higher will be the MIC
 MBC
The MBC is measured by inoculating the
broths used for MIC determinations onto
drug-free medium.
After incubation, MBC is the first dilution
at which no growth is observed
Cidal drugs have MBC values that are
close to the MIC value for particular
organisms.
static agents, the MIC is much lower
than the MBC.
MIC = 0.125 mg/L
MBC = 0.25 mg/L
 E test

A strip containing a exponential

gradient of antibiotic
concentration
elliptical zone of inhibition

MIC value = ellipse intercept with the

strip
E test
 When to do MIC?
Infective endocarditis, for determining
antibiotic regimen
eg MIC Strep. against Penicillin
:< 0.1 mg/L :sensitive - iv Pen + Gen x
2/52
then oral Pen x 2/52
: 0.1 mg/L :resistant iv Pen +Gen
x4/52
By clinical syndrome MIC mg/L
Strep. pneumoniae
& Penicillin route
infection
Susceptible Penicillin
Intermediat Resistant
e
RMeningitis; iv Penicillin 0.06 - 0.12
Non-meningitis; iv Pen 2 4 8
Non-meningitis; oral 0.06 0.12-1 2.0
 Antibiotic Assay

For antibiotics with toxicity near

therapeutic range eg Vancomycin,
aminoglycoside
Therapeutic Monitoring in renal failure pts
Take patients sera:
- pre-dose toxic level
- pos-dose therapeutic level
Change Antibiotic Regimen depending on
result.