Antimicrobial

antimicrobial agents
phg 423
part iii
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2
Microbial Origin of Antibiotics

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Microbial Origin of Antibiotics
Bacteria
Bacillus sp (Gramicidin, Bacitracin, Polymyxins)
Fungi
Penicillum sp (Penicillin)
Cephalosporium sp (Cephalosporins)
Fusidium sp (Fusidic acid)
Actinomycetes
Streptomyces sp (Amino glycoside, tetracycline,
macrolide)
Micromonospora sp. (Gentamycin and netilmicin)

Antibiotic Spectrum of Activity
No antibiotic is effective against all
microbes

MICROBIAL FERMENATION OF
ANNTIBIOTICS
MICROBIAL FERMENTATIONS OF ANTIBIOTICS

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Fermentation
Fermentation Derived from the latin word fervere which means to boil.
It is a metabolic process that consumes sugar in the absence of oxygen. The products
are organic acids, gases, or alcohol. The science of fermentation is known as
Zymology.
In microorganisms, fermentation is the primary means of producing energy by
the degradation of organic nutrients anaerobically. Humans have used
fermentation to produce drinks and beverages
In biotechnology, using of grown microorganisms on a large scale to produce
products in large quantities, to produce any type of useful materials, or to carry out
chemical transformation.
Production in huge amount is carried out using fermentors.
A primary metabolite is a kind of metabolite that is produced during active cell growth
and directly involved development, and reproduction. It usually performs a
physiological function in the organism. It is typically present in many organisms or
cells. Some common examples of primary metabolites include: ethanol; lactic acids,
and amino acids.
A secondary metabolite is not directly involved in those processes, but usually has an
important ecological function (i.e. a relational function). It produced near the onset of
stationary phase Some common examples of secondary metabolites include: ergot
alkaloids, antibiotics, naphthalenes, nucleosides, phenazines, quinolines, terpenoids,
peptides and growth factors.

Primary and Secondary Metabolites
Yeast Fermentation Antibiotic Production:
P. chrysogenum
Clicker Question:

Secondary metabolites
Not essential for growth
Formation depends on growth conditions
Produced as a group of related compounds
Often significantly overproduced
Often produced by spore-forming microbes during sporulation
Secondary metabolites are often large organic molecules that require a large
number of specific enzymatic steps for production
Synthesis of tetracycline requires at least 72 separate enzymatic steps

Major Products Of Microbial Fermentations

Some Antibiotics Produced Commercially

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PROPERTIES OF A USEFUL INDUSTRIAL MICROBE INCLUDE
Produces spores or can be easily inoculated
Grows rapidly on a large scale in inexpensive medium
Produces desired product quickly
Should not be pathogenic
Amenable to genetic manipulation.
Microbial products of industrial interest include
Microbial cells
Enzymes
Antibiotics, steroids, alkaloids
Food additives.
Vitamins.

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Growth Curve
Lag phase
Adaptation
Should be reduced to avoid the wastage of time and to reduce the
medium consumption
Reduced by using of previously inoculated cells
Log phase
Exponential growth
Growth rate >> death rate
Primary metabolites production (required either for growth or for
energy, e.g., acetic acid, ethanol, citric acid)
Stationary phase
Reduced level of nutrients, and accumulation of toxic metabolites
Growth rate = death rate
Secondary metabolites production (toxins, alkaloids, antibiotics,
steroids), which produced in response to depletion of nutrients
Death or decline phase

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Fermentor is where the microbiology process takes place (Figure
15.2a and b)
Any large-scale reaction is referred to as a fermentation
Most are aerobic processes
Fermentors vary in size from 5 to 500,000 liters
Aerobic and anaerobic fermentors
Large-scale fermentors are almost always stainless steel
Impellers and supply oxygen
(Figure 15.2c)

Figure 15.2b
Steam
Sterile
seal
Motor
pH pH controller
Acid base
reservoir and
pump
Viewing
port
Filter
Exhaust
Impeller
(mixing)
Cooling
jacket
External
cooling
water in
External
cooling
water out
Culture
broth
Steam in
Valve
Harvest
Sparger (high-
pressure air
for aeration)
Sterile air

Figure 15.3
© 2012 Pearson Education, Inc.

Industrial Production of Penicillin

INDUSTRIAL PRODUCTION OF PENICILLIN
The industrial production of penicillin was broadly
classified in to two processes namely:
Upstream processing
Downstream processing
Upstream processing encompasses any technology that
leads to the synthesis of a product. It includes the
exploration, development and production.
The extraction and purification of a biotechnological
product from fermentation is referred to as downstream
processing.

After Finding an Antibiotic Producing Microbe
1.
produce enough for the next steps.
2. Purification so that a highly pure crystalline product.
3. Chemical Identification, and testing tolerance in animal
models.

UPSTREAM PROCESSING
INOCULUM PREPARATION
The medium is designed to provide the
organism with all the nutrients that it
requires.
Inoculation method- submerged technique
Spores -major source of inoculum

RAW MATERIALS
CARBON SOURCES:
Lactose acts as a very satisfactory carbon compound, provided that
is used in a concentration of 6%. Others such as glucose & sucrose
may be used.
NITROGEN SOURCES:
Corn steep liquor (CSL)
Ammonium sulphate and ammonium acetate can be used as
nitrogenous sources.
MINERAL SOURCES:
Elements namely potassium, phosphorus, magnesium, sulphur, zinc
and copper are essential for penicillin production. Some of these are
applied by corn steep liquor.
Calcium can be added in the form of chalk to counter the natural
acidity of CSL
Phenylacetic acid (PAA)- precursor

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Growth Medium
Minerals & Vitamins (plant growth and differentiation)
Carbon/energy source (due to lack of photosynthesis)
Growth regulators (cell enlargement, division, and differentiation)

FERMENTATION PROCESS
The medium is inoculated with a suspension of
conidia of Penicillium chrysogenum.
The medium is constantly aerated and agitated,
and the mould grows throughout as pellets.
After about seven days, growth is complete, the
pH rises to 8.0 or above, and penicillin
production ceases

STAGES IN DOWNSTREAM PROCESSING
Downstream processing is relatively easy since penicillin is
secreted into the medium (to kill other cells), so there is
no need to break open the fungal cells.
However, the product needs to be very pure, since it being
used as a therapeutic medical drug, so it is dissolved and
then precipitated as a potassium salt to separate it from
other substances in the medium.
Removal of cells
The first step in product recovery is the separation of whole
cells and other insoluble ingredients from the culture broth
by technique such as filtration and centrifugation.

ISOLATION OF BENZYL PENICILLIN
The PH is adjusted to 2-2.5 with the help of phosphoric or sulphuric acids.
In aqueous solution at low PH values there is a partition coefficient in favor of
certain organic solvents such as butyl acetate.
This step has to be carried out quickly for penicillin is very unstable at low PH
values.
Antibiotic is then extracted back into an aqueous buffer at a PH of 7.5, the
partition coefficient now being strongly in favor of the aqueous phase. The
resulting aqueous solution is again acidified & re-extracted with an organic
solvent.
These shifts between the water and solvent help in the purification of penicillin.
The treatment of the crude penicillin extract varies according to the objective, but
involves the formation of an appropriate penicillin salt.
The solvent extract recovered in the previous stage is carefully extracted back
with aqueous sodium hydroxide.
This is followed by charcoal treatment to eliminate pyrogens and by sterilization.
Pure metal salts of penicillin can be safely sterilized by dry heat, if desired.
Thereafter, the aqueous solution of penicillin is subjected to crystallization

FURTHER PROCESSING
For parental use, the antibiotic is packed in sterile
vials as a powder or suspension.
For oral use, it is tabletted usually now with a film
coating.
Searching tests (ex: for purity, potency) are
performed on the appreciable number of random
samples of the finished product.
It must satisfy fully all the strict government
standards before being marketed

The main stages of Penicillin production are:

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The recovery and purification of fermentation products
The choice of recovery process is based on the following criteria:
1. The intracellular or extracellular location of the product.
2. The concentration of the product in the fermentation broth.
3. The physical and chemical properties of the desired product (as an aid to
select separation procedures).
4. The intended use of the product.
5. The minimal acceptable standard of purity.
6. The magnitude of biohazard of the product or broth.
7. The impurities in the fermenter broth.
8. The marketable price for the product.

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It may be possible to modify the handling characteristics of the broth so that it can be
handled faster with simpler equipment making use of a number of techniques:
1. Selection of a microorganism which does not produce pigments or
undesirable metabolites.
2. Modification of the fermentation conditions to reduce the production of
undesirable metabolites.
3. Precise timing of harvesting.
4. pH control after harvesting.
5. Temperature treatment after harvesting.
6. Addition of flocculating agents.
7. Use of enzymes to attack cell walls.

PRODUCTS:
The resulting penicillin (called penicillin G)
can be chemically and enzymatically
modified to make a variety of penicillins
with slightly different properties.
These semi-synthetic penicillins include
penicillin V, penicillin O, ampicillin and
amoxycillin.

PRODUCTION OF PENICILLIN V
Phenoxy methyl penicillin
Addition of different Acyl groups to the
medium.
Phenoxyacetic acid as precursor instead
of phenyl acetic acid.

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Optimization of fermentation process through:
Medium composition (carbon and nitrogen sours, C/N ratio,
vitamins, growth hormones and any additive).
Optimum condition of fermentation (temp., PH, aeration, stirring,
volume and inoculums size).
Uses of beneficial additives to the medium:
Methionine is added to Cephalsporium spp. To increases the production
of cephalosporines.
Phenylacetamide to Penicillum spp. For a high production of penicillin G.
In case of production of tetracycline using Sterpt. aureofaciens, addition
of mercaptothiazole (inhibits chlorination of tetracycline) to avoid
production of chlortetracycline (highly irritant and very difficult to
separated from tetracycline) and increase tetracycline production

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Isolation and purification of antibiotics: Most of
antibiotics are released into the culture medium except
nystatin, amphotericin B and griseofulvin remain inside
the microorganism cells they isolated by extraction of
microorganism cells.
Most of antibiotics are obtained from growth medium
by the following
Selective adsorption e.g. charcoal in case of amino
glycosides antibiotics.
Selective precipitation e.g. methyl orange in case of
polymyxin production.
Selective solvent - solvent extraction.

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The recovery and purification of many compounds may be achieved by a number
of alternative routes. The decision to follow a particular route involves comparing
the following factors to determine the most appropriate under a given set of
circumstances:
Capital and processing costs.
Throughput requirements.
Yield potential.
Product quality.
Technical expertise available.
Conformance to regulatory requirements.
Waste treatment needs.
Continuous or batch processing.
Automation.
Personnel health and safety

Fermenter Kinetics with
Penicillium chrysogenum

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Purification of Cephamycin C: Sequential Ion Exchange Process

Microbial
Sources of
Antibiotics

BACTERIAL RESISTANCE
TO ANTIBIOTICS
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Resistance describes the situation in which an antibiotic fails to kill or inhibit the
growth of a microorganism at concentrations that can safely be achieved at the site
of infection in the body.
Bacteria that are naturally resistant to many, or even most, of the commonly used
antibiotics and, for all practical purposes, it is true to say that they have always been
resistant, and are always likely to be in the future; this is described as innate or
intrinsic resistance.
What is much more of a problem, and the reason for the media attention, is the fact
that so many organisms that were originally sensitive to particular antibiotics when
the drugs were first discovered are sensitive no longer, so the antibiotics are
becoming less useful. This is described as acquired resistance because it originates
from the organisms acquiring new genes, either by mutations of those they already
possess or, more problematically, from other microorganisms.

This transmission of genes from one cell to another without reproduction or
increase in cell numbers is termed horizontal transmission, whereas the term
vertical transmission describes genes simply being passed through the generations
from each cell to its offspring.
Usually the concentrations required to inhibit the growth of the target organisms
rise slowly over the course of several years as a result of the cumulative effects
of minor increases in resistance arising from mutations; this phenomenon is known
as resistance creep and it illustrates another aspect of resistance.

Measurement of resistance
Antibiotic resistance (or susceptibility) is most commonly measured using antibiotic-
impregnated paper disks that are placed on the surface of inoculated petri dishes.
During incubation the antibiotic dissolves in the gel and diffuses outwards from the
disk to give a concentration gradient that produces a zone of growth inhibition
around the disk.
The size of the inhibition zone is measured and compared with published tables.
Some antifungal agents, for example, are poorly water soluble and are better
tested by minimum inhibitory concentration (MIC) tests, which, as their name
suggests, determine the lowest concentration of the antibiotic that is effective in
inhibiting growth of the test organism.
The MIC is the lowest concentration at which there is no growth after incubation. If
the antibiotic concentration that can safely be achieved at the infection site does
not exceed the measured MIC the organism is regarded as resistant and so-called
antibiotic breakpoints are used for the prediction of successful therapy. A
breakpoint is an MIC threshold, and organisms having an MIC below this threshold
value can be expected to be inhibited or killed by standard doses of the antibiotic.

Origins of antibiotic resistance
In addition to innate and acquired , there are other terms used to describe the
characteristics of antibiotic resistance, and a distinction is often made between
resistance of phenotypic and genotypic origin. A phenotypic change is, by
definition, one that does not arise from an alteration in the genes the organism
possesses; rather, it is one in which the cells in a population, such as pathogenic
bacteria at an infection site, modify their physical structure or biochemical
properties in response to an environmental stress, for example exposure to
antibiotic. This is sometimes referred to as adaptive resistance , and it is
characterized by a more-or-less simultaneous change in most, or all, of the cells,
which is usually reversed when the environmental stress is removed, so it is not a
permanently inherited trait.

Figure: origins of antibiotic resistance:

MECHANISMS OF RESISTANCE
Resistance to antimicrobial agents typically occurs by one
or more of the following mechanisms:
Inactivation of the drug
Alteration of the target
Reduced cellular uptake
Increased efflux.
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RESISTANCE TO -LACTAM ANTIBIOTICS
-lactam antibiotics act by inhibiting the carboxy-transpeptidase or penicillin-binding
proteins (pbps) involved in the late stages of peptidoglycan biosynthesis.
Resistance to many -lactam agents is common and is most often caused by -lactamases
or by mutation in the pbps resulting in reduced affinity.
A number of different -lactamases have been described, but all share the feature of
catalyzing the ring-opening of the -lactam moiety.
-lactamases may be chromosomal or plasmid borne, inducible or constitutive.
A number of classification systems have been proposed, including classes a d based on
peptide sequence. Classes A, C and D have a serine at the active site, whereas class B
enzymes have four zinc atoms at their active site and these are also called metallo- -
lactamases.
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Class A enzymes are highly active against benzylpenicillin.
Class b -lactamases are effective against cephalosporins
and penicillins.
Class c enzymes are usually inducible, but mutation can lead
to overexpression.
Class d can hydrolyze oxacillin.
Increasing resistance to -lactam agents, mainly by -
lactamase, prompted the discovery and introduction of agents
with greater -lactam stability such as cephalosporins,
carbapenems and monobactams.
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-Lactamase inhibitors:
Clavulanic acid, Sulbactam,
Tazobactam, Avibactam
Clavulanic acid is produced by a
Streptomyces and is a suicide inhibitor
of -lactamases from a number of
Gram-negative and Gram-positive
organisms.
Although these -lactamase inhibitors
have a little antimicrobial activityof
their own, but their combination with a
- lactam antibiotic has extended the
clinical usefulness of the latter.
Avibactam

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Clavulanic acid or clavulanate, usually combined with amoxicillin
(Augmentin) or ticarcillin (Timentin)
Sulbactam, usually combined with ampicillin (Unasyn) or Cefoperazone
(Sulperazon)
Tazobactam, usually combined with piperacillin (Zosyn) (Tazocin)
Avibactam, approved in combination with ceftazidime (Avycaz), currently
undergoing clinical trials for combination with ceftaroline

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Bacteria that can produce beta-lactamases include, but are not limited
to:
MRSA
Staphylococcus
Enterobacteriaceae
Haemophilus influenzae
Neisseria gonorrhoeae
Klebsiella pneumoniae
Citrobacter
Morganella

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Altered penicillin-binding proteins (PBPs) and methicillin-resistant
Staphylococcus aureus (MRSA)
Altered PBPs are responsible for reduced sensitivity to -lactam agents by
Streptococcus pneumoniae (PBP1a, PBP2b and PBP2x) and Haemophilus
influenzae
PBP2A is responsible for the methicillin resistant Staphylococcus aureus
(MRSA).
The acquisition and spread of plasmid encoded -lactamases had blunted the
effectiveness of penicillin for treating S. aureus infections such as boils,
carbuncles, pneumonia, endocarditis and osteomyelitis.
This was the result of S. aureus acquiring the mecA gene, which encodes an
altered PBP gene, PBP2a. PBP2a has low affinity for most -lactam antibiotics.

RESISTANCE TO GLYCOPEPTIDE ANTIBIOTICS
VANCOMYCIN and TEICOPLANIN are the two GLYCOPEPTIDES used clinically. They
bind the terminal d-alanyl- d-alanine side-chains of peptidoglycan and prevent cross-
linking in a number of gram-positive organisms.
They are not active against gram negative organisms due to the presence of the outer
membrane.
Vancomycin-resistant enterococci (vre) now account for more than 20% of all
enterococcal infections. Like E. Faecium and E. Faecalis.
Resistance to vancomycin is via a sensor histidine kinase (vans) and a response regulator
(vanr).
Vanh encodes a d-lactate dehydrogenase/ -keto acid reductase and generates d-lactate.
The result is cell wall precursors terminating in d-ala-d- lac to which vancomycin binds
with very low affinity.
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RESISTANCE TO AMINOGLYCOSIDE ANTIBIOTICS
The aminoglycosides are hydrophilic sugars possessing a number of amino and hydroxy substituents.
Aminoglycoside binding to the a site interferes with the accurate recognition of cognate trna by rrna during
translation and may also perturb translocation of the trna from the a site to the peptidyl-trna site (p site).
While high-level resistance in aminoglycoside-producing microorganisms is by methylation of the rrna,
this is not the mechanism of resistance in previously susceptible strains.
The most common mechanism for clinical aminoglycoside resistance is their structural modification by
enzymes expressed in resistant organisms, which compromises their ability to interact with rrna.
There are three classes of these enzymes: aminoglycoside phosphatases (aphs), aminoglycoside
nucleotidyl transferases (ants) and aminoglycoside acetyltransferases (aacs).
Attempts to circumvent theses modyfing enzymes have centered on stuctural modification. Examples
include tobramycin which lacks the 3 -hydroxyl group and is thus not a substrate for APH(3 ) and
amikacin which has an acylated N-1 group and is not a substrate for several modifying enzymes

RESISTANCE TO TETRACYCLINE ANTIBIOTICS
More than 60% of shigella flexneri isolates are resistant to tetracycline.
Resistant isolates of salmonella enterica serovar typhimurium are becoming more common
and among gram-positive species, approximately 90% of mrsa strains and 60% of
multiply resistant streptococcus pneumoniae are now tetracycline-resistant.
The major mechanisms of resistance are efflux and ribosomal protection.
One exception is the tet(x) gene that encodes an enzyme which modifies and inactivates
the tetracycline molecule, although this does not appear to be clinically significant.
The tet efflux proteins belong to the major facilitator superfamily (mfs). These proteins
exchange a proton for a tetracycline cation (usually mg2+) complex, reducing the
intracellular drug concentration and protecting the target ribosomes in the cell.
The widespread emergence of efflux- and ribosome protection-based resistance leads to
first- and second-generation tetracyclines has prompted the development of the 9-
glycinyltetracyclines (9-glycylcyclines). 71

RESISTANCE TO FLUOROQUINOLONE ANTIBIOTICS
Fluoroquinolones bind and inhibit two bacterial topoisomerase enzymes: DNA gyrase
(topoisomerase II) which is required for DNA supercoiling, and topoisomerase IV which is
required for strand separation during cell division.
Each topoisomerase is termed a heterotetramer, being composed of two copies of two
different subunits designated a and b.
The a and b subunits of dna gyrases are encoded by gyra and gyrb, respectively, while
topoisomerase iv is encoded by parc and pare (grla and grlb in s. Aureus).
Mutations in gyra, particularly involving substitution of a hydroxyl group with a bulky
hydrophobic group, induce conformational changes such that the fluoroquinolone can no
longer bind.
Topoisomerases are located in the cytoplasm and thus fluoroquinolones must cross the cell
envelope to reach their target.
Changes in outer-membrane permeability have been associated with resistance in gram-
negative bacteria, but permeability does not appear to be an issue with gram-positive
species.
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RESISTANCE TO MACROLIDE(MLS) ANTIBIOTICS
Although chemically distinct, members of the MLS group of antibiotics all inhibit bacterial
protein synthesis by binding to a target site on the ribosome.
Gram-negative bacteria are intrinsically resistant due to the permeability barrier of the outer
membrane, and three resistance mechanisms have been described in gram-positive bacteria.
Target modification, involving adenine methylation of domain v of the 23s ribosomal rna, is the
most common mechanism.
The second resistance mechanism is efflux. Expression of the mef gene confers resistance to
macrolides only, whereas msr expression results in resistance to macrolides and streptogramins.
Efflux-mediated resistance of S. Aureus to streptogramin A antibiotics is also conferred by vga
and vgab gene products.
A third resistance mechanism, involving ribosomal mutation, has been reported in a small number
of clinical isolates of s. Pneumoniae.
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RESISTANCE TO CHLORAMPHENICOL
Chloramphenicol inhibits protein synthesis by binding the 50S
ribosomal subunit and preventing the peptidyltransferase step.
Decreased outer membrane permeability and active efflux have been
identified in gram-negative bacteria.
The major resistance mechanism is drug inactivation by chloramphenicol
acetyl-transferase. This occurs in both gram-positive and gram-negative
species.
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Resistance to trimethoprim
Trimethoprim competitively inhibits dihydrofolate reductase
(DHFR) and resistance can be caused by overproduction of
host DHFR, mutation in the structural gene for DHFR and
acquisition of the dfr gene encoding a resistant form.

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Resistance to peptide antibiotics ( polymyxin)
Peptide antibiotics include the polymyxins, bacitracins and gramicidins as
well as the glycopeptides.
Polymyxins and other cationic antimicrobial peptides have a self
promoted uptake across the cell envelope and
perturb the cytoplasmic membrane barrier.
Addition of a 4-amino-4-deoxy-L-arabinose (L-Ara4N) moiety to the
phosphate groups on the lipid A component of Gram-negative
lipopolysaccharide has been implicated in resistance to polymyxin.

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RESISTANCE TO ANTI-MYCOBACTERIAL THERAPY
The nature of mycobacterial infections, particularly tuberculosis, means that
chemotherapy differs from other infections. Organisms tend to grow slowly (long
generation time) in a near dormant state with little metabolic activity. Hence, a
number of the conventional antimicrobial targets are not suitable.
Isoniazid is bactericidal, reducing the count of aerobically growing organisms.
Pyrazinamide is active only at low pH, making it well suited to killing organisms within
necrotic foci early in infection, but less useful later on when these foci have reduced in
number. Rifampicin targets slow growing organisms.
Problems most commonly occur in patients who receive inadequate therapy which provides a
serious selection advantage.
Resistance can occur to single agents and subsequently to multiple agents. Resistance to
rifampicin arises from mutation in the beta subunit of RNA polymerase encoded by rpoB and
resistant isolates show decreased growth rates.
Modification of the catalase gene katG results in resistance to isoniazid, mainly by reduced or
absent catalase activity.
Catalase activity is absolutely required to convert isoniazid to the active hydrazine derivative.

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Intrinsic resistance (innate)
Inadequate concentration of drug
Bacteria contain the drug receptors but do not respond because the
concentration of antibiotic at the target side is inadequate
Rifampin is not effective against fungi because it dose not readily pass
through the fungal cell envelope to its site of action and this intrinsic
resistance can be changed by using combination therapy with
amphotericin b which facilitate the entrance of rifampin in adequate
concentration inside the cell to inhibit DNApolymerase

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INTRINSIC RESISTANCE
Increased concentration of a metabolite antagonizing the drug action
Certain microbes require p-aminobenzoic acid (PABA) in order to synthesize
dihydrofolic acid which is required to produce purines and ultimately nucleic
acids.
Sulfonamides, chemical analogs of paba, are competitive inhibitors of
dihydropteroate synthetase
Overproduction of PABA helps the bacterium to utilize it as a precursors and
escape the inhibition mechanism of the sulphonamides

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Acquired resistance
Population of organisms that are initially sensitive to a drug undergo change so
that they become less sensitive or insensitive.
Decreased drug uptake
Tetracycline resistance
Enzymatic inactivation of drug
Acetylation of chloramphenicol by salmonella
Decreased conversion of a drug to the active growth inhibitor compound
The antifungal flucytosine must be converted in the organism to fluoro-
uracil which is further metabolized to the active metabolite form of the
drug, fungi become resistant to flucytosine by inhibition the activity of
enzyme along the activation pathway of the drug.

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Acquired resistance
Alter amount of drug receptor
Trimethoprim resistance by synthesizing large amount of DHFR (the
target of the drug action)
Decrease affinity of receptor for the drug
Sulfonamide, trimethoprim, streptomycin, erythromycin, and rifampicin
resistance
Mutation
In mutant organism the receptor proteins may be:
Altered so that it will no longer be able to bind the drug
Decreases receptor affinity for the drug, in this case the antibiotic is still
effective but at higher concentration is required for inhibition

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PREVENTION OF RESISTANCE
Judicious use of antibiotics
Carry out the antibiotic-sensitivity test before drug intake
Developing a new drug
Control the use of antibiotics in both animal and human
General public needs to be educated to decrease the antibiotic misuse
Developing a new vaccines to control bacterial infection
Antibiotic combination where resistance is decreased if two drugs with different
mechanisms of action are administered together
Blocking the specific resistance mechanism e.G. Clavulanic -
lactamase inhibitor
Complete therapy i.e. Sufficient dosage and long enough duration.
Do not dispense antibiotic without prescription.

Normal Flora and Bacterial
Pathogenesis

Normal Flora (Commensal Microbes)
Introduction
Significance of the
Normal Flora
Distribution of the
Normal Flora
Bacterial Pathogenesis
Introduction
Host Susceptibility
Pathogenic Mechanisms
Virulence Factors
Outline

Colonization vs. Infection
Colonization: establishment of a site of reproduction of
microbes on a person without necessarily resulting in tissue
invasion or damage.
Infection: growth and multiplication of a microbe in or on
the body of the host with or without the production of
disease.
Outcomes of exposure to a microorganism:
1. Transient colonization
2. Permanent colonization
3. Disease
Normal Flora and Pathogenesis

Introduction of Normal Flora
1. A diverse microbial flora =>
Human body Area: the skin and mucous membranes
Time: shortly after birth until death
Number: 1014 bacteria =>1013 host cells
2. Normal flora may:
a. Aid the host
b. Harm the host (in sometimes)
c. Exist as commensals (no effect to the host)
3. Viruses and parasites => NOT normal microbial flora
Most investigators consider that they are not
commensals and do not aid the host.

Significance of Normal Flora
Normal flora may aid the host in several ways:
Aid in digestion of food
Help the development of mucosa immunity
Protect the host from colonization with pathogenic
microbes.
106 pathogenic
microbes
GI infection
w/ normal flora
GI infection
w/ reduced flora after
Streptomycin treatment
10 pathogenic
microbes

Normal Flora competing with Invading Pathogens
Adopted from Samuel Baron Medical Microbiology

Normal flora may act as opportunistic pathogens
Especially in hosts rendered susceptible by:
1. Immuno-suppression (AIDS & SCID*)
2. Radiation therapy & Chemotherapy
3. Perforated mucous membranes
4. Rheumatic heart disease etc.
* Severe combined immunodeficiency, SCID, also known as alymphocytosis, Glanzmann
Riniker syndrome, severe mixed immunodeficiency syndrome, and thymic alymphoplasia is
a rare genetic disorder characterized by the disturbed development of functional T cells and B
cells caused by numerous genetic mutations that result in heterogeneous clinical presentations.
SCID involves defective antibody response due to either direct involvement with B lymphocytes
or through improper B lymphocyte activation due to non-functional T-helper cells

Respiratory tract and head
outer ear, eye, mouth, oropharynx, nasopharynx
Sterile sites: sinuses, middle ear, brain, lower respiratory tract
(trachea, bronchiole, lung)
Gastrointestinal tract
esophagus, stomach, small intestine, large intestine
Genitourinary system
anterior urethra, vagina
Sterile sites: bladder, cervix, uterus
Skin
Sites of human body that the normal flora microbes colonize

Adopted from Samue
Distribution of Normal Flora in Human
Body

1. Local Environment (pH, temperature, redox potential, O2, H2O,
and nutrient levels ).
2. Diet
3. Age
4. Health condition (immune activity )
5. Antibiotics, ..etc
Factors Influencing Normal Flora

Introduction
Host Susceptibility
Pathogenic Mechanisms
Virulence Factors

1. Infection: growth and multiplication of a microbe in or on the
body with or without the production of disease.
2. The capacity of a bacterium to cause disease reflects its relative
Pathogenicity.
3. Virulence is the measure of the pathogenicity of a
microorganism.
4. Pathogenesis refers both to (1) the mechanism of infection and
to (2) the mechanism by which disease develops.

Host Susceptibility
1. Susceptibility to bacterial infections => Host Defenses vs Bacterial
Virulence
2. Host Defenses:
Barriers (skin & mucus) the first line
Innate Immunity (complement, macrophages & cytokines) the early
stage
Adaptive Immunity (Ag-specific B & T cells) the later stage
3. Host defenses can be comprised by destructing barriers or defective
immune response.
e.g. Cystic Fibrosis => poor ciliary function => NOT clear
mucus efficiently from the respiratory tract =>
Pseudomonas aeruginosa => serious respiratory distress.

Strict pathogens
are more virulent and can
cause diseases in a normal
person.
Opportunistic pathogens
are typically members of normal
flora and cause diseases when
they are introduced into
unprotected sites; usually occur
in people with underlying
conditions.

Transmission of infection
Carrier: a person or animal with asymptomatic infection that
can be transmitted to another person or animal.
The clinical symptoms of diseases produced by microbes often
promote transmission of the agents.
Zoonosis: infectious diseases transmitted between animals and
men.
Hospital- (nosocomial) vs. community-acquired infections
Microorganisms that normally live in people enhance the
possibility of transmission from one person to another.

Entry into the human body
: infection : shedding
The most frequent
portals of entry- Mucus
- Skin
Routes:
Ingestion, Inhalation,
Trauma, Needlestick,
Catheters, Arthropod
bite, Sexual transmission

1. Transmissibility
2. Adherence to host cells
3. Invasion of host cells and tissue
4. Evasion of the host immune system
5. Toxigenicity
A bacterium may cause diseases by
1. Destroying tissue (invasiveness)
2. Producing toxins (toxigenicity)
3. Stimulating overwhelming host immune responses
Characteristics of Pathogenic Bacteria

Mechanisms of acquiring bacterial
virulence genes

Bacterial Virulence Mechanisms

Bacterial virulence factors
Adhesins
Pili (fimbriae)
Invasion of the host cells
Tissue damage
Growth byproducts
Tissue-degrading enzymes
Immunopathogenesis
Toxins
Exotoxins (cytolytic enzymes and A-B toxins).
Enterotoxins; superantigens; endotoxin
and other cell wall components
Resistance to antibiotics

Adhesion---
1. Adherence of bacterium to epithelial or endothelial cells allow them to
colonize the tissue.
2. Common adhesins: pili (fimbriae), slime, lipoteichoic acid, surface
proteins or lectins.
3. Biofilm, formed on a surface by the bacteria that are bound together
within a sticky web of polysaccharide, is a special bacterial adaptation
that facilitates colonization on the surgical appliances (e.g., artificial
valves or indwelling catheters) and dental plaque. It can protect the
bacteria from host defenses and antibiotics.

1. Fever,
2. Leukopenia followed by leukocytosis,
3. Activation of complement, thrombocytopenia,
4. Disseminated intravasacular coagulation,
5. Decreased peripheral circulation and perfusion to
major organs (multiple organ system failure),
6. Shock and death.
Endotoxin-mediated toxicity

The A-B toxins
Mode of action
Inhibition of
protein synthesis
Hypersecretion
Inhibition of
neurotransmitter
release
In many cases the
toxin gene is
encoded on a
plasmid or a
lysogenic phage
A chain has the inhibitory activity against some vital function
B chain binds to a receptor and promotes entry of the A chain

Encapsulation (Inhibition of phagocytosis and serum bactericidal effect)
Antigenic mimicry
Intracellular multiplication
Escape phagosome
Inhibition of phagolysosome fusion
Resistance to lysosomal enzymes
Production of anti-immunoglobulin protease
Inhibition of chemotaxis
Destruction of phagocytes
Microbial defenses against host
immunologic clearance

Mechanisms for escaping phagocytic
clearance and intracellular survival

Mechanisms for escaping
phagocytic clearance and
intracellular survival

Candidate compounds have to pass tests for:
Toxicity
Allergic effects
Mutagenicity
Carcinogenicity
The estimated cost of a new drug is between $100 million to
$500 million.
Development can take as long as 5-10 years.

Sensitivitytests
Susceptible or resistant to antibiotic
MIC = Minimum inhibitory concentration
MBC = Minimum bactericidal concentration
Minimum concentration required to inhibit growth
Disc diffusion
Agar dilution
E-test
Breakpoint MIC

Sensitivity of microorganisms to antibiotics
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 or not.

Diffusion of antibiotic from a paper disc
After
Incubation
Zone of
Sensitivity
Concentration of
antibiotic at
periphery of zone
equals the MIC
Disc
Area of
Bacterial
growth
Disc
Concentration
Gradient

a control sensitive bacterium is
inoculated on part of a plate and the
tested bacterium is plated on the
remainder.
Disks of antibiotics are placed at the
interface and the zones of inhibition
are compared.
The use of a sensitive control shows
that the antibiotic is active, so that if
the test organism grows up to the disk
it may safely be assumed that the test
organism is resistant to that drug.

The bacterium in the diagram is
susceptible to drug "x" but
resistant to drug "y". The disc
containing drug "y" contains
active antibiotic as shown by
the zone of inhibition it causes
in the control bacterium.

The effectiveness of the
antibiotic is relative to the
inhibition zones of the bacterial
growth, the more the diameter,
the more potent the tested
antibiotic.

118
1 ug/ml
MIC = 8 ug/ml
MBC = 16 ug/ml
Minimal Inhibitory Concentration (MIC)
vs.
Minimal Bactericidal Concentration (MBC)
32 ug/ml
16 ug/ml
8 ug/ml 4 ug/ml 2 ug/ml
Sub-culture to agar medium

The MIC is the lowest concentration of the antibiotic that
results in inhibition of visible growth (i.e. colonies on a plate
or turbidity in broth culture) under standard conditions.
The MBC is the lowest concentration of the antibiotic that
kills 99.9% of the original inoculum in a given time. OR
The lowest concentration of antibiotic that allows less
than 0.1% of the original inoculum to survive.

Development of new antibiotics follows a very stringent highly
regulated pathway.
Selective toxicity required to keep the public safe
The need for product safety two-edged sword.
It requires a great deal of time and money.
Several testing systems can be used to evaluate new compounds.
Kirby-Bauer is the most widely used.
The E-test is more advanced.

TESTING OFANTIBIOTICS:
Kirby-BauerTest
An agar plate is covered with known pathogen.
Filter-paper disks impregnated with known concentrations of the
compound.
They are placed on agar.
Zones of inhibition can be identified.
The method is also used to compare the relative effectiveness of
different compounds.
Zones are evaluated using standardized tables.

TESTING OF ANTIBIOTICS: Kirby-Bauer Test

TESTING OFANTIBIOTICS:
Kirby-BauerTest
The resistance of specific organisms can be classified as:
Sensitive
Intermediate
Resistant
The Kirby-Bauer test is inadequate for most clinical purposes.

The E test is a more advanced diffusion test.
Permits determination of the minimal
inhibitory concentration (MIC).
Uses plastic-coated strips containing
gradients of antibiotic concentrations.
After incubation MIC can be read from the scale
Kirby-Bauer and E-test show which compounds
inhibit pathogen growth.
They cannot determine between
microbicidal and microbistatic.
The broth dilution test used for this purpose.
TESTING OFANTIBIOTICS: E-test

E test Determination of MIC
16
8
4
32
24
12
6
3
2
1.5
1
CI

Broth Dilution Test:
96 wells/ plate: simultaneously
performed with many tests organisms/
specimens, less reagent required
A specific organism is incubated in
decreasing amounts of antibiotic.
Growth in this medium indicates the test
compound is microbistatic.
No growth in this medium indicates the test
compound is microbicidal.
Used to determine serum concentrations

Breakpoint MIC
Breakpoint:
The breakpoint is the highest plasma concentration of the
drug that can safely be achieved in the patient and defines
whether an organism is susceptible or resistant to the drug.
This number is derived from in vitro susceptibility testing,
used by clinical microbiologists to tell clinicians whether the
antibiotic will work, could work or will fail in vivo against a
given organism.
Clinical breakpoint values are determined by specific criteria
described by EUCAST (European Committee on Antimicrobial
Susceptibility Testing). Criteria include dose, target organism
and its resistance mechanisms, MICs, clinical indications,
PK/PD properties, toxicity and desired clinical outcome.

Evaluation of Laboratory Tests
MIC test on plates is the best
Time consuming and costly
Most detailed
Disc test/E-test is easiest
Requires more skill to interpret
Breakpoint
Least skill required
Technique must be exact
Can be read by computer
Large amounts of data

THEY ARE COMPLEXES CONSISTING OF PROTEIN AND AN RNA OR DNA GENOME.
THEY LACK BOTH CELLULAR STRUCTURE.
THEY HAVE NO METABOLIC SYSTEMS OF THEIR OWN BUT DEPEND ON THE HOST CELL.
INTRACELLULAR OBLIGATE PARASITES.
BIND TO RECEPTORS ON CELL MEMBRANES AND ENTER THE HOST CELL.
USE CELLULAR METABOLIC ACTIVITIES FOR REPLICATION.
MAY BE DNA OR RNA VIRUSES.
DNA VIRUSES INCORPORATE INTO CHROMOSOMAL DNA AND PRODUCE NEW VIRUSES.
RNA VIRUSES MUST BE CONVERTED TO DNA BY REVERSE TRANSCRIPTASE IN ORDER TO REPLICATE.
INDUCE ANTIBODIES AND IMMUNITY.
THE PROPORTION OF NUCLEIC ACID IN RELATION TO PROTEIN IN VIRUSES RANGES FROM ABOUT 1% TO
ABOUT 50%.
VIRUSES

CAPSID AND ENVELOPE
THE PROTEIN COAT SURROUNDING THE NUCLEIC ACID OF A VIRUS IS CALLED THE
CAPSID.
THE CAPSID IS COMPOSED OF SUBUNITS, CAPSOMERES, WHICH CAN BE A SINGLE
TYPE OF PROTEIN OR SEVERAL TYPES.
THE CAPSID OF SOME VIRUSES IS ENCLOSED BY AN ENVELOPE CONSISTING OF
LIPIDS, PROTEINS, AND CARBOHYDRATES.
SOME ENVELOPES ARE COVERED WITH CARBOHYDRATE-PROTEIN COMPLEXES
CALLED SPIKES.

VIRUSES AND CANCER
THE EARLIEST RELATIONSHIP BETWEEN CANCER AND VIRUSES WAS DEMONSTRATED IN THE EARLY 1900S,
WHEN CHICKEN LEUKEMIA AND CHICKEN SARCOMA WERE TRANSFERRED TO HEALTHY ANIMALS BY CELL-FREE
FILTRATES.
TRANSFORMATION OF NORMAL CELLS INTO TUMOR CELLS:
WHEN ACTIVATED, ONCOGENES TRANSFORM NORMAL CELLS INTO CANCEROUS CELLS.
VIRUSES CAPABLE OF PRODUCING TUMORS ARE CALLED ONCOGENIC VIRUSES.
SEVERAL DNA VIRUSES AND RETROVIRUSES ARE ONCOGENIC.
THE GENETIC MATERIAL OF ONCOGENIC VIRUSES BECOMES INTEGRATED INTO THE HOST CELL'S DNA.
TRANSFORMED CELLS LOSE CONTACT INHIBITION, CONTAIN VIRUS-SPECIFIC ANTIGENS (TSTA AND T ANTIGEN),
EXHIBIT CHROMOSOMAL ABNORMALITIES, AND CAN PRODUCE TUMORS WHEN INJECTED INTO SUSCEPTIBLE
ANIMALS.

VIRAL STRUCTURE
A VIRION IS A COMPLETE, FULLY DEVELOPED VIRAL
PARTICLE COMPOSED OF NUCLEIC ACID SURROUNDED BY
A COAT.
HELICAL VIRUSES (FOR EXAMPLE, EBOLA VIRUS) RESEMBLE
LONG RODS AND THEIR CAPSIDS ARE HOLLOW CYLINDERS
SURROUNDING THE NUCLEIC ACID.
POLYHEDRAL VIRUSES (FOR EXAMPLE, ADENOVIRUS) ARE
MANY-SIDED. USUALLY THE CAPSID IS AN ICOSAHEDRON.
ENVELOPED VIRUSES ARE COVERED BY AN ENVELOPE AND
ARE ROUGHLY SPHERICAL BUT HIGHLY PLEOMORPHIC (FOR
EXAMPLE, POXVIRUS). THERE ARE ALSO ENVELOPED
HELICAL VIRUSES (FOR EXAMPLE, INFLUENZA VIRUS) AND
ENVELOPED POLYHEDRAL VIRUSES (FOR EXAMPLE,
HERPESVIRUS).
COMPLEX VIRUSES HAVE COMPLEX STRUCTURES. FOR
EXAMPLE, MANY BACTERIOPHAGES HAVE A POLYHEDRAL
CAPSID WITH A HELICAL TAIL ATTACHED. BACTERIOPHAGE: A
VIRUS THAT INFECTS AND LYSES CERTAIN BACTERIA.

DNA VIRUSES
Gene expression is much like that of the host cell
DNA-dependent RNA polymerase synthesizes mRNA.
Host cell ribosomes and tRNAs used to translate viral mRNA
Unique viral proteins include structural proteins and replication enzymes for viral DNA.
Examples:
Herpesvirus,
Epstein-Barr virus
HBV.

RNA VIRUSES
Examples
Polioviruses
Rhinoviruses (Frequent Cause Of The Common "Cold")
Coronaviruses (Includes The Agent Of Severe Acute Respiratory
Syndrome (SARS)
Rubella (Causes "German" Measles)
Yellow Fever Virus
West Nile Virus
Dengue Fever Viruses
Equine Encephalitis Viruses
Hepatitis A ("Infectious Hepatitis") And Hepatitis C Viruses
Rabies
Ebola
Influenza

RETROVIRUSES
Virus has the enzyme reverse transcriptase as a part of the viral structure.
A double-stranded DNA copy of the viral genome is produced.
This copy can integrate into the host cell chromosome.
Some retroviruses can cause tumors in animals: oncogenes
Human immunodeficiency virus (HIV) is a retrovirus. This is the causative agent of
AIDS.

VIRAL REPLICATION
THE STEPS IN VIRAL REPLICATION ARE AS FOLLOWS:
Adsorption of the virus to specific receptors on the cell surface.
Penetration by the virus and intracellular release of nucleic acid.
Proliferation of the viral components: virus-coded synthesis of capsid and
noncapsid proteins, replication of nucleic acid by viral and cellular enzymes.
Assembly of replicated nucleic acid and new capsid protein.
Release of virus progeny from the cell.

HOST-CELL REACTIONS
Possible consequences of viral infection for the host cell:
1. Cytocidal infection (necrosis): viral replication results directly in cell destruction
(cytopathology, so-called cytopathic effect in cell cultures).
2. Apoptosis: the virus initiates a cascade of cellular events leading to cell death
( suicide ), in most cases interrupting the viral replication cycle.
3. Noncytocidal infection: viral replication does not destroy the host cell, although it
may be destroyed by secondary immunological reactions.
4. Latent infection: the viral genome is inside the cell, resulting in neither viral
replication nor cell destruction.
5. Tumor transformation: the viral infection transforms the host cell into a cancer cell,
whereby viral replication may or may not take place depending on the virus and/or
cell type involved.

Commonly encountered viral infections and the methods available to treat them:

Targets for antiviral agents:
In general the process of viral replication can be summarized as
follows:
1. Adsorption of virus to host cell and entry.
2. Uncoating to liberate viral genome.
3. Synthesis and/or replication of viral DNA.
4. Integration of DNA into host genome (for latent viruses).
5. Production and assembly of new viral components (nucleic acid and
protein).
6. Maturation.
7. Release of new virions.

Each of these stages represents a potential intervention site for
antiviral therapy, and their usefulness will be discussed in this chapter.
However, to date, most of the therapeutic strategies have been
directed towards interference with the replication of viral DNA and this
will be dealt with first.

1. Antiviral agents active against the herpes group of viruses:
Modern antiviral agents were developed initially for the treatment of infections
caused by the herpes group of viruses, in particular herpes simplex type I, and so
these will be considered first. The herpes group contains a range of latent viruses
as can be seen BELOW table . these viruses give rise to diseases which range in
severity from simply being a nuisance to being life-threatening.

1.1. Nucleoside analogues:
Antiviral therapy is aimed at preventing viral DNA from being replicated within the host
cell. The strategy is to use molecules which resemble natural nucleosides but which are
modified to make them nonfunctional. These are called nucleoside analogues.
Idoxuridine was one of the first nucleoside analogues and was synthesized in 1959 as
part of an intensive search for anticancer drugs. Its anticancer activity was quite weak
but it demonstrated potent anti-viral activity. It replaces thymidine in the growing DNA
chain and can still form DNA chains as it possesses oh groups at the 5 and 3 positions on
the pentose sugar. Important points to note about idoxuridine are as follows:
. Its main use is in the treatment of superficial herpes simplex infections of the skin and
eye;
. It is too toxic to be used systemically;
. It causes adverse effects to kidney, bone marrow and liver;
. It cannot kill latent viruses it only prevents replication;
. It has now been superseded by other less toxic drugs.

In the search for less toxic nucleoside analogues, aciclovir was
developed. Aciclovir is a substituted guanine derivative that lacks an
OH group at position 3 on the sugar so it cannot accommodate chain
elongation. Aciclovir itself is inactive and it must be phosphorylated to
form the nucleoside triphosphate in order to be utilized by DNA
polymerase. In virus-infected cells aciclovir is phosphorylated by the
viral enzyme thymidine kinase to form acyclovir monophosphate.
Subsequent phosphorylation to di and tri- phosphates is carried out
by cellular kinases.
In noninfected cells thymidine kinase does not exist and so aciclovir is
not phosphorylated and remains inactive.

Points to note about the uses of aciclovir are as follows:
. It is used in the treatment of herpes infections of the skin and mucous membranes:
Herpes simplex virus types I and II;
Herpes simplex keratitis;
Varicella zoster (chicken pox and shingles).
. It can be given orally, topically, or by intravenous infusion.
. Its solubility is poor, hence it should not be given by bolus injection it is infused slowly over
1 hour.
. The ph of the solution is high (11) and may lead to irritation at the site of infusion.
. Poor solubility leads to poor absorption by mouth so high doses are needed:
200 mg 400 mg five times per day for HSV type I.
800 mg five times per day for varicella and herpes zoster.
. Clinically, it is highly effective with minor side effects. . Newer drugs such as famciclovir and
valaciclovir are similar to aciclovir and differ mainly in their improved oral absorption.

A major problem with aciclovir is its relative lack of activity against a formidable
member of the herpes group called cytomegalovirus. This does not cause many
problemsin otherwise healthy people but can cause blindness or even be life
threatening in immunocompromised patients.
Ganciclovir is very similar in structure to aciclovir and its mode of action is the
same. However, it is a much more efficient substrate than aciclovir for viral
thymidine kinase and is converted to di- and tri- phosphates much more rapidly
(10more ganciclovir triphosphate than aciclovir triphosphate in equivalently
treated cells). Ganciclovir is broken down and eliminated more slowly from
infected cells and it does not lead to chain termination. As a consequence of
these factors, ganciclovir is much more toxic than acyclovir.
As a result ganciclovir is recommended only for then treatment of life-threatening
or sight-threatening cytomegalovirus infections.

1.2. Inorganic pyrophosphate mimics:
When DNA polymerase adds a nucleoside triphosphate to a growing DNA chain, two
of the phosphates are cleaved (inorganic pyrophosphate) to provide energy. From this
reaction we can see that if the inorganic pyrophosphate was present in excess then the
action of the DNA polymerase would be inhibited. The antiviral agent foscavir has
structural similarities to pyrophosphate and inhibits the enzyme by mimicking the end
product of the reaction. Its interaction is fairly specific for viral DNA polymerases but it
does interact with cellular DNA polymerases at higher concentrations.
Consequently, it is a very toxic drug and renal impairment can occur in up to 50% of
patients. It is therefore only indicated for the treatment of CMV retinitis in patients with
AIDS and in whom ganciclovir is contraindicated or inappropriate.

2. Anti-HIV drugs
It is called a retrovirus because its genome is in the form of RNA, and on entering the
host cell this RNA is converted to DNA by the viral enzyme reverse transcriptase, which
is the equivalent enzyme to DNA polymerase. The DNA is then integrated into the host
cell genome where it resides permanently as a latent virus. The HIV has surface proteins
which act as binding sites; these are called gp120 and gp41. In addition, there are
receptor sites on the lymphocyte surface. The main receptor on the lymphocyte is CD4,
to which the gp120 protein attaches. Other receptors are CXCR4 or CCR5, to which the
gp41 protein binds. An HIV infection of lymphocytes requires attachment at both sites
and tight attachment of the virus to the host surface receptors leads to membrane
fusion. The HIV RNA contains nine genes, which code for structural proteins including
capsid proteins but also three key enzymes:
. Reverse transcriptase; Integrase; and Protease.
These enzymes have provided useful targets for antiviral chemotherapy.

2.1. Nucleoside reverse transcriptase inhibitors
Those nucleoside analogues acting at this target site are referred to as nucleoside
reverse transcriptase inhibitors (NRTIs). Zidovudine otherwise known as 3-azido-3-
deoxythymidine (AZT) was the first of these to be developed.
Zidovudine is well absorbed from the gut and so can be taken orally. However, it does
have profound toxic effects, particularly causing blood disorders, so patients should be
assessed for haematological toxicity and their dosage adjusted accordingly.
In an attempt to find less toxic nrtis, didanosine and zalcitabine were developed in the
1990s.
Facts about didanosine:
. It is acid labile often given on an empty stomach or after antacids;
. Patients show raised CD4 cell counts and decreased hiv-1 RNA counts;
. Its spectrum of adverse reactions differs from AZT:
Haematological toxicity is minimal;
There are problems with peripheral neuropathy (damage to nerves) and pancreatitis.

Facts about zalcitabine:
. Its absorption rate is decreased by the presence of food;
. Improvements are shown in surrogate markers;
. Significant clinical improvements arise in those who can tolerate the drug;
. Haematological toxicity is rare, but peripheral neuropathy common;
. Pancreatitis may occur and is serious but rare.
Lamivudine is a more recently developed nrti, which has a number of significant
advantages:
. It is a potent antiviral agent;
. It is rapidly absorbed bioavailability >80%;
. It causes much less peripheral neuropathy than previous drugs;
. It is clinically well tolerated drug with the most common side effects being
headache, fatigue and diarrhoea

2.2. Nucleotide reverse transcriptase inhibitors
These are nucleotide analogues (as distinct fromnucleoside analogues),
which means that they are already phosphorylated. Other than that
they work in the same way as nrtis and include the antiviral agent
tenofovir.

2.3. Non-nucleoside reverse transcriptase inhibitors (nnrtis)
Another group of drugs acting on reverse transcriptase are the non-
nucleoside reverse transcriptase inhibitors. As the name suggests, these are
not nucleoside analogues and their structures are completely different. They
bind at a different site on the enzyme to nucleoside analogues and there is
no equivalent receptor on human enzymes.
Examples include nevirapine and efavirenz.

2.4. Protease inhibitors (PIs)
It was noted above that HIV produces a number of enzymes one of which is protease.
This is responsible for modifying the newly formed proteins produced during viral
replication so that they are in the correct configuration for incorporation into the
developing viral progeny. This was recognized as a potential target, and protease
inhibitors were first introduced in 1996. Unlike nucleoside analogues, they do not require
phosphorylation for activity. They prevent post translational modification of viral proteins
leading to production of noninfectious virus particles. Fortunately, these inhibitors do not
interfere with activity of human proteases, for example trypsin and pepsin.
There are problems with lipodystrophy abnormal distribution of body fat and they can
also give elevated plasma lipid levels. Caution is needed in patients with haemophilia who
are at increased risk of bleeding. There are now numerous examples in clinical use
including ritonavir, saquinavir and lopinavir.

2.5. Fusion inhibitors
The HIV is an enveloped virus and has to fuse membranes with the host cell in order to bring
about an infection. We have already said that two receptors on HIV (gp120 and gp41)
bind to host cell receptors (CD4 and CXCR4) respectively and are responsible for infection.
If these receptors could be blocked then the infection process will be stalled.
Enfuvirtide is a 36 amino acid peptide derived from HIV gp41 and inhibits gp41-
mediated fusion. It works extracellularly and has to be self-administered by subcutaneous
injection. The most common side effect is injection-site reaction. It is synergistic with NRTIs,
NNRTIs, PIs and some other antiretrovirals.
Maraviroc is an orally active fusion inhibitor that has recently been released onto the
market. It is only active against viruses binding to the CCR5 co-receptor; not CXCR4. The
reason for the difference in virus receptor binding is unclear but CXCR4 binding variants
may emerge as the disease progresses. Patients on maraviroc have been shown to be twice
as likely to achieve undetectable HIV-1 RNA levels and double the gain in their CD4 T cells.
The profile of adverse effects is also acceptable.

2.6. Integrase inhibitors
After insertion of HIV viral RNA into the host cell, the genetic material
is converted to dsDNA by the enzyme reverse transcriptase produced
by the virus. This DNA is then further processed by another viral
enzyme called integrase which produces sticky ends on the DNA of
virus and host. The viral DNA is then spliced into the host DNA.
Raltegravir is an integrase inhibitor that has recently been released
for clinical use.

2.7. Maturation inhibitors:
Prior to release from the host cell all newly constructed HIV particles
undergo a maturation process and this is a key step in viral replication.
Development is underway on a group of drugs called maturation
inhibitors which disrupt this late stage viral maturation process. One of
the potential targets is the HIV Gag protein, which forms the capsid
shell of the virus. Maturation inhibitors cause Gag protein, and hence
the capsid, to be defective and noninfectious. These drugs have been
shown to be active against drug-resistant strains of HIV because they
act at a different site but as yet none has been licensed for use.

3. Viral hepatitis
Hepatitis is inflammation of the liver primarily caused by hepatitis A,
B or C viruses. Hepatitis A mainly occurs in epidemics among children
and young adults in institutions. Hepatitis B virus (HBV) infection occurs
by direct inoculation into blood or contamination of mucous
membranes and hepatitis C resembles a mild form of hepatitis B,
commonly associated with transfusion of blood or blood products.
Hepatitis b may be self-limiting but chronic liver disease can develop
in 10% of patients and chronic infection is associated with an
increased risk of hepatic carcinoma.

Facts about hepatitis B:
. It is estimated that there are 300 million chronic carriers worldwide.
. Carriers may be asymptomatic or have chronic active hepatitis, cirrhosis and/or
carcinoma.
. Immunization is available against hepatitis a and b infections.
. Antiviral chemotherapy for hepatitis b includes:
Interferon a limited usefulness (only 50% response rate).
Lamivudine used for initial treatment; resistance may occur on long-term treatment.
Entecavir nucleoside analogue. Not active against HIV, but stops HBV from
replicating in the liver.
Tenofovir a nucleotide analogue used as first-line therapy.
Adefovir dipivoxil a nucleotide analogue effective in lamivudine-resistant chronic
hepatitis B.

4. Influenza
Influenza is a respiratory illness caused by an influenza virus and the symptoms
include headache, fever, cough, sore throat, aching muscles and joints. There are
three main types: influenza A, B and C, of which influenza A is the most important.
Infections occur mostly in the winter and peak around december to march in the
northern hemisphere. Illnesses occurring in summer are usually due to other viruses.
For most patients influenza is an unpleasant illness but they will recover fully; for
others it can be life threatening. Common complications are bronchitis and secondary
bacterial pneumonia, which are a particular problem for the elderly, asthmatics and
those in poor health.
Influenza viruses a and b are antigenically unstable. Their viral surface contains
haemagglutinins and neuraminidase, which constantly mutate. The population
develops immunity to the viruses currently in circulation but occasionally a major
antigenic change occurs and epidemics or even pandemics result.

Oseltamivir and zanamivir inhibit the neuraminidase enzyme on the influenza viral
envelope. Neuraminidase is essential for the release of virus from its host and the
enzyme hydrolyses the sialic acid end of glycoproteins and glycolipids present on
the surface of host-cell membranes.
Oseltamivir is a sialic acid analogue mimicking the substrate and binding to the
active site on neuraminidase.
Oseltamivir (tamiflu) is orally active while zanamivir (relenza) is available only for
administration by inhalation.
Both are licensed for treatment if started within 48 hours and will reduce the
duration of symptoms by between 1 and 1.5 days.

HUMAN RESPIRATORY SYNCYTIAL VIRUS (HRSV)
Causing respiratory disease
Virazole (ribavirin)
Administered by inhalation
Can cause bronchospasm

ANTIRETROVIRALS
Always used in combination
Target enzymes or receptor sites
Specific guidelines for pregnancy

COMBINATION ANTIRETROVIRALS
Combovir (stavudine and zidovudine)
Trizivir (abacavir, lamivudine and zidovudine)
Decrease pill burden

Summary of antiviral chemotherapy:

Antimicrobial

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