Lecture 11 - Antimicrobial Chemotherapy

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 Blactamases 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 ( Blactamases) • They may also influence virulence

Mechanism of acquired resistance A. B.

Blockage of transport of drug into bacterial cell 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

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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)

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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

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Monitoring: ( toxicity): –

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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 Blactamases

Quiz • 2. 3. 4. 5. 6.

Answer T or F: Chloramphenicol is widely used due to its safety Serum levels of gentamicin and vancomycin can be monitored in the laboratory to minimize the risk of toxicity The use of two antibiotics is always more effective than monotherapy MIC and MBC determination is the most accurate way of determining an organism’s antibiotic susceptibility 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

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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

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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

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Transduction of genetic material- bacteriophage Transformation of DNA Transposons – Carry multiple resistance genes – Integrated into plasmids or chromosomes

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Acuisition of new chromosomal genes – Methicillin-resistant S.aureus (MRSA) e.g. mecA

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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 •