Antibiotics I: Overview

Question 1

MRSA) is an

organism which is resistant

Answer 1

resistant to many beta‐lactam antibiotics

Question 2

Clostridium difficile is

Answer 2

Clostridium difficile is, as the name suggests, difficult to treat. It resides in the lower regions of the GI tract and is most often associated with elderly patients and those with sedentary lifestyles. Again, this bacterium is associated with poor hygiene in healthcare settings.

Question 3

Glycopeptide‐resistant Enterococci are

Answer 3

Glycopeptide‐resistant Enterococci are of particular concern, since they are resistant to those antibiotics which were once considered to be the most powerful, the drugs of last resort. Enterococci inhabit our GI tract and are part of the normal microflora. However, enterococcal infections are potentially fatal and there is a high morbidity rate associate with them. Resistance to glycopeptide antibiotics, e.g. vancomycin, in enterococci is on the increase and there is a limited range of antibiotics that can be used to treat such infections.

Question 4

Classic antibiotic discovery pathway

Answer 4

The first step was to test a range of compounds against standard set of screens in order to find any active compounds; the use of combinatorial chemistry methodology and high throughput screening facilitates this stage. Once activity had been detected the structure of the compound was determined (if unknown), the target and mechanism of action was deduced, PK and toxicology studies were undertaken and the results of all of these investigations were fed into the medicinal chemistry programme. At this stage a range of analogues of the active compound would be prepared and screened, with the results again feeding back and informing the analogue design. When a clinical candidate had been found this was then put through clinical trials, etc and post‐market surveillance. Of course, when resistance was observed the process started again, either through further analogue synthesis or with a completely new compound.

Question 5

the essential molecular machinery in prokaryotes is located..

Answer 5

The essential molecular machinery in prokaryotes is located within the cytosol and is unconstrained by any intracellular boundary layers.

Question 6

Eukaryotes have a much more..

Answer 6

Eukaryotes have a much more complex cellular structure but lack the rigid cell wall that is a characteristic of
prokaryotic cells.

Question 7

the replicative cycle of prokaryotes is

Answer 7

the replicative cycle of prokaryotes is notable because it is radically different from that observed in eukaryotic cells.

Question 8

Prokaryotes contain a..

Answer 8

Prokaryotes contain a single chromosome comprising double‐stranded DNA that is formed into a circle. During replication the two strands separate to form a ‘replication
bubble’ and complementary DNA replication occurs to form identical copies of the chromosome which are then passed on to daughter cells.

Question 9

gram-ve cell wall

Answer 9

The periplasmic space is an aqueous gel‐like region between two membranes of Gram –ve bacteria. It contains enzymes associated with nutrient acquisition and transport, also defence enzymes used to resist antibiotics, e.g. B‐lactamase.
Porins in the cell surface allow passive diffusion of molecules into the periplasmic space.
Lipopolysaccharides play an important role in cell defence and the immunological response.

Question 10

To cause disease the organism must:

Answer 10

• Encounter and gain access to host
• Evade defence mechanisms
• Colonise and maintain colony
• Carriage and spread of infection
• Asymptomatic vs disease e.g. TB

Question 11

Antibacterial drug target

Answer 11

There are five main antibacterial drug targets in bacteria: cell‐wall synthesis, DNA gyrase, metabolic enzymes, DNA‐directed RNA polymerase and protein synthesis. The figure shows the antimicrobial agents that are directed against each of these targets.

Question 12

Which antimicrobial agents are directed against protein synthesis

Answer 12

aminoglycosides and tetracyclines inhibit 30S RNA, and macrolides, chloramphenicol and clindamycin inhibit 50S RNA. mRNA, messenger RNA

Question 13

discovery of penicillin

Answer 13

Since the discovery of penicillin by Fleming in 1928

Question 14

Mechanisms of genetic resistance to antimicrobial agents

Answer 14

Bacteria have developed, or will develop, genetic resistance to all known antimicrobial agents that are
now in the marketplace. The five main mechanisms that bacteria use to resist antibacterial drugs are shown in the figure.
a | The site of action (enzyme, ribosome or
cell‐wall precursor) can be altered. Eg, acquiring a plasmid or transposon that codes for a resistant dihydrofolate reductase confers trimethoprim resistance to bacteria.
b | The inhibited steps can be by‐passed.
c | Bacteria can reduce the intracellular conc of the antimicrobial agent, either by reducing membrane permeability, for example, as shown by Pseudomonas aeruginosa, or by active efflux of the agent.
d | They can inactivate the drug. For example, some bacteria produceβ‐lactamase, which destroys the penicillinβ‐lactam ring50,51 (FIG. 1).
e | The target enzyme can be overproduced by the bacteria.

Question 15

Prokaryote DNA - Plasmids

Answer 15

• Small loops of extrachromosomal DNA
• Not essential for survival a priori
• May contain genes that are expressed in response to certain situations
• May be used to transfer genes between cells, e.g. resistance to antibiotics
• Can be incorporated into the chromosome
• Single DNA strand transferred between cells by conjugation using ‘rolling circle’ replication

Question 16

Horizontal Gene Transfer

Answer 16

a | Transformation occurs when naked DNA is released on lysis of an organism and is taken up by another organism. The antibiotic-resistance gene can be integrated into the chromosome or plasmid of the recipient cell.
b | In transduction, antibiotic-resistance genes are transferred from one bacterium to another by means of bacteriophages and can be integrated into the chromosome of the recipient cell (lysogeny).
c | Conjugation occurs by direct contact between two bacteria: plasmids form a mating bridge across the bacteria and DNA is exchanged, which can result in acquisition of antibiotic-resistance genes by the recipient cell. Transposons are sequences of DNA that carry their own recombination enzymes that allow for transposition from one location to another; transposons can also carry antibiotic-resistance genes.

Question 17

beta‐lactam class of antibiotics

Answer 17

The beta‐lactam class of antibiotics contains the penicillins, cephalospoirins, carbapenems, monobactams and other compound that contain the four‐membered
lactam ring (the beta‐lactam ring; a lactam is a cyclic amide in which the amide bond forms part of the ring). The majority of beta‐lactam antibiotics act as substrate
analogues for D‐Ala‐D‐Ala and associate with transpeptidase enzymes that form the crosslinks within peptidoglycan, thus inhibiting crosslink formation.

Question 18

B-Lactam Antibiotics - 2 points

Answer 18

• Substrate analogues of D-Ala-D-Ala

* Inhibit crosslinking step of peptidoglycan synthesis

Question 19

Methicillin Resistant S. aureus (MRSA)

Answer 19

• MRSA contains novel PBP2a; substitutes for native PBPs and has low affinity for all B-lactams
• MRSA chromosome contains ~ 50kb mec region not present in MSSA
• PBP2a is encoded by mecA gene; expression controlled by mecI, mecR1 and other factors
• Most MRSA are multi-drug resistant (MDR-MRSA) but are susceptible to vancomycin
• Epidemic strains EMRSA-15 and -16 most prevalent

Question 20

Schematic circular diagrams of the MRSA252 and MSSA476 chromosomes.

Answer 20

Where appropriate, categories are shown as pairs of concentric circles representing both coding strands. The outer colored segments on the gray outer ring represent genomic islands and horizontally acquired DNA (see figure for key). Inside the gray outer ring, the rings from outside to inside represent scale in Mbp, annotated CDS (colored according to predicted function), tRNA and rRNA
(green), additional DNA compared to the other S. aureus strain described here (MSSA476 or MRSA252 where appropriate; red), additional DNA compared to other sequenced S. aureus strains [N315 (5), Mu50 (5), and MW2 (6); blue], percentage of G + C content, and G + C deviation (>0%, olive; <0%, purple).
Color coding for CDSs is as follows: dark blue, pathogenicity/adaptation; black, energy metabolism; red, information transfer; dark green, surface-associated;
cyan, degradation of large molecules; magenta, degradation of small molecules; yellow, central/intermediary metabolism; pale green, unknown; pale blue, regulators; orange, conserved hypothetical; brown, pseudogenes; pink, phage plus insertion sequence elements; gray, miscellaneous.

Question 21

Tn1546 and the vanA Operon

Answer 21

A TWO-COMPONENT REGULATORY system VanR–VanS regulates vancomycin resistance in vancomycin-resistant ENTEROCOCCI (VRE) and vancomycin-resistant Staphylococcus aureus (VRSA) strains. VanS is a
membrane-associated sensor (of vancomycin) that controls the level of phosphorylation of VanR. VanR is a transcriptional activator of the operon encoding VanH, VanA and VanX. VanH is a dehydrogenase that reduces pyruvate to D-Lac, whereas VanA is a ligase that catalyses the formation of an ester bond between D-Ala and D-Lac. Vancomycin does not bind to D-Ala-D-Lac, which leads to vancomycin resistance. VanX is a dipeptidase that hydrolyses the normal peptidoglycan component D-Ala-D-Ala, which prevents it from causing vancomycin sensitivity. VanY is a D,D-carboxypeptidase that hydrolyses the terminal D-Ala residue of late peptidoglycan precursors that are produced if elimination of D-Ala-D-Ala by VanX is not complete. So, D-Ala-D-Lac replaces the normal dipeptide D-Ala-D-Ala in peptidoglycan synthesis resulting in vancomycin resistance. VanZ confers resistance to teicoplanin by an unknown mechanism.

Question 22

VRSA – A Real and Present Danger

Answer 22

• S. aureus with reduced susceptibility to vancomycin
• First reported in 1997 in the USA
• Isolates typically obtained from patients with chronic VRE infection
• Decreased susceptibility may be due to increased levels of peptidoglycan and precursors

Question 23

Biochemical targets for chemotherapy include

Answer 23

the synthesis of peptidoglycan, proteins and nucleic acids

Question 24

The most challenging and lengthy phase in antibacterial drug discovery

Answer 24

Lead optimization is the most challenging and lengthy phase in antibacterial drug discovery.
Probability of success during lead optimization is strongly linked to the size of the medicinal chemistry effort devoted to the project. GSK committed large teams of chemists for an uninterrupted period of 5 years to achieve a 50% success rate. Lead optimization efforts with less resources could easily take considerably longer.

Question 25

Example of glycopeptide antibiotic

Answer 25

vancomycin