-LACTAMS

Bacterial Cell Wall Composition

Structurally, most bacteria consist of a cell membrane surrounded by a cell wall, and for some bacteria, an additional outer layer.
Internal to the cell membrane is the cytoplasm which contains ribosomes, a nuclear region and in some cases granules and/or
vesicles. Depending on the bacterial species, a number of different external structures may be found such as a capsule, flagella and
pili. In gram-negative bacteria, the gap between the cell membrane and the cell wall, is known as the periplasmic space. Most
gram-positive bacteria do not possess a periplasmic space, but have only periplasm where metabolic digestion occurs and new cell
peptidoglycan is attached. Peptidoglycan, the most important component of the cell wall is a polymer made of N-acetyl muramic
acid (NAM) alternating with N-acetyl glucosamine (NAG) which are cross-linked by chains of four amino acids. The function of
the bacterial cell wall is to maintain the characteristic shape of the organism and to prevent the bacterium from bursting when
fluid flows into the organism by osmosis. Synthesis of the peptidoglycan and ultimately the bacterial cell wall occurs in a number
of stages. One of the first stages is the addition of five amino acids to N-acetyl muramic acid (NAM). Next, N-acetyl glucosamine
(NAG) is added to the N-acetyl muramic acid (NAM) to form a precursor of peptidoglycan. This peptidoglycan precursor is then
transported across the cell membrane to a cell wall acceptor in the periplasm. Once in the periplasm, the peptidoglycan precursors
bind to cell wall acceptors and undergo extensive cross-linking. Two major enzymes are involved in cross-linking: transpeptidase
and D-alanyl carboxypeptidase. These enzymes are also known as penicillin binding proteins (PBPs) because of their ability to
bind to penicillins and cephalosporins. Eventually several layers of peptidoglycan are formed all of which are cross-linked to
create the cell wall. Gram-positive bacteria may have more layers then gram-negative bacteria and thus have a much thicker cell
wall.
-Lactams: Mechanism of Action
-Lactam antibiotics include all penicillins and cephalosporins that contain a chemical structure called a beta-lactam ring. This
structure is capable of binding to the enzymes that cross-linked peptidoglycans. -Lactams interfere with cross-linking by binding
to transpeptidase and D-alanyl carboxypeptidase enzymes, thus preventing bacterial cell wall synthesis. By inhibiting cell wall
synthesis, the bacterial cell is damaged. Gram-positive bacteria have a high internal osmotic pressure. Without a normal rigid cell
wall, these cells burst when subjected to the low osmotic pressure of their surrounding environment. As well, the antibiotic
penicillin binding protein complex stimulates the release of autolysins that are capable of digesting the existing cell wall. -lactam
antibiotics are therefore considered bactericidal agents.
-Lactams: Mechanisms of Resistance
Bacterial resistance to -lactam antibiotics may be acquired by several routes:
- Alteration of the Penicillin Binding Proteins (PBPs): One of the most important mechanisms is through a process known as
transformation. During transformation, chromosomal genes are transferred from one bacterium to another. When a bacterium
containing a resistance gene dies, naked DNA is released into the surrounding environment. If a bacterium of sufficient
similarity to the dead one is in the vicinity, it will be able to uptake the naked DNA containing the resistance gene. Once
inside the bacterium, the resistance gene may be transferred from the naked DNA to the chromosome with the host bacteria
by a process known as homologous transformation. Over time, the bacterium may acquire enough of these resistance genes to
result in a remodelling of the segment of the host DNA. If this remodelled DNA segment codes for cross-linking enzymes (i.e.
penicillin binding proteins), the result is the production of altered penicillin binding proteins. These altered penicillin binding
proteins can still cross-link the peptidoglycan layers of the cell wall but have a reduced affinity for -lactam antibiotics, thus
rendering the bacterium resistant to the effects of penicillin and other -lactam agents. This transfer process has resulted in
penicillin resistant S. pneumonia, through the acquisition of genes from other naturally occurring penicillin resistant
Streptococcus species.
- Production of -Lactamases: A second important mechanism by which bacteria become resistance to beta-lactam antibiotics,
is by the production of enzymes capable of inactivating or modifying the drug before it has a chance to exert its effect on the
bacteria. Depending on the bacterial species, the gene coding for these enzymes may be found as part of the host DNA or on
plasmids, which are small self-replicating units of genetic material. Bacteria are capable of passing these resistance plasmids
to each other by conjugation. When two bacteria come into close contact with each other, a small channel is created between
them which allows one bacteria to pass a copy of the resistance plasmid to the other. If the plasmid is transcribed and
translated, the bacteria will begin to produce inactivating enzymes. These enzymes capable of destroying -lactam antibiotics
are known as -lactamases. In gram-positive bacteria, the -lactamase enzyme is generally inducible, resulting in a large
amount of enzyme being produced in the presence of the drug. In gram-negative bacteria the -lactamase enzymes are
produced constitutively (i.e. even when the antibiotic is not present). Gram-positive bacteria release the -lactamase enzyme
from the cell into the extracellular environment where it inactivates the drug before it enters the bacterial cell. In contrast,
gram-negative bacteria retain the -lactamase enzyme within the periplasmic space resulting in a more efficient mechanism
than gram-positive bacteria. Ultimately, the destruction of the -lactam ring of the antibiotic, renders it incapable of binding
to the penicillin binding protein and thus the bacteria become resistant to that drug or class of drugs.

FLUOROQUINOLONES
Bacterial DNA Synthesis
In this animation we demonstrate the biology of DNA replication leading to bacterial cell division in a gram-positive bacterium
such as S. pneumonia. The DNA is shown as a circular double-stranded within the bacterial cell. Like the DNA of all living
organisms, it contains the unique genetic code for all the proteins required for bacterial cell survival. Bacteria replicate by a
process known as binary fission, whereby one bacterium separates into two new daughter cells. However before this can occur,
the bacterium must make an identical copy of its complete circular DNA. DNA replication requires that the two strands of DNA
separate so that the genetic code of the bacterium can be read and a new complementary strand can be created for each of the
original strands. To accomplish this, various enzymes, known as helicases, break the hydrogen bonds between the bases in the two
DNA strands, unwind the strands from each other and stabilise the exposed single strands, preventing them from joining back
together. The points at which the two strands of DNA separate to allow replication of DNA, are known as replication forks. The
enzymes DNA polymerase then move along each strand of DNA behind each replication fork, synthesizing new DNA strands
complementary to the original ones. As a replication forks move forward, positive super helical twists in the DNA begin to
accumulate ahead of them. In order for DNA replication to continue, these super helical twists must be removed. The bacterial
enzyme, DNA gyrase, also known as topoisomerase II, is responsible for removing the positive super helical twists so that DNA
replications can proceed. DNA gyrase is an essential bacterial enzyme composed of two A and two B subunits, which are products
of the gyrA and gyrB genes. This enzyme has other important functions which affect the initiation of DNA replication and
transcriptional many genes. With the combined involvement of these enzymes, an entire duplicate copy of the bacterial genome is
produced as the two replication forks move in opposite directions around the circular DNA genome. Eventually as the two
replication forks meet, two new complete chromosomes have been made, each consisting of one old and one new strand of DNA.
This is referred to as semi-conservative replication. In order to allow the two new interlinked chromosomes to come apart, another
bacterial enzyme is needed which is known as topoisomerase IV. This enzyme is structurally related to DNA gyrase and is coded
for by the parC and parE genes. Topoisomerase for allows for the two new interlinked chromosomes to separate, so that they can
be segregated into two new daughter bacterial cells.
Fluoroquinolones: Mechanism of Action
This animation will demonstrate two mechanisms a fluoroquinolone action.
- Fluoroquinolone antibiotics, are synthetic molecules that are bactericidal. The potency of these drugs is greatly improved by
the addition of a fluorine molecule at position 6 and thus the term fluoroquinolones. Fluoroquinolones rapidly inhibit bacterial
DNA synthesis resulting in bacterial cell death. Fluoroquinolones act by inhibiting the activity of both the DNA gyrase and
the topoisomerase IV enzymes. For most gram-negative bacteria, DNA gyrase is the primary fluoroquinolone target.
Fluoroquinolones have been shown to bind specifically to the complex of DNA gyrase and DNA, rather than the DNA gyrase
alone. As a result of this binding, quinolones appear to stabilize the enzyme-DNA complexes which in turn results in breaks
in the DNA that are fatal to the bacterium.
- A second mechanism of fluoroquinolone action is shown here. With some exceptions, topoisomersase IV is the primary target
a fluoroquinolone action in most gram-positive bacteria such as staphylococcus and streptococci with DNA gyrase being a
secondary target. The separation of two new interlinked daughter strands of circular DNA is disrupted. The final result on the
bacteria however is the same. Bacterial replication is disrupted and the bacterium breaks apart. The relative potency of
different fluoroquinolone antibiotics and thus the spectrum of activity is depended in part on their affinity for either DNA
gyrase or topoisomerase IV or both.
Fluoroquinolones: Mechanisms of Resistance
One of the most common mechanisms by which bacteria acquire resistance to fluoroquinolones, is by spontaneously occurring
mutations in chromosomal genes that alter the target enzymes, DNA gyrase and topoisomerase IV or both. The frequency with
which the spontaneous mutations occurs may be in the range of 10 -6. The effective mutations on the activity of an individual
fluoroquinolone will vary depending on the number of mutations, the location of the mutations and which target enzyme is
affected. If a mutation occurs either in the gyrA or gyrB gene that alters DNA gyrase and results in a reduced affinity the
fluoroquinolone antibiotic for this enzyme, the organism will become resistant. Similarly a mutation may occur that alters
topoisomerase IV either in the parC or parE gene and results in a reduced affinity of the fluoroquinolone antibiotic for this enzyme,
and the bacterial organism will become resistant. It is important to note that for some fluoroquinolones that have similar affinity
and potency against both target enzymes, mutations in both DNA gyrase and topoisomerase IV will be needed for resistance to
occur. In resistant organisms semi-conservative replication continues.

MACROLIDES
Bacterial Protein Synthesis
The DNA is shown as a circular double-strand within the bacterial cell. Like the DNA of all living organisms, it contains the
unique genetic code for all other proteins required for bacterial survival. These include the proteins required for reproduction,
growth, repair and regulation of metabolism. It also codes for the three kinds of RNA that are essential for carrying out protein
synthesis. These are known as Ribosomal RNA (rRNA), messenger RNA (mRNA), and transfer RNA (tRNA). In order for the
bacteria to begin protein synthesis, the double-stranded DNA molecule must first unwind and separate in the region which codes
for the specific protein that is to be made. Only one strand of the DNA serves as a template for this process known as transcription.
Transcription results in the formation of messenger RNAs (mRNA) which is a mirror copy of the DNA segment. Once the strand
of mRNA is complete, it will detach from the DNA template and in turn, become attached to ribosomes. Bacterial ribosomes are
made up a small 30s ribosomal subunit and a large 50s ribosomal subunit. After the two sub units joined together around the
strand of mRNA, synthesis of the polypeptide chain begins. This step involves the aligning of transfer RNA (tRNA) molecules
and sequence along the mRNA. Each tRNA carries a unique amino acid determined by the sequence of the tRNA, which when
aligned along the mRNA and ribosome, join together to form the polypeptide chain. This step is known as translation. The
ribosomes will continue to add amino acids to the growing polypeptide chain until it reaches a point along the mRNA that signals
it to stop. At this point, it releases the finished protein molecule.
Macrolides: Mechanism of Action
Macrolide antibiotics, such as erythromycin, act as inhibitors of protein synthesis by attaching to the 50s ribosomal subunit. As a
result they block the ability of the ribosome to synthesize the polypeptide chain. By inhibiting protein synthesis, macrolides are
considered bacteriostatic antibiotics. However, at higher concentrations and with lower bacterial density, or during rapid bacterial
growth, macrolides may be bactericidal.
Macrolides: Mechanisms of Resistance
Target Site Modification: Changes or modifications to the 50s ribosomal subunit (i.e. the target binding site for macrolide
antibiotics) will confer resistance to macrolides and sometimes other classes of antibiotics. This type of resistance may be of
a high-level. This mechanism of resistance is mediated by the ERM (erythromycin ribosome methylation) gene, which is
found on plasmids or transposons (i.e. small genetic elements which are capable of moving from one bacterium to another
and integrating into the host chromosomal DNA). Copies of the ERM gene are transported to other bacteria via plasmids or
transposons through pili-like channels. The ERM gene is incorporated into the new bacterial genome. During the process of
protein synthesis, this bacterium will transcribe and translate the genetic code of the ERM gene, resulting in the production of
a protein enzyme capable of methylating the 50s ribosomal subunit at a specific position. This altered 50s subunit results in
decreased binding affinity for macrolides and other antibiotics such as lincosamides and Streptogramin B. This pattern of
resistance is referred to as the MLS phenotype. Because the macrolide antibiotic is unable to bind into the 50s ribosomal
subunit, it is unable to inhibit protein synthesis and thus the bacteria itself is not harmed, continuing to produce polypeptide
chains of amino acids.
Energy-dependent Efflux: A second mechanism a bacterial resistance to macrolide antibiotics is mediated by efflux pumps.
These efflux pumps are encoded by the mef(A) gene, which is a transposable element. Because they confer resistance to only
macrolides and not lincosamides or Streptogramin B, they're referred to as the M phenotype. The efflux pumps are energydependent and for S. pneumoniae, result in moderate levels of resistance. These pumps traverse the cell membrane of the
bacteria and function to pump out the macrolide antibiotic after it has entered the bacterium. It should be noted that for other
bacteria such as Staphylococcus aureus, for example, a different efflux system which is plasma-mediated and encoded by the
msrA, gene result in macrolide resistance as well as lilincosamide and Streptogramin B resistance in some cases. Despite the
presence of these efflux pumps, macrolide antibiotics continue to enter the bacteria. However once inside the cytoplasm other
bacteria these efflux pumps actively remove the macrolide antibiotics before they have a chance to reach their target; the 50s
ribosomes subunit, and bacterial protein synthesis is unaffected.