Antimicrobial

Chemotherapy II
Dr. Eiman Mokaddas MD, FRCPath
Associate Professor
Microbiology Department
 Mechanism of Resistance
Clinical Definition of resistance:
• An organism is considered resistant to a
given drug when it is unlikely to respond to
attainable levels of that drug in tissues.
• Measurement of the antibiotic sensitivity of
an organism in the laboratory is designed
to predict whether an infection will respond
to treatment with that antibiotic
 A. Inherent or intrinsic
Resistance
• In some cases all strains of a given species are
naturally resistant to an antibiotic and is
unrelated to contact with the antibiotic e.g.
Streptococci are always resistant to
aminoglycosides and Gram-negatives are
always resistant to vancomycin
• This is due to inability of the drug to penetrate
the bacterial cell wall to exert its action
• This is mainly due to either absence of the target
or permeability barrier
• No need for laboratory testing as it is usually
predictable
 B. Acquired resistance
The genetic mechanism of resistance:
• Chromosomal mutations: sensitive bacteria may mutate to resistant
forms under the influence of an antibiotic (antibiotic pressure). The rate of
mutation development is influenced by the presence of drug mutants
which will survive in the presence of the antibiotic and can spread and
become predominant
• Types:
• Single large-step mutation: The target of the drug is altered by mutation,
unable to bind the drug
• Multiple drug-mutation: selection of the resistant mutant, more resistant
organisms will appear
• Examples
1. Streptomycin, rifampicin and isoniazid in M.tuberculosis
( combination therapy)
2. Mutation in genes contolling the production of chromosomally encoded B-
lactamases in Gram-negative bacteria ( resistance to cephalosporins)
2. Extra-chromosomal ( transferable antibiotic resistance):
• Mainly by plasmid-mediated conjugation
• Plasmids are pieces of extra chromosomal DNA that may carry
resistance or R factors
• Can be transferred easily among bacteria
• Common and wide spread
• Transposons accelerate the gene transfer through plasmids
• It is the most important mechanism clinically as it spreads easily
from strain to strain.
• They control enzymese that destroy the antibiotics ( B-
lactamases)
• They may also influence virulence
 Mechanism of acquired
resistance
A. Blockage of transport of drug into bacterial cell
B. Alteration of target site:
e.g.
• Changes in bacterial ribosome (erythromycin)
• Changes in PBP ( MRSA)
C. Metabolic bypass: Plasmid mediated
E. Destruction or inactivation of the drug:
• This is the most important mechanism of resistance and mediated
via production of inactivating enzymes ( plasmid or
chromosomally coded)
e.g.
• B-lactamases are the commonest
• Attack the B-lactam ring of penicillins ( penicillinases) or
cephalosporines ( cephalosporinases)
• Acetyltransferases, phosphorylases in aminoglycosiges
 General principles of clinical usage
A. patient Characteristics
• Age: Certain drugs are contraindicated in children
( ciprofloxacin)
• Renal Function: Many antibiotics are excreted in the
kidneys and tend to accumulate and cause renal failure
( aminoglycosides and vancomycin) should be
reduced in renal impairment
• Liver function: If hepatotoxic should be reduced in
hepatic failure
• Pregnancy:
– Some antibiotics are contraindicated in pregnancy ( mutagenic
or teratogenic)e.g. trimethoprim
– Some antibiotics are safe e.g. penicillins, cephalosporins
 General principles of clinical usage
B. Indication for antimicrobials
• Prophylaxis: The administration of
antimicrobials to prevent the future occurrence
of infection.
• Medical indications:
1. Meningococcal meningitis rifampicin or ciprofloxacin is
given to the contacts to eradicate the nasal carriage
2. Rheumatic fever: penicillin is given to prevent recurrent
infection to prevent rheumatic heart disease
• Surgical indications:
1. Abdominal surgeries( contaminated with bowel flora)
2. Give antibiotics shortly before the operation and 1-2 doses
after)
General principles of clinical usage
B. Indication for antimicrobials
2. Therapy:
• Empirical ( blind or best guess): The
organism is not known
• Microbiologically guided; according to the
bacterial isolate and the antimicrobial
susceptibility pattern
 General principles of clinical usage
C. Drug related consideration
• Spectrum of antimicrobial agent: narrow vs. broad
• Monotherapy vs. combination:
– Mixed infections ( more than one organism)
– Enhanced effect of the combination ( synergy)
• Additive, synergy and antagonism
• Bactericidal vs. bacteriostatic
– To minimize the development of drug resistance ( Treatment of tuberculosis)
• Penetration to site of infection:
– Meningitis and penetration into BBB: excellent penetration
( chloramphenicol), well penetration if inflammed meningies (penicillin,
cephalosporins), poor penetration ( aminoglycosides)
– Osteomyelitis; clindamycin and fusidic acid
• Monitoring: ( toxicity):
– some antibiotics have a low therapeutic index such as aminoglycosides,
vancomycin and chloramphenicol. This means that the difference between the
toxic dose and the therapeutic dose is small. Thus should measure the serum
levels
• Dose and duration of therapy:
 Role of the Laboratory
1. Advice on choice of antimicrobial:
– Given by microbiologist especially the blind
treatment
2. Monitoring of efficacy and toxicity:
– Two reasons to monitor serum levels:
• To ensure that therapeutic levels have been
achieved
• To ensure that the levels are not so high to be
toxic
– Peak and trough levels
 Role of Laboratory
3. Susceptibility Testing:
• Disc diffusion
• MIC/ MBC measurement
Disc Diffusion method
MIC/ MBC Method
 Quiz
• Answer T or F:
2. All antibiotic resistance is caused by genes
carried on plasmids
3. An infection should ideally be treated with an
antibiotic will always be killed by it
4. All penicillins are inactivated by B-lactamases
5. Penicillin penetrates well into CSF only in the
presence of meningeal inflammation
6. All antibiotic resistance is caused by B-
lactamases
 Quiz
• Answer T or F:
2. Chloramphenicol is widely used due to its safety
3. Serum levels of gentamicin and vancomycin can be
monitored in the laboratory to minimize the risk of
toxicity
4. The use of two antibiotics is always more effective
than monotherapy
5. MIC and MBC determination is the most accurate way
of determining an organism’s antibiotic susceptibility
6. Antibiotic therapy should never be started before the
results of laboratory sensitivity tests are available
 Quiz 1
• A diabetic patient developed carbuncle (boil) in the axilla.
He was treated with ampicillin orally in the clinic and sent
home. Three days later, the lesion instead of improving
had become enlarged and very painful.
• Question 1:
• What is the likely causative agent?
• Staphylococcus aureus
• Why did the lesion become larger?
• Ampicillin is not effective
• What class of antibiotics is penicillin?
• B-lactam antibiotic
• Question 2:
• By which mechanism did the organism
become resistant to ampicillin?
• By producing an inactivating enzyme
called B-lactamase which break down the
B-lactam ring and renders the antibiotic
inactive
• Question 3:
• What is the mechanism of antimicrobial
resistance in bacteria?
– 1. Intrinsic resistance
– 2. Acquired resistance
• Question 4:
• What are the main mechanisms of acquired
resistance?
– 1. Mutation
– 2. Transfer of resistance genes among bacteria
• Question 5:
• What is mutational resistance?
• Give examples.
• A change in the normal sequence or number of
chromosomal genes
• Examples:
– Alteration in 30s subunit ribosome as in MTB resistance to
streptomycin
– Change in porin subunit leading to impaired transport of the
antibioic into the cell
– Alteration in the penicillin binding protein PBP e.g.
Streptococcus pneumoniae resistance to penicillin
• Question 6:
• By which method are resistance genes acquired by bacteria?
• Resistance R plasmids:
– Code for synthesis of a drug- inactivating or a drug- modifying enzyme
– Transmitted by conjugation
• Transduction of genetic material- bacteriophage
• Transformation of DNA
• Transposons
– Carry multiple resistance genes
– Integrated into plasmids or chromosomes
• Acuisition of new chromosomal genes
– Methicillin-resistant S.aureus (MRSA) e.g. mecA
• Question 7:
• What is a plasmid?
• What is a transposon?
• Plasmid:
– A piece of double stranded circular DNA that replicates in
synchrony with but independent of the chromosome such that a
ratio of 1:1 is always maintained between the two of them
• Transposon:
– A small promiscuous piece of plasmid that can incorporate into
plasmids DNA or jump into chromosomal DNA ( jumping gene)
– Carries multiple genetic information
• What are the mechanisms of bacterial resistance in
general?
• Enzymatic inactivation of antibacterial agents
– B-lactamases
– Acetyltransferases, phosphorylases and nucleotidases(
aminoglycosides)
– Chloramphenicol acetyltransferases
• Modification of cell wall permiability
• Alteration of target molecule: PBP, OMP
• Development of alternative pathway Active exclusion
of antimicrobial agent from bacteria (eflux)
• Development of tolerence
•