Introduction to Antibiotics

Professor Syed Ziaur Rahman

Department of Pharmacology

Jawaharlal Nehru Medical College, AMU, Aligarh

Email: rahmansz@yahoo.com

Content in this presentation

• Few characteristic properties of AB • Principles of AB Dosing • AB Resistance • Classification of Pathogens

Principles of AB Dosing

•AB Therapy goal - clear tissues of the microbial pathogens by achieving AB levels in infected area = or > Minimum Inhibitory Concentration (MIC) • MIC of the antibiotic is lowest concentration that prevents

visible growth of bacteria in culture plates using serial dilutions after 24 hr incubation period with a standard organism inoculation of 104 to 103 cfu/ml

•Minimum Bactericidal Concentration (MBC) • Lowest Concentration of AB that causes complete destruction of

organism or permits survival of 0.1% of the inoculum (kills 99.9% of bacteria)

Inference of MIC and MBC

•Small difference between MIC and MBC indicates that AB primarily bactericidal whereas large difference indicates bacteriostatic action. •Serum AB concentration and MIC serve as surrogate

markers to assess or quantify the AB activity

Concentration Dependent Killing? and Time Dependent Killing?

Principles of AB Dosing

Concentration Dependent Killing

• Some ABs are much more effective if higher blood concentrations are reached periodically. These exhibits Concentration Dependent Killing (CDK)

• Killing effect of drug is high when the ratio of peak concentration to MIC is more.

• These drugs produce better action when used as a large single dose as compared to same daily dose divided into 2-3 portions.

• Example: Aminoglycosides, Fluoroquinolones and Metronidazole is CDK effect

Time Dependent Killing

• AB more effective if blood level is maintained above MIC for as long a duration as possible - TDK • AB depends on the length of time the concentration remains above

MIC

• For these drugs multiple daily doses are preferred over single dose. • Example B-lactams and Vancomycin is TDK effect. • Macrolides and Clindamycin also possess time dependent activity.

However these are static drugs not TDK but Time dependent inhibitory activity drugs. (Remember most common)

Post Antibiotic Effect (PAE)

•After exposure of organism to AB, its growth stops.

•When it is placed in a antibiotic free medium, the growth resumes but only after a lag period. • Signifies inhibitory effect of AB is present even when

concentration is below MIC. This period is known as PAE.

•Most of antimicrobials have long PAE >1.5 hrs against gram positive bacteria.

•Carbapenems, Chloramphenicol, Aminoglycosides, Tetracyclines, Fluroquinolones and Rifampicin have long PAE against gram –ve bacteria.

Post Antibiotic Effect (PAE)

•Rifampicin prolongs the PAE of Isoniazid. • Due to this reason isoniazid given thrice weekly when given

in combination with rifampicin • short course chemotherapy of TB

• Needs to be administered daily if used alone.

Post Antibiotic Effect (PAE)

Mechanism of resistance

•Population of AB gets doubled in just 30 min - several generations of same bacteria in couple of hours - provides ample opportunity for genetic adaptations

•Drug resistance in the bacteria may be?

•Natural or Acquired

AB Resistance

Genetic Methods of AB Resistantt

•Chromosomal Methods: Mutations

•Extrachromosomal Mechanism: Plasmids

Chromosomal Methods: Mutations (Acquired resistance)

•Spontaneous Mutation in bacterial cells occur at frequency of approx one per million cells •Mutation rate is slow • Selection of Mutants

• Reduced pathogenecity • Clinical Problems: Mycobacteria (TB & Leprosy) & MRSA

•Development of acquired resistance may be due to • Single step mutation - Streptomycin and Rifampicin. •Multiple step mutation - Erythromycin, Tetracycline and

Chloramphenicol

Extrahromosomal Methods: Plasmids (Infectious Resistance)

•Plasmids • Carry genes resistant to AB (r-genes) are called R-Plasmids

•Plasmid-mediated resistance can be transferred by r-genes from one microorganism to other called Infectious resistance (Gene Transfer) via: •Gene Conjugation •Gene Transduction •Gene Transformation

Conjugation

•Due to physical contact between bacteria and is responsible for multidrug resistance. • If r-genes transferred between different species • Conjugative plasmids containing r-genes, make connecting

tube (sex pili) between 2 bacteria through which plasmid can pass

•Main mechanism of for spread of resistance specially at high density as in gut • Important mechanism for the development of resistance

against - Streptomycin and Chloramphenicol

Transduction

• Less common

• Transfer of Plasmid DNA enclosed in bacterial virus (bacteriophage) • Transfer of resistance gene to another bacterium of the same species

• Observed in transmission of resistant genes between strains of staphylococcus and between strains of streptococcus - Penicillin, Erythromycin and Chloramphenicol.

Transformation

• Least clinical problem

•Certain bacteria pick up free DNA from the environment (from a cell belonging to closely related or the same strain)

•New DNA incorporated into the genome of the bacteria which then becomes resistant e.g. Penicillin G

Transfer of r-genes between Plasmids within the bacterium

• By Transposon

• Transposons are DNA segments that cannot self-replicate but can self transfer between plasmids or from plasmid to chromosomes

• Donor plasmid containing a transposons cointegrates with the acceptor (or target) plasmid

• During the process of cointegration, the transposon can now replicate

• Both the plasmids then separate and each contain r-genes carrying transposon

• Strains of staphylococcus and enterococcus acquire resistance transferred by transposons – Nosocomial Infections

Transfer of r-genes between Plasmids within the bacterium

•By Integrons • Multidrug resistance can also spread by larger mobile DNA unit

called integrons, which can be located on transposons • Each integron is packed with multiple gene casettes (each

consisting of resistant gene attached to a small recognition site) • Gene casettes are encoded with several bacterial functions

inclduing resistance and virulance • Currently gene casettes have been identified for all AB except

FQs

Biochemical Mechanism of Resistance to AB

•Elaboration of enzymes

• Acetyl transferase (Chloramphenicol AT)

• Beta Lactamase (Penicillin, Cephalosporins)

• Acetyl transferase, Phosphotransferases and adenyltransferases (Aminoglycoside)

Prevention of Drug Accumulation in the bacterium (Decreased Permeability)

•Decreased influx • Inhibit the plasmid mediated synthesis of porin

channels, which obstructs the influx of drugs •Decreased drug permeability due to loss of specific

channels •Aminoglycosides and Tetracyclines attain much lower

concentration in resistant organisms than in the sensitive organisms.

•Promoting Efflux Pumps •Chromosomal or plasmid-mediated efflux transporter

proteins protect the bacterial cell from foreign chemical invasion and regulated by a number of genes

•Development of efflux pumps - Tetracyclines, erythromycin and fluoroquinolones •Active extrusion of drug from the resistant organisms.

Prevention of Drug Accumulation in the bacterium (Decreased Permeability)

By modification/protection of the target site

•Resistance resulting from altered target sites. •Ribosomal point mutations (replacement of only one

base pair in the DNA structure) for tetracycline, macrolides, clindamycin • Change of only one Aa in the β-subunit of DNA directed RNA

polymerase in Mycobacterium for Rifampicin •Altered DNA gyrase and topoisomerase for FQs •Altered penicillin binding proteins in Pneumococci and

Staphylococci

Alternative Metabolic Pathway

•Development of alternative metabolic pathway

•Overproduction of PABA • Sulfonamide resistant organisms start utilizing

preformed Folic Acid in place of synthesizing it from PABA

•Overproduction of β-lactamases

Qourum Sensing

• Regulation of gene expression in response to fluctuations in cell-population density.

• Quorum sensing bacteria produce and release chemical signal molecules (autoinducers) that increase in concentration as a function of cell density.

• Detection of minimal threshold stimulatory concentration of an autoinducer leads to alteration in gene expression.

• Gram-positive and Gram-negative bacteria use quorum sensing communication circuits to regulate diverse array of physiological activities. • These processes include symbiosis, virulence, competence, conjugation, antibiotic

production, motility, sporulation, and biofilm formation.

• In general, Gram-negative bacteria use acylated homoserine lactones (AHL) as autoinducers, and Gram-positive bacteria use processed oligo-peptides to communicate.

Figure (Taken from Wikipedia): Gram-negative bacteria , it is most commonly AHL.

Qourum Sensing

• The evolution of quorum sensing systems in bacteria could, therefore, have been one of the early steps in the development of multicellularity.

• Number of compounds have been synthesised which inhibit QS-system and include AHL, AIP, AI-2 Analogues

Qourum Sensing

Bacteriocins

Normal microbial flora contributes to host defense by development of bacteriocins

Superinfection

•Refers to appearance of new infection as a result of antimicrobial therapy.

•Broad spectrum antibiotics Aminoglycosides, Ampicillin, Chloramphenicol, Clindamycin and Tetracyclines may kill normal flora and result in development of new infections.

•Pathogens also have to compete with normal flora for nutrients.

Superinfection

• Superinfection more commonly seen in immunocompromised patients.

•Oropharynx, intestine, respiratory and genitourinary tract common sites for development of new infection.

•Organisms frequently involved Candida albicans, Clostridium difficle, Staphylococci, Proteus and Pseudomonas.

•Clostridium difficile superinfection may result in pseudomembranous colitis most commonly due to third generation cephalosporins for which metronidazole is drug of choice alternative drug is Vancomycin. •Further due to loss of commensal flora, there may be decreased formation of vitamin K leading to enhanced coagulation effects of warfarin.

Superinfection

•Two bacteriostatic agents often show additive effect.

•Two bactericidal agents are additive if organism is sensitive to both e.g. isoniazid and rifampicin in TB.

•Combination of bactericidal drug with bacteriostatic drug is additive if organism has low sensitivity to cidal drug e.g. streptomycin + tetracycline for brucellosis.

Clinical Significance

•Combination of bactericidal drug with bacteriostatic drug is additive if organism has low sensitivity to cidal drug e.g. streptomycin + tetracycline for brucellosis.

•Combination of bactericidal with bacteriostatic agent is antagonistic if organism has high sensitivity to cidal drug. e.g. penicillin + tetracycline or chloramphenicol for pneumococci.

Clinical Significance

•In immunocompromised patients, patients with HIV, on steroid therapy, neutropenic etc, only the bactericidal drugs should be used. •Bactericidal drugs are must in immunocompromised patients.

Clinical Significance

•Antimicrobials producing hemolysis in glucose-6-phosphate dehydrogenase deficient patients are primaquine, chloroquine, quinine, chloramphenicol, nitrofurantoin, fluoroquinolones, dapsone and sulfonamides.

Clinical Significance

WHO priority pathogens list for R&D of new antibiotics (2020)

Priority 1: CRITICAL

• Acinetobacter baumannii, carbapenem-resistant

• Pseudomonas aeruginosa, carbapenem-resistant

• Enterobacteriaceae, carbapenem-resistant, ESBL-producing

Priority 2: HIGH

• Enterococcus faecium, vancomycin-resistant

• Staphylococcus aureus, methicillin-resistant, vancomycin-intermediate and resistant

• Helicobacter pylori, clarithromycin-resistant

• Campylobacter spp., fluoroquinolone-resistant

• Salmonellae, fluoroquinolone-resistant

• Neisseria gonorrhoeae, cephalosporin-resistant, fluoroquinolone-resistant

Priority 3: MEDIUM

• Streptococcus pneumoniae, penicillin-non-susceptible

• Haemophilus influenzae, ampicillin-resistant

• Shigella spp., fluoroquinolone-resistant

Next classes

• In the next class, we will cover • Sulphonamide in detail

If any query, please email or call me!

Thank you…

The notes were prepared after consulting books by HL Sharma/KK Sharma, R.S. Satoskar/Nirmala Rege & Sparsh