1. Introduction to Microbiology
Anas Abu-Humaidan M.D. Ph.D.
Lecture 21

2. Overview The following topics will be discussed:
History of antibiotic use
Sources of antimicrobials
Mechanism of action of antibiotics
Susceptibility and resistance
Mechanism of resistance

3. History of antibiotic use
Antibiotics have been used since ancient times, Egyptians for example, applied mouldy bread to infected wounds. Nevertheless, until the 20th century, infections that we now consider straightforward to treat – such as pneumonia and diarrhoea – that are caused by bacteria, were the number one cause of human death in the developed world.
Paul Ehrlich, a German physician, noted that certain chemical dyes coloured some bacterial cells but not others. He concluded that, according to this principle, it must be possible to create substances that can kill certain bacteria selectively without harming other cells
The first mass produced antibiotic (penicillin) in 1940s have changed life in industrialized countries. Alexander Fleming and others have been instrumental in this effort.
Alexander Fleming

4. Sources of antimicrobials
There are three main sources of antimicrobial agents:
First are antibiotics, which are molecules of biological origin. Penicillin, for example, is produced by several molds of the genus Penicillium, and the first cephalosporin antibiotics were derived from other molds. Another source of naturally occurring antibiotics is the genus Streptomyces, which are Gram-positive, branching bacteria found in soil and freshwater sediments
Second are the chemically synthesized antimicrobial agents. These were initially discovered among compounds synthesized for other purposes (e.g sulphonamides), or active compounds that have been synthesized with structures tailored to be effective inhibitors or competitors of known metabolic pathways (e.g Trimethoprim)
A third source of antimicrobials arises from the molecular manipulation of previously discovered antibiotics to broaden their range and degree of activity against microorganisms or to improve their pharmacologic characteristics. (e.g. penicillinase-resistant penicillens)

5. Mechanism of action
Clinically effective antimicrobial agents exhibit selective toxicity toward the microbe rather than the host, a characteristic that differentiates them from the disinfectants.
Ideally, selective toxicity is based on the ability of an antimicrobial agent to attack a target present in bacteria but not humans
Some antimicrobials, such as penicillin, are essentially nontoxic to the host, unless hypersensitivity develops, while for aminoglycosides, the effective therapeutic dose is relatively close to the toxic dose

6. Mechanism of action- Cell wall synthesis
The peptidoglycan component of the bacterial cell wall provides its shape and rigidity. This giant molecule is formed by the linear glycans N-acetylglucosamine and N-acetylmuramic acid (NAG and NAM). Mature peptidoglycan is held together by cross-linked short peptide side chains hanging off the long glycan molecules.
This cross-linking process is the target of two of the most important groups of antimicrobials, the β-lactams and the glycopeptides (vancomycin and teicoplanin)

7. Mechanism of action- Cell wall synthesis
Penicillinase is one of a family of bacterial enzymes called β-lactamases that inactivate β-lactam antimicrobials
β-Lactamase inhibitors are β-lactams that bind β-lactamases Penicillin activity is enhanced in the presence of β-lactamase inhibitors (e.g. clavulanic acid, sulbactam, and tazobactam).

8. Mechanism of action-
Protein synthesis

9. Mechanism of action-
Targeting nucleic acid

10. SUSCEPTIBILITY AND RESISTANCE
Minimal inhibitory concentration (MIC)—a laboratory term that defines the lowest concentration (μg/mL) able to inhibit growth of the microorganism in vitro.
Resistant, nonsusceptible—term applied when organisms are not inhibited by clinically achievable concentrations of an antimicrobial agent.
Sensitive, susceptible—term applied to microorganisms indicating that they will be inhibited by concentrations of the antimicrobial that can be achieved clinically.
Spectrum—an expression of the categories of microorganisms against which an antimicrobial is typically active. A narrow-spectrum agent has activity against only a few organisms. A broad-spectrum agent has activity against organisms of diverse types (eg, Gram-positive and Gram-negative bacteria).
Pharmacologic properties such as absorption, distribution, metabolism, and elimination affect the usefulness of antimicrobials

11. SUSCEPTIBILITY AND RESISTANCE
Drug selection should include susceptibility, pharmacology, and clinical experience.
Dilution tests determine the MIC directly by using serial dilutions of the antimicrobial agent in broth that span a clinically significant range of concentrations
Diffusion Tests Antimicrobial in disks produces a circular concentration gradient, or in strips produces an elliptical concentration gradient Inhibition zone is a measure of the drug’s effect.

12. Mechanism of resistance
Resistance may be inherent to the organism or appear in a previously susceptible species by mutation or the acquisition of new genes.
The major mechanisms of bacterial resistance are:
(1) Exclusion of the antimicrobial from the bacterial cell due to impermeability or active efflux;
(2) alterations of an antimicrobial target, which render it insusceptible
(3) inactivation of the antimicrobial agent by an enzyme produced by the microorganism.

13. Mechanisms of resistance- Exclusion
Cell wall and outer membrane are barriers to antimicrobials
Outer membrane protein porins restrict access to interior, this is a major reason for inherent resistance to antimicrobial agents by gram negatives.

14. Mechanisms of resistance- Altered target
Once in the cell, antimicrobials act by binding and inactivating their target, which is typically a crucial enzyme or ribosomal site. If the target is altered in a way that decreases its affinity for the antimicrobial, the inhibitory effect will be proportionately decreased.

15. Mechanisms of resistance- Enzymatic inactivation
Hundreds of distinct enzymes produced by resistant bacteria may inactivate the antimicrobial in the cell, in the periplasmic space, or outside the cell.
β-Lactamase Enzymes break open the β-lactam ring. Other enzymes can inactivate aminoglycosides for example through modification.

16. Genetics of Resistance
Intrinsic Resistance: The resistant species have features such as permeability barriers, a lack of susceptibility of the cell wall, or ribosomal targets that make them inherently insusceptible.
Mutational Resistance: Acquired resistance may occur when there is a crucial mutation in the target of the antimicrobial or in proteins related to access to the target (ie, permeability).
Genetic exchange through horizontal gene transfer:
The transfer of plasmids by conjugation was the first discovered mechanism for the acquisition of new resistance genes, and it continues to be the most important. Species may carry multiple or no plasmids.
Transposon resistance genes move between chromosomes and plasmids, Transposition and conjugation combine for resistance spread

17. ANTIMICROBIAL STEWARDSHIP
Stewardship is the rational, optimal use of antimicrobials. All medical providers should behave as antimicrobial stewards
Bacteria will always evolve resistance in response to selective pressure.
So healthcare providers need to do their part in preserving this precious resource through following proper guidelines for antibiotic use, and through educating the public on this important subject.

18. Further reading: Sherris Medical Microbiology, sixth edition
Chapter 23: Antibacterial agents and resistance