Control of microbial growth
Growth may be defined as an increase in cellular constituents. It leads
to a rise in cell number when microorganisms reproduce. Growth also results
when cells simply become longer or larger. If the microorganism is coenocytic;
that is, a multinucleate organism in which chromosomal replication is not
accompanied by cell division— growth results in an increase in cell size but
not cell number. It is usually not convenient to investigate the growth and
reproduction of individual microorganisms because of their small size. Therefore,
when studying growth, microbiologists normally follow changes in the total
population number.
BACTERIAL CELL CYCLE
The cell cycle is the complete sequence of events
extending from the formation of a new cell through the next division. It is of
intrinsic interest to microbiologists as a fundamental biological process.
However, understanding the cell cycle has practical importance as well. For
instance in bacteria, the synthesis of peptidoglycan are the target of numerous
antibiotics. Antibacterial drugs: Inhibitors of cell wall synthesis. Although
some prokaryotes reproduce by budding, fragmentation, and other means, most prokaryotes
reproduce by binary fission. Binary fission is a relatively simple type of cell
division: the cell elongates, replicates its chromosome, and separates the
newly formed DNA molecules so there is one chromosome in each half of the cell.
Finally, a septum (cross wall) is formed at mid cell, dividing the parent cell
into two progeny cells, each having its own chromosome and a complement of
other cellular constituents. Despite the apparent simplicity of the prokaryotic
cell cycle, it is poorly understood. The cell cycles of several bacteria—
Escherichia coli, Bacillus subtilis, and the aquatic bacterium Caulobacter
crescentus— have been examined extensively, and our understanding of the
bacterial cell cycle is based largely on these studies. Two pathways function
during the bacterial cell cycle: one pathway replicates and partitions the DNA
into the progeny cells, the other carries out cytokinesis—formation of the
septum and progeny cells. Although these pathways overlap, it is easiest to
consider them separately.
Chromosome Replication and Partitioning
Recall that most bacterial
chromosomes are circular. Each circular chromosome has a single site at which
replication starts called the origin of replication or simply the origin. Replication is completed at the terminus,
which is located directly opposite the origin. In a newly formed E. coli cell,
the chromosome is compacted and organized so that the origin and terminus are
in opposite halves of the cell. Early in the cell cycle, the origin and
terminus move to midcell and a group of proteins needed for DNA synthesis
assemble at the origin to form the replisome. DNA replication proceeds in both
directions from the origin, and the parent DNA is thought to spool through the
replisomes. As progeny chromosomes are synthesized, the two newly formed
origins move toward opposite ends of the cell, and the rest of the chromosome follows
in an orderly fashion. Although the process of DNA synthesis and movement seems
rather straightforward, the mechanism by which chromosomes are partitioned to
each daughter cell is not well understood. Surprisingly, a picture is emerging
in which components of the cytoskeleton are involved. For many years, it was
assumed that prokaryotes were too small for eukaryotic, like cytoskeletal
structures. However, a protein called MreB, which is similar to eukaryotic
actin, seems to be involved in several processes, including determining cell
shape and chromosome movement.
MreB polymerizes to form a spiral around the inside periphery of the
cell. One model suggests that the origin of each newly replicated chromosome
associates with MreB, which then moves them to opposite poles of the cell. The
notion that prokaryotic chromosomes may be actively moved to the poles is
further suggested by the fact that if MreB is mutated so that it can no longer
hydrolyze ATP, its source of energy, chromosomes fail to segregate
properly. Prokaryotic cytoplasm: Prokaryotic
cytoskeleton.
Cytokinesis
Septation is the process of
forming a cross wall between two daughter cells. Cytokinesis, a term that has traditionally
been used to describe the formation of two eukaryotic daughter cells, is now
used to describe this process in prokaryotes as well. Septation is divided into
several steps:
(1) Selection of the site where the septum will be formed;
(2) Assembly of a specialized structure called the Z ring, which
divides the cell in two by constriction;
(3) Linkage of the Z ring to the plasma membrane and perhaps
components of the cell wall;
(4) Assembly of the cell wall-synthesizing machinery (i.e., for
synthesis of peptidoglycan and other cell wall constituents); and
(5) Constriction of the cell
and septum formation. Synthesis of sugars and polysaccharides: Synthesis of
peptidoglycan. The assembly of the Z ring is a critical step in septation, as
it must be formed if subsequent steps are to occur. The FtsZ protein, a tubulin
homologue found in most bacteria and many archaea, forms the Z ring. FtsZ, like
tubulin, polymerizes to form fi laments, which are thought to create the
meshwork that constitutes the Z ring. Numerous studies show that the Z ring is
very dynamic, with portions being exchanged constantly with newly formed, short
FtsZ polymers from the cytosol. Another protein, called MinCD, is an inhibitor
of Z-ring assembly. Like FtsZ, it is very dynamic, oscillating its position
from one end of the cell to the other, forcing Z-ring formation only at the
center of the cell. Once the Z-ring forms, the rest of the division machinery
is constructed. First one or more anchoring proteins link the Z ring to the
cell membrane. Then the cell wall-synthesizing machinery is assembled. The
final steps in division involve constriction of the cell by the Z ring,
accompanied by invagination of the cell membrane and synthesis of the septal
wall. Several models for Z-ring function have been proposed. One model holds
that the FtsZ filaments are shortened by losing FtsZ subunits (i.e.,
depolymerization) at sites where the Z ring is anchored to the plasma membrane.
This model is supported by the observation that Z rings of cells producing an
excessive amount of FtsZ subunits fail to constrict the cell. The preceding
discussion of the cell cycle describes what occurs in slowly growing E. coli cells.
In these cells, the cell cycle takes approximately 60 minutes to complete.
However, E.coli can reproduce at a much more rapid rate, completing the entire
cell cycle in about 20 minutes, despite the fact that DNA replication always
requires at least 40 minutes. E. coli accomplishes
this by beginning a second round of DNA replication (and sometimes even a third
or fourth round) before the first round of replication is completed. Thus the
progeny cells receive two or more replication forks, and replication is
continuous because the cells are always copying their DNA.
GROWTH
CURVE
Binary fission and other cell
division processes bring about an increase in the number of cells in a
population. Population growth is studied by analyzing the growth curve of a
microbial culture. When microorganisms are cultivated in liquid medium, they
usually are grown in a batch culture, i.e.; they are incubated in a closed
culture vessel with a single batch of medium. Because no fresh medium is
provided during incubation, nutrient concentrations decline and concentrations
of wastes increase. The growth of microorganisms reproducing by binary fission
can be plotted as the logarithm of the number of viable cells versus the
incubation time. The resulting curve has four distinct phases.
Lag Phase
When microorganisms are introduced into fresh culture medium, usually
no immediate increase in cell number occurs. This period is called the lag phase. However, cells in the culture are
synthesizing new components. A lag phase can be necessary for a variety of
reasons. The cells may be old and depleted of ATP, essential cofactors, and
ribosomes; these must be synthesized before growth can begin. The medium may be
different from the one the microorganism was growing in previously. Here new
enzymes would be needed to use different nutrients. Possibly the microorganisms
have been injured and require time to recover. Whatever the causes, eventually
the cells begin to replicate their DNA, increase in mass, and finally
divide.
Exponential Phase
During the exponential (log) phase,
microorganisms are growing and dividing at the maximal rate possible given
their genetic potential, the nature of the medium, and the environmental conditions.
Their rate of growth is constant during the exponential phase; that is, they
are completing the cell cycle and doubling in number at regular intervals. The
population is most uniform in terms of chemical and physiological properties
during this phase; therefore exponential phase cultures are usually used in
biochemical and physiological studies. Exponential (logarithmic) growth is balanced
growth. That is, all cellular
constituents are manufactured at constant rates relative to each other. If
nutrient levels or other environmental conditions change, unbalanced growth
results. During unbalanced growth, the rates of synthesis of cell components
vary relative to one another until a new balanced state is reached. Unbalanced
growth is readily observed in two types of experiments: shift-up, where a
culture is transferred from a nutritionally poor medium to a richer one; and
shift-down, where a culture is transferred from a rich medium to a poor one. In
a shift-up experiment, there is a lag while the cells first construct new
ribosomes to enhance their capacity for protein synthesis. In a shift-down
experiment, there is a lag in growth because cells need time to make the
enzymes required for the biosynthesis of unavailable nutrients. Once the cells
are able to grow again, balanced growth is resumed and the culture enters the
exponential phase. These shift-up and shift-down experiments demonstrate that
microbial growth is under precise, coordinated control and responds quickly to
changes in environmental conditions. When microbial growth is limited by the
low concentration of a required nutrient, the final net growth or yield of
cells increases with the initial amount of the limiting nutrient present. The
rate of growth also increases with nutrient concentration but in a hyperbolic
manner much like that seen with many enzymes. The shape of the curve seems to
reflect the rate of nutrient uptake by microbial transport proteins. At sufficiently
high nutrient levels, the transport systems are saturated, and the growth rate
does not rise further with increasing nutrient concentration.
Stationary Phase
In a closed system such as a
batch culture, population growth eventually ceases and the growth curve becomes
horizontal. This stationary phase usually is attained by bacteria at a population
level of around 10^6 cells per ml. Other microorganisms normally do not reach
such high population densities. For instance, protist cultures often have
maximum concentrations of about 10^6 cells per ml. Final population size depend
on nutrient availability and other factors, as well as the type of
microorganism being cultured. In the stationary phase, the total number of
viable microorganisms remains constant. This may result from a balance between
cell division and cell death or the population may simply cease to divide but
remain metabolically active. Microbial populations enter the stationary phase
for several reasons. One obvious factor is nutrient limitation; if an essential
nutrient is severely depleted, population growth will slow. Aerobic organisms
often are limited by O2 availability. Oxygen is not very soluble and
may be depleted so quickly that only the surface of a culture will have an O2 concentration adequate for growth. The cells
beneath the surface will not be able to grow unless the culture is shaken or
aerated in another way. Population growth also may cease due to the
accumulation of toxic waste products. This factor seems to limit the growth of
many anaerobic cultures (cultures growing in the absence of O2). For example, streptococci can pro- duce so
much lactic acid and other organic acids from sugar fermentation that their
medium becomes acidic and growth is inhibited. Finally, some evidence exists
that growth may cease when a critical population level is reached. Thus
entrance into the stationary phase may result from several factors operating in
concert. As we have seen, bacteria in a
batch culture may enter stationary phase in response to starvation. This
probably occurs often in nature because many environments have low nutrient
levels. Prokaryotes have evolved a number of strategies to survive starvation.
Some bacteria respond with obvious morphological changes such as endospore
formation, but many only decrease somewhat in overall size. This is often
accompanied by protoplast shrinkage and nucleoid condensation. The more
important changes during starvation are in gene expression and physiology.
Starving bacteria frequently produce a variety of starvation proteins, which
make the cell much more resistant to damage. Some increase peptidoglycan
crosslinking and cell wall strength. The DBP (DNA-binding protein from starved
cells) protein protects DNA. Proteins called chaperone proteins prevent protein
denaturation and renature damaged proteins. Because of these and many other
mechanisms, starved cells become harder to kill and more resistant to
starvation, damaging temperature changes, oxidative and osmotic damage, and
toxic chemicals such as chlorine. These changes are so effective that some
bacteria can survive starvation for years. There is even evidence that
Salmonella enterica serovar Typhimurium (S.typhimurium) and some other bacterial
pathogens become more virulent when starved. Clearly, these considerations are
of great practical importance in medical and industrial microbiology.
Senescence and Death
For many years, the decline in viable cells following the stationary
phase was described simply as the “death phase.” It was assumed that
detrimental environmental changes such as nutrient deprivation and the buildup
of toxic wastes caused irreparable harm and loss of viability. That is, even
when bacterial cells were transferred to fresh medium, no cellular growth was
observed. Because loss of viability was often not accompanied by a loss in
total cell number, it was assumed that cells died but did not lyse. This view is currently under debate. There
are two alternative hypotheses. Some microbiologists think starving cells that
show an exponential decline in density have not irreversibly lost their ability
to reproduce. Rather, they suggest that microbes are temporarily unable to
grow, at least under the laboratory conditions used. This phenomenon, in which
the cells are called viable but non culturable (VBNC) is thought to be the
result of a genetic response triggered in starving, stationary phase cells.
Just as some bacteria form endospores as a survival mechanism, it is argued
that others are able to become dormant without changes in morphology. Once the
appropriate conditions are available (for instance, a change in temperature or
passage through an animal), VBNC microbes resume growth. VBNC microorganisms
could pose a public health threat, as many assays that test for food and
drinking water safety are culture-based. The second alternative to a simple
death phase is programmed cell death.
In contrast to the VBNC hypothesis whereby cells are genetically programmed to
survive, programmed cell death predicts that a fraction of the microbial
population is genetically programmed to die after growth ceases. In this case,
some cells die and the nutrients they leak enable the eventual growth of those
cells in the population that did not initiate cell death. The dying cells are
thus “altruistic”—they sacrifice themselves for the benefit of the larger
population. Long-term growth experiments reveal that an exponential decline in
viability is sometimes replaced by a gradual decline in the number of culturable
cells. This decline can last months to years. During this time, the bacterial
population continually evolves so that actively reproducing cells are those
best able to use the nutrients released by their dying brethren and best able
to tolerate the accumulated toxins. This dynamic process is marked by
successive waves of genetically distinct variants. Thus natural selection can
be witnessed within a single culture vessel.
MEASUREMENT OF MICROBIAL GROWTH
There are many ways to measure microbial growth to determine growth
rates and generation times. Either population number or mass may be followed
because growth leads to increases in both. Here the most commonly employed
techniques for deter- mining population size are examined briefly and the
advantages and disadvantages of each noted. No single technique is always best;
the most appropriate approach depends on the experimental situation.
Measurement of Cell Numbers
The most obvious way to
determine microbial numbers is by direct counts. Using a counting chamber is easy,
inexpensive, and relatively quick; it also gives information about the size and
morphology of microorganisms. Petroff-Hausser counting chambers can be used for
counting procaryotes; hemocytometers can be used for both procaryotes and
eucaryotes. Both of these specially designed slides have chambers of known
depth with an etched grid on the chamber bottom. Procaryotes are more easily
counted if they are stained or when a phase-contrast or a fluorescence
microscope is employed. The number of micro- organisms in a sample can be
calculated by taking into account the chamber’s volume and any sample dilutions
required. One disadvantage is that to determine population size accurately, the
microbial population must be relatively large because only a small volume of
the population is sampled. Larger
microorganisms such as protists and yeasts can be directly counted with
electronic counters such as the Coulter Counter, although the flow cytometer is
increasingly used. In the Coulter Counter, the microbial suspension is forced
through a small hole. Electrical current flows through the hole, and electrodes
placed on both sides of the whole measure electrical resistance. Every time a
microbial cell passes through the hole, electrical resistance increases (i.e.,
the conductivity drops), and the cell is counted. Flow cytometry gives accurate
results with larger cells, and is extensively used in hospital laboratories to
count red and white blood cells. It is not as useful in counting bacteria
because of interference by small debris p articles, the formation of fi
laments, and other problems. The number of bacteria in aquatic samples is
frequently determined from direct counts after the bacteria have been trapped
on membrane filters. In the membrane filter technique, the sample is first
filtered through a black polycarbonate membrane filter. Then the bacteria are
stained with a fluorescent dye such as acridine orange or the DNA stain DAPI
and observed microscopically. The stained cells are easily observed against the
black back- ground of the membrane filter and can be counted when viewed with
an epifluorescence microscope. Traditional methods for directly counting
microbes in a sample usually yield cell densities that are much higher than the
plating methods described next because direct counting procedures do not
distinguish dead cells from culturable cells. Newer methods for direct counts
avoid this problem. Commercial kits that use fluorescent reagents to stain live
and dead cells differently are now available, making it possible to count
directly the number of live and dead microorganisms in a sample.
Several plating methods can be
used to determine the number of viable microbes in a sample. These are referred
to as viable counting methods (plate counts) because they count only
those cells that are able to reproduce when cultured. Two commonly used procedures
are the spread-plate and the pour-plate techniques. In both of these methods, a
diluted sample of microorganisms is dispersed over or within agar. If each cell
is far enough away from other cells, then each cell will develop into a
distinct colony. The original number of viable microorganisms in the sample can
be calculated from the number of colonies formed and the sample dilution. For
example, if 1.0 ml of a 1 × 10^6 dilution yielded 150 colonies, the original sample
contained around 1.5 × 10^8 cells per ml. usually the count is made more
accurate by use of a colony counter. In this way the spread- plate and
pour-plate techniques may be used to find the number of microorganisms in a
sample. Another commonly used plating method first traps bacteria in aquatic
samples on a membrane filter. The filter is then placed on an agar medium or on
a pad soaked with liquid media and incubated until each cell forms a separate
colony. A colony count gives the number of microorganisms in the filtered
sample, and selective media can be used to select for specific microorganisms.
This technique is especially useful in analyzing water purity. Plating techniques are simple, sensitive, and
widely used for viable counts of bacteria and other microorganisms in samples
of food, water, and soil. Several problems, however, can lead to inaccurate
counts. Low counts will result if clumps of cells are not broken up and the
microorganisms well dispersed. Because it is not possible to be certain that each
colony arose from an individual cell, the results are often expressed in terms
of colony forming units (CFU) rather
than the number of microorganisms. The samples should yield between 30 and 300
colonies for most accurate counting (counts of 25 to 250 are used in some
applications). Of course the counts will also be low if the medium employed
cannot support growth of all the viable microorganisms present. The hot agar
used in the pour-plate technique may injure or kill sensitive cells; thus
spread plates sometimes give higher counts than pour plates.
Measurement of Cell Mass
Techniques for measuring changes in cell mass also can be used to
follow growth. The most direct approach is the determination of microbial dry
weight. Cells growing in liquid medium are collected by centrifugation, washed,
dried in an oven, and weighed. This is an especially useful technique for measuring
the growth of filamentous fungi. It is time-consuming, however, and not very
sensitive. Because bacteria weigh so little, it may be necessary to centrifuge
several hundred milliliters of culture to collect a sufficient quantity. A more rapid and sensitive method for
measuring cell mass is spectrophotometry. Spectrophotometry depends on the fact
that microbial cells scatter light that strikes them. Because microbial cells
in a population are of roughly constant size, the amount of scattering is
directly proportional to the biomass of cells present and indirectly related to
cell number. When the concentration of bacteria reaches about a million (10^6) cells
per ml, the medium appears slightly cloudy or turbid. Further increases in
concentration result in greater turbidity, and less light is transmitted
through the medium. The extent of light scattering (i.e., decrease in transmitted
light) can be measured by a spectrophotometer and is called the absorbance
(optical density) of the medium. Absorbance is almost linearly related to cell
concentration at absorbance levels less than about 0.5. If the sample exceeds
this value, it must first be diluted and then absorbance measured. Thus
population growth can be easily measured as long as the population is high
enough to give detectable turbidity. Cell mass can also be estimated by
measuring the concentration of some cellular substance, as long as its
concentration is constant in each cell. For example, a sample of cells can be
analyzed for total protein or nitrogen. An increase in the microbial population
will be reflected in higher total protein levels. Similarly, chlorophyll
determinations can be used to measure phototrophic protist and cyanobacteria
populations, and the quantity of ATP can be used to estimate the amount of
living microbial mass.
CONTINUOUS CULTURE OF MICROORGANISMS
Thus far, our focus has been on closed systems called batch cultures
in which nutrients are not renewed nor wastes removed. Exponential
(logarithmic) growth lasts for only a few generations and soon stationary phase
is reached. However, it is possible to grow microorganisms in a system with
constant environmental conditions maintained through continual provision of
nutrients and removal of wastes. Such a system is called a continuous culture system. These systems can maintain a microbial
population in exponential growth, growing at a known rate and at a constant
biomass concentration for extended periods. Continuous culture systems make
possible the study of microbial growth at very low nutrient levels,
concentrations close to those present in natural environments. These systems
are essential for research in many areas, including ecology. For example,
interactions between microbial species in environ- mental conditions resembling
those in a freshwater lake or pond can be modeled. Continuous culture systems
also are used in food and industrial microbiology. Two major types of continuous
culture systems commonly are used: chemostats and turbidostats.
Chemostats
A chemostat is constructed so
that the rate at which sterile medium is fed into the culture vessel is the
same as the rate at which the media containing microorganisms is removed. The
culture medium for a chemostat possesses an essential nutrient (e.g., a
vitamin) in limiting quantities. Because one nutrient is limiting, growth rate
is determined by the rate at which new medium is fed into the growth chamber;
the final cell density depends on the concentration of the limiting nutrient.
The rate of nutrient exchange is expressed as the dilution rate (D), the rate at
which medium flows through the culture vessel relative to the vessel volume,
where f is the flow rate (ml/hr.) and V is the vessel volume (ml). D = f / V
For example, if f is 30 ml/hr. and
V is 100 ml, the dilution rate is 0.30 hr 1.
Both population size and generation time are related to the dilution
rate, and population density remains unchanged over a wide range of dilution
rates. The generation time decreases (i.e., the rate of growth increases) as
the dilution rate increases. The limiting nutrient will be almost completely
depleted under these balanced conditions. If the dilution rate rises too high,
microorganisms can actually be washed out of the culture vessel before
reproducing because the dilution rate is greater than the maximum growth rate.
This occurs because fewer microorganisms are present to consume the limiting
nutrient. At very low dilution rates, an increase in D causes a rise in both
cell density and the growth rate. This is because of the effect of nutrient
concentration on the growth rate, sometimes called the Monod relationship. When
dilution rates are low, only a limited supply of nutrient is available and the
microbes can conserve only a limited amount of energy. Much of that energy must
be used for cell maintenance, not for growth and reproduction. As the dilution
rate increases, the amount of nutrients and the resulting cell density rise
because energy is available for both maintenance and reproduction. The growth
rate increases when the total available energy exceeds the maintenance
energy.
Turbidostats
The second type of continuous culture system, the turbidostat, has a
photocell that measures the turbidity (absorbance) of the culture in the growth
vessel. The flow rate of media through the vessel is automatically regulated to
maintain a predetermined turbidity. Because turbidity is related to cell
density, the turbidostat maintains a desired cell density. The turbidostat
differs from the chemostat in several ways. The dilution rate in a turbidostat
varies rather than remaining constant, and a turbidostat’s culture medium
contains all nutrients in excess. That is, none of the nutrients is limiting.
The turbidostat operates best at high dilution rates; the chemostat is most
stable and effective at lower dilution rates.
INFLUENCES OF ENVIRONMENTAL FACTORS ON
GROWTH
As we have seen, microorganisms must be able to respond to variations
in nutrient levels. Microorganisms also are greatly affected by the chemical
and physical nature of their surroundings. An understanding of environmental
influences aids in the control of microbial growth and the study of the
ecological distribution of microorganisms. The adaptations of some
microorganisms to extreme and inhospitable environments are truly remarkable.
Microbes are present virtually everywhere on Earth. Many habitats in which
microbes thrive would kill most other organisms. Bacteria such as Bacillus
infernus are able to live over 2.4 kilometers
below Earth’s surface, without oxygen and at temperatures above 60°C. Other
microbes live in acidic hot springs, at great ocean depths, or in lakes such as
the Great Salt Lake in Utah (USA) that have high sodium chloride
concentrations. Microorganisms that grow in such harsh conditions are
called extremophiles. We briefly
review the effects of the most important environmental factors on microbial
growth. Major emphasis is given to solutes and water activity, pH, temperature,
oxygen level, pressure, and radiation. Microorganisms are categorized in terms
of their response to these factors. It is important to note that for most
environmental factors, a range of levels supports growth of a microbe. For
example, a microbe might exhibit optimum growth at pH 7 but grows, though not
optimally, at pH values down to pH 6 (its pH minimum) and up to pH 8 (its pH
maximum). Furthermore, outside this range, the microbe might cease reproducing
but remain viable for some time. Clearly, each microbe must have evolved
adaptations that allow it to adjust its physiology within its preferred range,
and it may also have adaptations that protect it in environments outside this
range. These adaptations also are discussed in this section.
Solutes and Water Activity
Because a selectively permeable plasma membrane separates
microorganisms from their environment, they can be affected by changes in the
osmotic concentration of their surroundings. If a microorganism is placed in a
hypotonic solution (one with a lower osmotic concentration), water will enter
the cell and cause it to burst unless something is done to prevent the influx
or inhibit plasma membrane expansion. Conversely if it is placed in a hypertonic
solution (one with a higher osmotic concentration), water will flow out of the
cell. In microbes that have cell walls, the membrane shrinks away from the cell
wall a process called plasmolysis. Dehydration of the cell in hypertonic
environments may damage the cell membrane and cause the cell to become
metabolically inactive. Clearly it is
important that microbes be able to respond to changes in the osmotic
concentrations of their environment. Microbes in hypotonic environments can
reduce the osmotic con- centration of their cytoplasm. This can be achieved
using inclusion bodies or other mechanisms. For example, some prokaryotes have
mechanosensitive (MS) channels in their plasma membrane. In a hypotonic
environment, the membrane stretches due to an increase in hydrostatic pressure
and cellular swelling. MS channels then open and allow solutes to leave. Thus,
MS channels act as escape valves to protect cells from bursting. Because many
protists do not have a cell wall, they must use contractile vacuoles to expel
excess water. Many microorganisms, whether in hypotonic or hypertonic
environments, keep the osmotic concentration of their cytoplasm somewhat above
that of the habitat by the use of compatible solutes, so that the plasma
membrane is always pressed firmly against their cell wall. Compatible solutes are
solutes that do not interfere with metabolism and growth when at high
intracellular concentrations. Most procaryotes increase their internal osmotic
concentration in a hypertonic environment through the synthesis or uptake of
choline, betaine, proline, glutamic acid, and other amino acids; elevated
levels of potassium ions may also be used. Photosynthetic protists and fungi
employ sucrose and polyols for example, arabitol, glycerol, and mannitol for
the same purpose. Polyols and amino acids are ideal compatible solutes because
they normally do not disrupt enzyme structure and function.
Some microbes are adapted to extreme hypertonic environments. Halophiles grow optimally in the presence of NaCl or
other salts at a concentration above about 0.2 M. Extreme halophiles have
adapted so completely to hypertonic, saline conditions that they require high
levels of sodium chloride to grow concentrations between about 2 M and saturation
(about 6.2 M). The archaeon Halobacterium can be isolated from the Dead Sea (a
salt lake between Israel and Jordan), the Great Salt Lake in Utah, and other
aquatic habitats with salt concentrations approaching saturation. Halobacterium
and other extremely halophilic procaryotes accumulate enormous quantities of
potassium in order to remain hypertonic to their environment; the internal
potassium concentration may reach 4 to 7 M. Furthermore, their enzymes,
ribosomes, and transport proteins require high potassium levels for stability
and activity. In addition, the plasma membrane and cell wall of Halobacterium
is stabilized by high concentrations of sodium ion. If the sodium concentration
decreases too much, the wall and plasma membrane disintegrate. Extreme
halophiles have successfully adapted to environmental conditions that would
destroy most organisms. In the process, they have become so specialized that
they have lost ecological flexibility and can prosper only in a few extreme
habitats. Because the osmotic concentration of a habitat has such pro- found
effects on microorganisms, it is useful to express quantitatively the degree of
water availability. Microbiologists generally use water activity (a w) for this purpose (water avail-ability also
may be expressed as water potential, which is related to a w). The water
activity of a solution is 1/100 the relative humidity of the solution (when
expressed as a percent). It is also equivalent to the ratio of the solution’s
vapor pressure (P soln) to that of pure water (P water).
pH
pH is a measure of the relative acidity of a solution and is defined
as the negative logarithm of the hydrogen ion concentration (expressed in terms
of molarity).
pH = −log [H +] = log (1/[H+ ])
The pH scale extends from pH
0.0 (1.0 M H +) to pH 14.0 (1.0 × 10 −14 M H +), and each pH unit represents a tenfold change in hydrogen
ion concentration. Microbial habitats vary widely in pH from pH 0 to 2 at the acidic end to alkaline lakes and soil with pH
values between 9 and 10. Each species has a definite pH growth range and pH growth
optimum. Acidophiles have their growth optimum between pH 0 and
5.5; neutrophiles, between pH 5.5 and 8.0; and alkalophiles (alkaliphiles),
between pH 8.0 and 11.5. Extreme alkalophiles have growth optima at pH 10 or
higher. In general, different microbial groups have characteristic pH
preferences. Most bacteria and protists are neutrophiles. Most fungi prefer
more acidic surroundings, about pH 4 to 6; photosynthetic protists also seem to
favor slight acidity. Many archaea are acidophiles. For example, the archaeon Sulfolobus
acidocaldarius is a common inhabitant of acidic hot springs; it grows well from
pH 1 to 3 and at high temperatures. The archaea Ferroplasma acidarmanus and Picrophilus
oshimae can actually grow very close to pH 0. Alkalophiles are distributed
among all three domains of life. They include bacteria belonging to the genera Bacillus,
Micrococcus, Pseudomonas, and Streptomyces; yeasts and filamentous fungi; and
numerous archaea. Although
microorganisms often grow over wide ranges of pH and far from their optima,
there are limits to their tolerance. When the external pH is low, the
concentration of H + is greater outside than inside, and H + will move into the
cytoplasm and lower the cytoplasmic pH. Drastic variations in cytoplasmic pH
can harm microorganisms by disrupting the plasma membrane or inhibiting the
activity of enzymes and membrane transport proteins. Most procaryotes die if
the internal pH drops much below 5.0 to 5.5. Changes in the external pH also
might alter the ionization of nutrient molecules and thus reduce their availability
to the organism. Microorganisms respond
to external pH changes using mechanisms that maintain a neutral cytoplasmic pH.
Several mechanisms for adjusting to small changes in external pH have been
proposed. Neutrophiles appear to exchange potassium for protons using an
antiport transport system. Internal buffering also may contribute to pH
homeostasis. However, if the external pH becomes too acidic, other mechanisms
come into play. When the pH drops below about 5.5 to 6.0, Salmonella enterica serovar
Typhimurium and E. coli synthesize an array of new proteins as part of what has
been called their acidic tolerance response. A proton-translocating ATPase
enzyme contributes to this protective response, either by making more ATP or by
pumping protons out of the cell. If the external pH decreases to 4.5 or lower,
acid shock proteins and heat shock proteins are synthesized. These prevent the
denaturation of proteins and aid in the refolding of denatured proteins in
acidic conditions. What about microbes that live at pH extremes? Extreme
alkalophiles such as Bacillus alcalophilus maintain their internal pH close to
neutrality by exchanging internal sodium ions for external protons. Acidophiles
use a variety of measures to maintain a neutral internal pH. These include the
transport of cations (e.g., potassium ions) into the cell, thus decreasing the
movement of H + into the cell; proton transporters that pump H + out if they
get in; and highly impermeable cell membranes.
Temperature
Microorganisms are particularly
susceptible to external temperatures because they cannot regulate their
internal temperature. An important factor influencing the effect of temperature
on growth is the temperature sensitivity of enzyme-catalyzed reactions. Each
enzyme has a temperature at which it functions optimally. At some temperature
below the optimum, it ceases to be catalytic. As the temperature rises from
this low point, the rate of catalysis increases to that observed for the
optimal temperature. The velocity of the reaction roughly doubles for every
10°C rise in temperature. When all enzymes in a microbe are considered
together, as the rate of each reaction increases, metabolism as a whole becomes
more active, and the microorganism grows faster. However, beyond a certain
point, further increases actually slow growth, and sufficiently high
temperatures are lethal. High temperatures denature enzymes, transport
carriers, and other proteins. Temperature also has a significant effect on
microbial membranes. At very low temperatures, membranes solidify. At high
temperatures, the lipid bilayer simply melts and disintegrates. Thus when organisms
are above their optimum temperature, both function and cell structures are
affected. If temperatures are very low, function is affected but not
necessarily cell chemical composition and structure. Because of these opposing temperature influences,
microbial growth has characteristic temperature dependence with distinct cardinal
temperatures minimum, optimum, and maximum growth temperatures. Although the
shape of temperature dependence curves varies, the temperature optimum is
always closer to the maximum than to the minimum. The cardinal temperatures are
not rigidly fixed. Instead they depend to some extent on other environmental
factors such as pH and avail- able nutrients. For example, Crithidia
fasciculate, a flagellated protist living in the gut of mosquitoes, grows in a
simple medium at 22 to 27°C. However, it cannot be cultured at 33 to 34°C
without the addition of extra metals, amino acids, vitamins, and lipids. The
cardinal temperatures vary greatly among microorganisms. Optima usually range
from 0°C to 75°C, whereas microbial growth occurs at temperatures extending
from less than 20°C to over 120°C. Some archaea even grow at 121°C (250°F), the
temperature normally used in autoclaves. A major factor determining growth
range seems to be water. Even at the most extreme temperatures, microorganisms
need liquid water to grow. The growth temperature range for a particular
microorganism usually spans about 30 degrees. Some species (e.g., Neisseria
gonorrhoeae) have a small range; others, such as Enterococcus faecalis, grow
over a wide range of temperatures. The major microbial groups differ from one
another regarding their maximum growth temperatures. The upper limit for
protists is around 50°C. Some fungi grow at temperatures as high as 55 to 60°C.
Procaryotes can grow at much higher temperatures than eucaryotes. It has been
suggested that eucaryotes are not able to manufacture stable and functional organelles
membranes at temperatures above 60°C. The photosynthetic apparatus also appears
to be relatively unstable because photosynthetic organisms are not found
growing at very high temperatures.
Microorganisms such as those listed in t able 7.4 can be placed in one
of five classes based on their temperature ranges for growth.
1. Psychrophiles grow well at 0°C and have an optimum growth
temperature of 10°C or lower; the maximum is around 15°C. They are readily
isolated from Arctic and Antarctic habitats. Oceans constitute an enormous habitat
for psychrophiles because 90% of ocean water is 5°C or colder. The psychrophilic
protist Chlamydomonas nivalis can actually turn a snow field or glacier pink
with its bright red spores. Psychrophiles are widespread among bacterial taxa
and are found in such genera as Pseudomonas, Vibrio, Alcaligenes, Bacillus,
Photobacterium, and Shewanella. A
psychrophilic archaeon, Methanogenium, has been isolated from Ace Lake in
Antarctica. Psychrophilic microorganisms have adapted to their environment in
several ways. Their enzymes, transport systems, and protein synthetic machinery
function well at low temperatures. The cell membranes of psychrophilic
microorganisms have high levels of unsaturated fatty acids and remain semifluid
when cold. Indeed, many psychrophiles begin to leak cellular constituents at
temperatures higher than 20°C because of cell membrane disruption.
2. Psychrotrophs (facultative
psychrophiles) grow at 0 to 7°C even
though they have optima between 20 and 30°C, and maxima at about 35°C.
Psychrotrophic bacteria and fungi are major causes of refrigerated food
spoilage, as described in chapter 34.
3. Mesophiles are microorganisms with growth
optima around 20 to 45°C. They often have a temperature minimum of 15 to 20°C,
and their maximum is about 45°C or lower. Most microorganisms probably fall
within this category. Almost all human pathogens are mesophiles, as might be
expected because the human body is a fairly constant 37°C.
4. Thermophiles grow at temperatures between 55 and 85°C.
Their growth minimum is usually around 45°C, and they often have optima between
55 and 65°C. The vast majority are procaryotes, although a few photosynthetic
protists and fungi are thermophilic. These organisms flourish in many habitats
including composts, self-heating hay stacks, hot water lines, and hot
springs.
5. Hyperthermophiles have growth optima
between 85°C and about 113°C. They usually do not grow well below 55°C. Pyrococcus abyssi and Pyrodictium occultum are
examples of marine hyperthermophiles found in hot areas of the seafloor.
Oxygen Concentration
The importance of oxygen to the
growth of an organism correlates with its metabolism in particular, with the
processes it uses to conserve the energy supplied by its energy source. Almost
all energy-conserving metabolic processes involve the movement of electrons
through a series of membrane-bound electron carriers called the electron
transport chain (ETC). For chemotrophs, an externally supplied terminal
electron acceptor is critical to the functioning of the ETC. The nature of the
terminal electron acceptor is related to an organism’s oxygen requirement.
An organism able to grow in the
presence of atmospheric O2 is an aerobe, whereas
one that can grow in its absence is an anaerobe. Almost all multicellular organisms are
completely dependent on atmospheric O2 for growth that is, they are obligate aerobes. Oxygen serves as the
terminal electron acceptor for the ETC in the metabolic process called aerobic
respiration. In addition, aerobic eucaryotes employ O2 in the synthesis of sterols and unsaturated
fatty acids. Microaerophiles such as
Campylobacter are damaged by the normal atmospheric level of O2 (20%) and require O2 levels in the range of 2 to 10% for growth. Facultative anaerobes do not require O2 for growth but grow better in its presence. In
the presence of oxygen, they use O2 as the terminal electron acceptor during aerobic respiration.
Aerotolerant anaerobes such as Enterococcus faecalis simply ignore O2 and grow equally well whether it is present or
not; chemotrophic aerotolerant anaerobes are often described as having strictly
fermentative metabolism. In contrast, strict or obligate anaerobes (e.g., Bacteroides,
Clostridium pasteurianum, Methanococcus) are usually killed in the presence of O2. Strict anaerobes cannot generate energy through
aerobic respiration and employ other metabolic strategies such as fermentation
or anaerobic respiration, neither of which requires O2. The nature of bacterial O2 responses can be readily determined by growing the bacteria in culture
tubes filled with a solid culture medium or a medium such as thioglycollate
broth, which contains a reducing agent to lower O2levels. A microbial group may
show more than one type of relation- ship to O2. All five types are found among the procaryotes and protists. Fungi
are normally aerobic, but a number of species particularly among the yeasts are
facultative anaerobes. Photosynthetic protists are usually obligate aerobes.
Although obligate anaerobes are killed by O2, they may be recovered from habitats that appear to be toxic. In such
cases they associate with facultative anaerobes that use up the available O2and thus make the growth of strict anaerobes
possible. For example, the strict anaerobe Bacteroides gingivalis lives in the
mouth where it grows in the anoxic crevices around the teeth. Clearly the
ability to grow in both toxic and anoxic environments provides considerable flexibility
and is an ecological advantage. The
different relationships with O2 are due to several
factors, including the inactivation of proteins and the effect of toxic O2 derivatives. Enzymes can be inactivated when
sensitive groups such as sulfhydryls are oxidized. A notable example is the
nitrogen fixation enzyme nitrogenase, which is very oxygen sensitive. Toxic O2 derivatives are formed when proteins such as
flavoproteins promote oxygen reduction. The reduction products are superoxide radical, hydrogen peroxide, and hydroxyl radical.
O2 + e −→ O2 • (superoxide radical)
O2 • + e − + 2H + −→ H2 O2 (hydrogen peroxide)
H2O2+ e − + H + −→H 2 O + OH• (hydroxyl radical)
These products are extremely
toxic because they oxidize and rapidly destroy cellular constituents. A
microorganism must be able to protect itself against such oxygen products or it
will be killed. Indeed, neutrophils and macrophages, two important immune
system cells, use these toxic oxygen products to destroy invading
pathogens. Many microorganisms possess
enzymes that protect against toxic O2 products. Obligate aerobes and facultative anaerobes usually contain
the enzymes superoxide dismutase (SOD and catalase, which catalyze the
destruction of superoxide radical and hydrogen peroxide, respectively.
Peroxidase also can be used to destroy hydrogen peroxide.
Aerotolerant microorganisms may lack catalase but usually have
superoxide dismutase. The aerotolerant bacterium Lactobacillus plantarum uses
manganous ions instead of superoxide dismutase to destroy the superoxide
radical. All strict anaerobes lack both enzymes or have them in very low
concentrations and therefore cannot tolerate O2. However, some microaerophilic bacteria and anaerobic archaea protect
themselves from the toxic effects of O2 with the enzymes superoxide reductase and peroxidase. Superoxide
reductase reduces superoxide to H2O2 without producing O2. The H2O2 is then converted to water by
peroxidase.
Because aerobes need O2and anaerobes are
killed by it, radically different approaches must be used when they are cultured.
When large volumes of aerobic microorganisms are cultured, either they must be
shaken to aerate the culture medium or sterile air must be pumped through the
culture vessel. Precisely the opposite problem arises with anaerobes all O2 must be excluded. This is accomplished in several
ways.
(1) Special anaerobic media containing reducing agents such as
thioglycollate or cysteine may be used. The medium is boiled during preparation
to dissolve its components and drive off oxygen. The reducing agents eliminate
any residual dissolved O2 in the medium so
that anaerobes can grow beneath its surface.
(2) Oxygen also may be eliminated by removing air with a vacuum pump
and flushing out residual O2with nitrogen gas.
Often CO2 as well as nitrogen is added to the chamber
since many anaerobes require a small amount of CO2 for best growth.
(3) One of the most popular
ways of culturing small numbers of anaerobes is by use of a GasPak jar, which
uses hydrogen and a palladium catalyst to remove O2. (4) A similar approach uses plastic bags or
pouches containing calcium carbonate and a catalyst, which produce an anoxic,
carbon dioxide rich atmosphere.
Pressure
Organisms that spend their lives on land or the surface of water are
always subjected to a pressure of 1 atmosphere (atm) and are never affected
significantly by pressure. It is thought that high hydrostatic pressure affects
membrane fluidity and membrane- associated function. Yet many procaryotes live
in the deep sea (ocean depths of 1,000 m or more) where the hydrostatic
pressure can reach 600 to 1,100 atm and the temperature is about 2 to 3°C. Many
of these procaryotes are barotolerant :
increased pressure adversely affects them but not as much as it does
nontolerant microbes. Some procaryotes are truly barophilic they grow more rapidly at high
pressures. A barophile recovered from the Mariana trench near the Philippines
(depth about 10,500 m) grows only at pressures between about 400 to 500 atm
when incubated at 2°C. Barophiles may play an important role in nutrient
recycling in the deep sea. Thus far, they have been found among several
bacterial genera (e.g., Photobacterium, Shewanella, and Colwellia). Some archaea
are thermobarophiles (e.g., Pyrococcus spp., Methanocaldococcus jannaschii).
Radiation
Our world is bombarded with electromagnetic
radiation of various types. Radiation behaves as if it were composed of waves
moving through space like waves traveling on the surface of water. The distance
between two wave crests or troughs is the wavelength. As the wavelength of
electromagnetic radiation decreases, the energy of the radiation increases;
gamma rays and X rays are much more energetic than visible light or infrared
waves. Electromagnetic radiation also acts like a stream of energy packets
called photons, each photon having a quantum of energy whose value depends on
the wavelength of the radiation. Sunlight is the major source of radiation on
Earth. It includes visible light, ultraviolet (UV) radiation, infrared rays,
and radio waves. Visible light is a most conspicuous and important aspect of
our environment: most life depends on the ability of photosynthetic organisms
to trap the light energy of the sun. Almost 60% of the sun’s radiation is in
the infrared region rather than the visible portion of the spectrum. Infrared
is the major source of Earth’s heat. At sea level, one finds very little
ultraviolet radiation below about 290 to 300 nm. UV radiation of wavelengths
shorter than 287 nm is absorbed by O2 in Earth’s atmosphere; this process forms a layer of ozone between 40
and 48 kilometers above Earth’s surface. The ozone layer absorbs somewhat
longer UV rays and reforms O2. The even
distribution of sunlight throughout the visible spectrum accounts for the fact
that sunlight is generally “white. Many
forms of electromagnetic radiation are very harmful to microorganisms. This is
particularly true of ionizing radiation, radiation of very short wavelength and
high energy, which can cause atoms to lose electrons (ionize). Two major forms
of ionizing radiation are
(1) X rays, which are
artificially produced, and
(2) Gamma rays, which are
emitted during radioisotope decay. Low levels of ionizing radiation may pro-
duce mutations and may indirectly result in death, whereas higher levels are
directly lethal. Although microorganisms are more resistant to ionizing
radiation than larger organisms, they are still destroyed by a sufficiently
large dose. Ionizing radiation can be used to sterilize items. Some procaryotes
(e.g., Deinococcus radiodurans) and bacterial endospores can survive large
doses of ionizing radiation. Ionizing
radiation causes a variety of changes in cells. It breaks hydrogen bonds,
oxidizes double bonds, destroys ring structures, and polymerizes some
molecules. Oxygen enhances these destructive effects, probably through the
generation of hydroxyl radicals (OH•). Although many types of constituents can
be affected, the destruction of DNA is probably the most important cause of
death. Ultraviolet (UV)
radiation can kill microorganisms due
to its short wavelength (approximately from 10 to 400 nm) and high energy. The
most lethal UV radiation has a wavelength of 260 nm, the wavelength most effectively
absorbed by DNA. The primary mechanism of UV damage is the formation of thymine
dimers in DNA. Two adjacent thymines in a DNA strand are covalently joined to
inhibit DNA replication and function. The damage caused by UV light can be
repaired by several DNA repair mechanisms, as discussed in chapter 14.
Excessive expo- sure to UV light outstrips the organism’s ability to repair the
damage and death results. Longer wavelengths of UV light (near- UV radiation;
325 to 400 nm) can also harm microorganisms because they induce the breakdown
of tryptophan to toxic photo- products. It appears that these toxic tryptophan
photoproducts plus the near-UV radiation itself produce breaks in DNA strands.
The precise mechanism is not known, although it is different from that seen
with 260 nm UV. Even visible light, when present in sufficient intensity, can damage
or kill microbial cells. Usually pigments called photo- sensitizers and O2 are involved. Photosensitizers include pigments
such as chlorophyll, bacteriochlorophyll, cytochromes, and flavins, which can
absorb light energy and become excited or activated. The excited
photosensitizer (P) transfers its energy to O2, generating singlet
oxygen (1 O2).
P P (activated) light
P (activated) + O2 → P 1 O2
Singlet oxygen is a very reactive, powerful oxidizing agent that
quickly destroys a cell. M any
microorganisms that are airborne or live on exposed surfaces use carotenoid
pigments for protection against photo- oxidation. Carotenoids effectively
quench singlet oxygen that is, they absorb energy from singlet oxygen and
convert it back into the unexcited ground state. Both photosynthetic and non-photosynthetic
microorganisms employ pigments in this way.
DEFINITIONS OF FREQUENTLY USED TERMS
Terminology is especially important when the control of micro-
organisms is discussed because words such as disinfectant and antiseptic often
are used loosely. The situation is even more con- fusing because a particular
treatment can either inhibit growth or kill, depending on the conditions. Sterilization
(Latin sterilize, unable to produce offspring or barren) is the process by
which all living cells, spores, and acellular entities (e.g., viruses, viroids,
and prions) are either destroyed or removed from an object or habitat. A
sterile object is totally free of viable microorganisms, spores, and other
infectious agents. When sterilization is achieved by a chemical agent, the
chemical is called a sterilant. In contrast, disinfection is the killing, inhibition, or
removal of microorganisms that may cause disease; disinfection is the
substantial reduction of the total microbial population and the destruction of
potential pathogens. Disinfectants are agents, usually chemical, used to carry
out dis- infection and normally used only on inanimate objects. A disinfectant
does not necessarily sterilize an object because viable spores and a few
microorganisms may remain. Sanitization is closely related to disinfection. In
sanitization, the microbial population is reduced to levels that are considered
safe by public health standards. The inanimate object is usually cleaned as
well as partially disinfected. For example, sanitizers are used to clean eating
utensils in restaurants. It also is frequently necessary to control
microorganisms on or in living tissue with chemical agents. Antisepsis (Greek anti, against, and sepsis, putrefaction)
is the prevention of infection or sepsis and is accomplished with antiseptics .
These are chemical agents applied to tissue to prevent infection by
killing or inhibiting pathogen growth; they also reduce the total microbial
population. Because they must not destroy too much host tissue, antiseptics are
generally not as toxic as disinfectants.
Chemotherapy is the use of
chemical agents to kill or inhibit the growth of microorganisms within host
tissue. A suffix can be employed to denote the type of antimicrobial agent.
Substances that kill organisms often have the suffix –cide (Latin cida, to
kill); a germicide kills pathogens
(and many non-pathogens) but not necessarily endospores. A disinfectant or
antiseptic can be particularly effective against a specific group, in which
case it may be called a bactericide, fungicide or viricide. Other chemicals do
not kill but rather prevent growth. If these agents are removed, growth will
resume. Their names end in –static (Greek statikos, causing to stand or
stopping) for example, bacteriostatic
and fungistatic.
THE PATTERN OF MICROBIAL DEATH
A microbial population is not killed instantly when exposed to a
lethal agent. Population death is generally exponential (logarithmic) that is,
the population will be reduced by the same fraction at constant intervals. If
the logarithm of the population number remaining is plotted against the time of
exposure of the microorganism to the agent, a straight-line plot will result. When
the population has been greatly reduced, the rate of killing may slow due to
the survival of a more resistant strain of the microorganism. It is essential
to have a precise measure of an agent’s killing efficiency. One such measure is
the decimal reduction time (D) or D value. The decimal reduction time is the
time required to kill 90% of the microorganisms or spores in a sample under
specified conditions. For example, in a semilogarithmic plot of the population
remaining versus the time of heating, the D value is the time required for the
line to drop by one log cycle or tenfold. The D value is usually written with a
subscript to indicate the temperature for which it applies. To study the
effectiveness of a lethal agent, one must be able to decide when microorganisms
are dead, which may pre- sent some challenges. A microbial cell is often defined
as dead if it does not grow and reproduce when inoculated into culture medium
that would normally support its growth. In like manner, an inactive virus
cannot infect a suitable host. This definition has flaws, however. It has been
demonstrated that when bacteria are exposed to certain conditions, they can
remain alive but are temporarily unable to reproduce. When in this state, they
are referred to as viable but non culturable (VBNC). In conventional tests to
demonstrate killing by an antimicrobial agent, VBNC bacteria would be thought
to be dead. This is a serious problem because after a period of recovery, the
bacteria may regain their ability to reproduce and cause infection.
CONDITIONS INFLUENCING THE EFFECTIVENESS OF ANTIMICROBIAL AGENTS
Destruction of microorganisms and inhibition of microbial growth are
not simple matters because the efficiency of an antimicrobial agent (an agent
that kills microorganisms or inhibits their growth) is affected by at least six
factors.
1. Population size. Because an equal fraction of a microbial
population is killed during each interval, a larger population requires a
longer time to die than a smaller one.
2. Population composition. The effectiveness of an agent varies greatly
with the nature of the organisms being treated because microorganisms differ
markedly in susceptibility. Bacterial spores are much more resistant to most
antimicrobial agents than are vegetative forms, and younger cells are usually
more readily destroyed than mature organisms. Some species are able to withstand
adverse conditions better than others. For instance, Mycobacterium
tuberculosis, which causes tuberculosis, is much more resistant to
antimicrobial agents than most other bacteria.
3. Concentration or intensity
of an antimicrobial agent. Often, but
not always, the more concentrated a chemical agent or intense a physical agent,
the more rapidly microorganisms are destroyed. However, agent effectiveness
usually is not directly related to concentration or intensity. Over a short
range, a small increase in concentration leads to an exponential rise in
effectiveness; beyond a certain point, increases may not raise the killing rate
much at all. Sometimes an agent is more effective at lower concentrations. For
example, 70% ethanol is more bacteriocidal than 95% ethanol because the
activity of ethanol is enhanced by the presence of water.
4. Duration of exposure. The longer a population is exposed to a microbicidal
agent, the more organisms are killed. To achieve sterilization, exposure should
be long enough to reduce the probability of survival to 10^-6 or less.
5. Temperature. An increase in the temperature at which a
chemical acts often enhances its activity. Frequently a lower concentration of
disinfectant or sterilizing agent can be used at a higher temperature.
6. Local environment. The population to be controlled is not
isolated but surrounded by environmental factors that may either offer protection
or aid in its destruction. For example, because heat kills more readily at an
acidic pH, acidic foods and beverages such as fruits and tomatoes are easier to
pasteurize than more alkaline foods such as milk. A second important
environmental factor is organic matter, which can protect microorganisms
against physical and chemical disinfecting agents. Biofilms are a good example.
The organic matter in a biofilm protects the biofilm’s microorganisms. Furthermore, it has been clearly documented that bacteria
in biofilms are altered physiologically, and this makes them less susceptible
to many antimicrobial agents. Because of the impact of organic matter, it may
be necessary to clean objects, especially medical and dental equipment, before
they are disinfected or sterilized.
THE USE OF PHYSICAL METHODS IN
CONTROL
Heat and other physical agents are normally used to control microbial
growth and sterilize objects, as can be seen from the continual operation of
the autoclave in every microbiology laboratory. The most frequently employed
physical agents are heat, filtration, and radiation.
Heat
Moist heat readily destroys viruses, bacteria, and fungi. Moist heat
kills by degrading nucleic acids and denaturing enzymes and other essential
proteins. It also disrupts cell membranes. Exposure to boiling water for 10
minutes is sufficient to destroy vegetative cells and eukaryotic spores.
Unfortunately the temperature of boiling water (100°C or 212°F at sea level) is
not sufficient to destroy bacterial spores, which may survive hours of boiling.
Therefore boiling can be used for disinfection of drinking water and objects
not harmed by water, but boiling does not sterilize to destroy bacterial
spores, moist heat sterilization must be carried out at temperatures above
100°C, and this requires the use of saturated steam under pressure. Steam
sterilization is carried out with an
autoclave, a device somewhat like a fancy pressure cooker. The
development of the autoclave by Chamberland in 1884 tremendously stimulated the
growth of microbiology. Water is boiled to produce steam, which is released
into the autoclaves chambered. The air initially present in the chamber is forced
out until the chamber is filled with saturated steam and the outlets are
closed. Hot, saturated steam continues to enter until the chamber reaches the
desired temperature and pressure, usually 121°C and 15 pounds of pressure. At
this temperature saturated steam destroys all vegetative cells and spores in a
small volume of liquid within 10 to 12 minutes. Treatment is continued for at
least 15 minutes to provide a margin of safety. Of course larger containers of
liquid such as flasks and carboys require much longer treatment times. Autoclaving
must be carried out properly or the processed materials will not be sterile. If
all air has not been flushed out of the chamber, it will not reach 121°C, even
though it may reach a pressure of 15 pounds. The chamber should not be packed
too tightly because the steam needs to circulate freely and contact everything
in the autoclave. Bacterial spores will be killed only if they are kept at
121°C for 10 to 12 minutes. When a large volume of liquid must be sterilized,
an extended sterilization time is needed because it takes longer for the center
of the liquid to reach 121°C; 5 liters of liquid may require about 70 minutes.
In view of these potential difficulties, a biological indicator is often
autoclaved along with other material. This indicator commonly consists of a
culture tube containing a sterile ampule of medium and a paper strip covered
with spores of Geobacillus stearothermophilus.
After autoclaving, the ampule is aseptically broken and the culture
incubated for several days. If the test bacterium does not grow in the medium,
the sterilization run has been successful. Sometimes either special indicator
tape or paper that changes color upon sufficient heating is autoclaved with a
load of material. These approaches are convenient and save time but are not as
reliable as the killing of bacterial spores. Any heat-sensitive substances,
such as milk, are treated with controlled heating at temperatures well below
boiling, a process known as
pasteurization in honor of its
developer, Louis Pasteur. In the 1860s the French wine industry was plagued by
the problem of wine spoilage, which made wine storage and shipping difficult.
Pasteur examined spoiled wine under the microscope and detected microorganisms
that looked like the bacteria responsible for lactic acid and acetic acid
fermentations. He then discovered that a brief heating at 55 to 60°C would
destroy these microorganisms and preserve wine for long periods. In 1886 the
German chemists V. H. and F. Soxhlet adapted the technique for preserving milk
and reducing milk- transmissible diseases. Milk pasteurization was introduced
into the United States in 1889. Milk, beer, and many other beverages are now
pasteurized. Pasteurization does not sterilize a beverage, but it does kill any
pathogens present and drastically slows spoilage by reducing the level of
nonpathogenic spoilage microorganisms.
Some materials cannot withstand the high temperature of the autoclave,
and spore contamination precludes the use of other methods to sterilize them.
For these materials, a process of intermittent sterilization, also known as
tyndallization (for John Tyndall, the British physicist who used the technique
to destroy heat-resistant microorganisms in dust) is used. The process also
uses steam (30–60 minutes) to destroy vegetative bacteria. However, steam
exposure is repeated for a total of three times with 23 to 24 hour incubations
between steam exposures. The incubations permit remaining spores to germinate
into heat-sensitive vegetative cells that are then destroyed upon subsequent
steam exposures. Many objects are best
sterilized in the absence of water by dry heat sterilization. Some items are
sterilized by incineration. For instance, inoculating loops, which are used
routinely in the laboratory, can be sterilized in a small, bench-top incinerator.
Other items are sterilized in an oven at 160 to 170°C for 2 to 3 hours.
Microbial death results from the oxidation of cell constituents and
denaturation of proteins. Dry air heat is less effective than moist heat. The
spores of Clostridium botulinum, the cause of botulism, are killed in 5 minutes
at 121°C by moist heat but only after 2 hours at 160°C with dry heat. However,
dry heat has some definite advantages. It does not corrode glassware and metal
instruments as moist heat does, and it can be used to sterilize powders, oils,
and similar items. Despite these advantages, dry heat sterilization is slow and
not suitable for heat-sensitive materials such as many plastic and rubber
items.
Filtration
Filtration is an excellent way to reduce the microbial population in
solutions of heat-sensitive material, and sometimes it can be used to sterilize
solutions. Rather than directly destroying contaminating microorganisms, the filter
simply removes them. There are two types of filters. Depth filters consist of
fibrous or granular materials that have been bonded into a thick layer filled
with twisting channels of small diameter. The solution containing
microorganisms is sucked through this layer under vacuum, and microbial cells
are removed by physical screening or entrapment and by adsorption to the
surface of the filter material. Depth filters are made of diatomaceous earth
(Berke field filters), unglazed porcelain (Chamberlain filters), asbestos, or
other similar materials. Membrane filters have replaced depth filters for many
purposes. These circular filters are porous membranes, a little over 0.1 mm
thick, made of cellulose acetate, cellulose nitrate, polycarbonate,
polyvinylidene fluoride, or other synthetic materials. Although a wide variety
of pore sizes are available, membranes with pores about 0.2 μm in diameter are
used to remove most vegetative cells, but not viruses, from solutions ranging
in volume from less than 1 ml to many liters. The membranes are held in special
holders and are often preceded by depth filters made of glass fibers to remove
larger particles that might clog the membrane filter. The solution is pulled or
forced through the filter with a vacuum or with pressure from a syringe,
peristaltic pump, or nitrogen gas, and collected in previously sterilized
containers. Membrane filters remove microorganisms by screening them out much
as a sieve separates large sand particles from small ones. These filters are
used to sterilize pharmaceuticals, ophthalmic solutions, culture media, oils,
antibiotics, and other heat-sensitive solutions. Air also can be sterilized by
filtration. Two common examples are N-95 disposable masks used in hospitals and
labs, and cotton plugs on culture vessels that let air in but keep microorganisms
out. N-95 masks exclude 95% of particles that are larger than 0.3 m. Other
important examples are laminar flow biological safety cabinets, which employ
high efficiency particulate air (HEPA) filters (a type of depth filter) to
remove 99.97% of particles 0.3 m or larger. Laminar flow biological safety cabinets
or hoods force air through HEPA filters, and then project a vertical curtain of
sterile air across the cabinet opening. This protects a worker from
microorganisms being handled within the cabinet and prevents contamination of
the room. A person uses these cabinets when working with dangerous agents such
as M. tuberculosis and tumor viruses. They are also employed in research labs
and industries, such as the pharmaceutical industry, when a sterile working
environment is needed.
Radiation
Ultraviolet (UV)
radiation around 260 nm is quite
lethal. UV radiation causes thiamine-thiamine dimerization of the microbial
DNA, preventing polymerase-mediated replication and transcription. However, UV
does not penetrate glass, dirt films, water, and other substances very
effectively. Because of this disadvantage, UV radiation is used as a sterilizing
agent only in a few specific situations. UV lamps are sometimes placed on the
ceilings of rooms or in biological safety cabinets to sterilize the air and any
exposed surfaces. Because UV radiation burns the skin and damages eyes, people
working in such areas must be certain the UV lamps are off when the areas are
in use. Commercial UV units are available for water treatment. Pathogens and
other microorganisms are destroyed when a thin layer of water is passed under
the lamps. Ionizing radiation is an
excellent sterilizing agent and penetrates deep into objects. It will destroy
bacterial spores and vegetative cells, both prokaryotic and eukaryotic; however,
ionizing radiation is not always effective against viruses. Gamma radiation
from a cobalt 60 source and accelerated electrons from high-voltage electricity
are used in the cold sterilization of antibiotics, hormones, sutures, and
plastic disposable supplies such as syringes. Gamma radiation and electron
beams have also been used to sterilize and “pasteurize” meat and other foods.
Irradiation can eliminate the threat of such pathogens as Escherichia coli O157:H7,
Staphylococcus aureus, and Campylobacter jejuni. Based on the results of numerous studies,
both the U.S. Food and Drug Administration and the World Health Organization
have approved irradiated food and declared it safe for human consumption.
Currently irradiation is being used to treat poultry, beef, pork, veal, lamb,
fruits, vegetables, and spices.
THE USE OF CHEMICAL AGENTS IN
CONTROL
Physical agents are generally used to sterilize objects. Chemicals, on
the other hand, are more often employed in disinfection and antisepsis. The
proper use of chemical agents is essential to laboratory and hospital safety.
Chemicals also are employed to prevent microbial growth in food, and certain
chemicals are used to treat infectious disease. Next we discuss chemicals used
outside the body. Many different chemicals are available for use as disinfectants,
and each has its own advantages and disadvantages. Ideally the disinfectant
must be effective against a wide variety of infectious agents (gram-positive
and gram-negative bacteria, acid-fast bacteria, bacterial spores, fungi, and
viruses) at low concentrations and in the presence of organic matter. Although
the chemical must be toxic for infectious agents, it should not be toxic to
people or corrosive for common materials. In practice, this balance between
effectiveness and low toxicity for animals is hard to achieve. Some chemicals
are used despite their low effectiveness because they are relatively nontoxic.
The ideal disinfectant should be stable upon storage, odorless or with a
pleasant odor, soluble in water and lipids for penetration into microorganisms,
have a low surface tension so that it can enter cracks in surfaces, and be
relatively inexpensive. One potentially
serious problem is the overuse of antiseptics. For instance, the antibacterial
agent triclosan is found in products such as deodorants, mouthwashes, soaps,
cutting boards, and baby toys. Unfortunately, the emergence of
triclosan-resistant bacteria has become a problem. For example, Pseudomonas
aeruginosa actively pumps the antiseptic out of the cell. There is now evidence
that extensive use of triclosan also increases the frequency of bacterial
resistance to antibiotics. Thus overuse of antiseptics can have unintended
harmful consequences. The properties and uses of several groups of common disinfectants
and antiseptics are surveyed next. Many of the characteristics of disinfectants
and antiseptics.
Phenolics:
Phenol was the first widely used antiseptic and disinfectant. In 1867
Joseph Lister employed it to reduce the risk of infection during surgery. Today
phenol and phenolics (phenol derivatives) such as cresols, xylenols, and
orthophenylphenol are used as disinfectants in laboratories and hospitals. The
commercial disinfectant Lysol is made of a mixture of phenolics. Phenolics act
by denaturing proteins and disrupting cell membranes. They have some real
advantages as disinfectants: phenolics are tuberculocidal, effective in the
presence of organic material, and remain active on surfaces long after
application. However, they have a disagreeable odor and can cause skin
irritation.
Alcohols
Alcohols are among the most widely used disinfectants and antiseptics.
They are bactericidal and fungicidal but not sporicidal; some lipid-containing
viruses are also destroyed. The two most popular alcohol germicides are ethanol
and isopropanol, usually used in about 70 to 80% concentration. They act by
denaturing proteins and possibly by dissolving membrane lipids. A 10 to 15
minute soaking is sufficient to disinfect small instruments.
Halogens
A halogen is any of the five elements (fluorine, chlorine, bromine,
iodine, and astatine) in group VIIA of the periodic table. They exist as
diatomic molecules in the Free State and form salt like compounds with sodium
and most other metals. The halogens iodine and chlorine are important
antimicrobial agents. Iodine is used as a skin antiseptic and kills by
oxidizing cell constituents and iodinating cell proteins. At higher
concentrations, it may even kill some spores. Iodine often has been applied as
tincture of iodine, 2% or more iodine in a water-ethanol solution of potassium
iodide. Although it is an effective antiseptic, the skin may be damaged, a
stain is left, and iodine allergies can result. Iodine has been complexed with
an organic carrier to form an iodophor.
Iodophors are water soluble, stable, and non-staining, and release
iodine slowly to minimize skin burns and irritation. They are used in hospitals
for cleansing preoperative skin and in hospitals and laboratories for
disinfecting. Some popular brands are Wesco dyne for skin and laboratory
disinfection and Betadine for wounds. Chlorine is the usual disinfectant for
municipal water sup- plies and swimming pools and is also employed in the dairy
and food industries. It may be applied as chlorine gas (Cl 2), sodium
hypochlorite (bleach, NaClO), or calcium hypochlorite [Ca(OCl)2], all of which yield hypochlorous acid (HClO)
(see chemical reactions below). The result is oxidation of cellular materials
and destruction of vegetative bacteria and fungi.
Cl 2 + H 2 O → HCl + HClO
NaOCl + H 2 O → NaOH + HClO
Ca(OCl) 2 + 2H 2 O → Ca(OH) 2 + 2HClO
Death of almost all
microorganisms usually occurs within 30 minutes. One potential problem is that
chlorine reacts with organic compounds to form carcinogenic trihalomethanes,
which must be monitored in drinking water. Ozone has been used successfully as
an alternative to chlorination in Europe and Canada.
Chlorine is also an excellent disinfectant for individual use because
it is effective, inexpensive, and easy to employ. Small quantities of drinking
water can be disinfected with halazone tablets. Halazone (parasulfone
dichloramidobenzoic acid) slowly releases chloride when added to water and
disinfects it in about a half hour. It is frequently used by campers lacking
access to uncontaminated drinking water.
Heavy Metals
F or many years the ions of heavy metals such as mercury, silver,
arsenic, zinc, and copper were used as germicides. These have now been
superseded by other less toxic and more effective germicides (many heavy metals
are more bacteriostatic than bactericidal). There are a few exceptions. In some
hospitals, a 1% solution of silver nitrate is added to the eyes of infants to
prevent ophthalmic gonorrhea. Silver sulfadiazine is used on burns. Copper
sulfate is an effective algicide in lakes and swimming pools. Heavy metals
combine with proteins, often with their sulfhydryl groups, and inactivate them.
They may also precipitate cell proteins.
Quaternary Ammonium Compounds
Quaternary ammonium compounds are detergents that have antimicrobial
activity and are effective disinfectants.
Detergents (Latin detergere, to wipe away) are organic cleansing agents
that are amphipathic, having both polar hydrophilic and nonpolar hydrophobic
components. The hydrophilic portion of a quaternary ammonium compound is
positively charged quaternary nitrogen; thus quaternary ammonium compounds are
cationic detergents. Their antimicrobial activity is the result of their
ability to disrupt microbial membranes; they may also denature proteins. Cationic
detergents such as benzalkonium chloride and acetyl-pyridinium chloride kill
most bacteria but not M. tuberculosis or spores. They have the advantages of
being stable and nontoxic, but they are inactivated by hard water and soap.
Cationic detergents are often used as disinfectants for food utensils and small
instruments and as skin antiseptics.
Aldehydes
Both of the commonly used aldehydes, formaldehyde and glutaraldehyde
are highly reactive molecules that combine with nucleic acids and proteins, and
inactivate them, probably by cross-linking and alkylating molecules. They are
sporicidal and can be used as chemical sterilants. Formaldehyde is usually
dissolved in water or alcohol before use. A 2% buffered solution of
glutaraldehyde is an effective disinfectant. It is less irritating than
formaldehyde and is used to disinfect hospital and laboratory equipment.
Glutaraldehyde usually disinfects objects within about 10 minutes but may
require as long as 12 hours to destroy all spores.
Sterilizing Gases
Many heat-sensitive items such
as disposable plastic petri dishes and syringes, heart-lung machine components,
sutures, and catheters are sterilized with ethylene oxide gas. Ethylene oxide
(EtO) is both microbicidal and sporicidal. It is a very strong alkylating agent
that kills by reacting with functional groups of DNA and proteins to block
replication and enzymatic activity. It is a particularly effective sterilizing
agent because it rapidly penetrates packing materials, even plastic wraps. Sterilization is carried out in a special
ethylene oxide sterilizer, very much resembling an autoclave in appearance,
that controls the EtO concentration, temperature, and humidity. Because pure
EtO is explosive, it is usually supplied in a 10 to 20% concentration mixed
with either CO2 or dichloro-difluoromethane. The
ethylene oxide concentration, humidity, and temperature influence the rate of
sterilization. A clean object can be sterilized if treated for 5 to 8 hours at
38°C or 3 to 4 hours at 54°C when the relative humidity is maintained at 40 to
50% and the EtO concentration at 700 mg/l. Because it is so toxic to humans,
extensive aeration of the sterilized materials is necessary to remove residual
EtO.
Betapropiolactone (BPL) is occasionally employed as a sterilizing gas.
In the liquid form it has been used to sterilize vaccines and sera. BPL
decomposes to an inactive form after several hours and is therefore not as
difficult to eliminate as EtO. It also destroys microorganisms more readily
than ethylene oxide but does not penetrate materials well and may be carcinogenic.
For these reasons, BPL has not been used as extensively as EtO. Vaporized hydrogen peroxide can be used to
decontaminate biological safety cabinets, operating rooms, and other large
facilities. These systems introduce vaporized hydrogen peroxide into the
enclosure for some time, depending on the size of the enclosure and the
materials within. Hydrogen per- oxide and its oxy-radical byproducts are toxic
and kill a wide variety of microorganisms. During the course of the
decontamination process, it breaks down to water and oxygen, both of which are
harmless. Other advantages of these systems are that they can be used at a wide
range of temperatures (4 to 80°C) and they do not damage most materials.
EVALUATION OF ANTIMICROBIAL AGENT
EFFECTIVENESS
Testing of antimicrobial agents is a complex process regulated by two different
federal agencies. The U.S. Environmental Protection Agency regulates disinfectants,
whereas agents used on humans and animals are under the control of the Food and
Drug Administration. Testing of antimicrobial agents often begins with an
initial screening to see if they are effective and at what concentrations. This
may be followed by more realistic in-use testing. The best-known disinfectant
screening test is the phenol coefficient test in which the potency of a
disinfectant is compared with that of phenol. A series of dilutions of phenol
and the disinfectant being tested are prepared. Standard amounts of Salmonella
enterica Typhi and Staphylococcus aureus
are added to each dilution; the dilutions are then placed in a 20 or
37°C water bath. At 5-minute intervals, samples are withdrawn from each
dilution and used to inoculate a growth medium, which is incubated for two or
more days and then examined for growth. If there is no growth in the medium,
the dilution at that particular time of sampling killed the bacteria. The
highest dilution (i.e., the lowest concentration) that kills the bacteria after
a 10-minute exposure but not after 5 minutes is used to calculate the phenol
coefficient. This is done by dividing the reciprocal of the appropriate
dilution for the disinfectant being tested by the reciprocal of the appropriate
phenol dilution. For instance, if the phenol dilution was 1/90 and maximum effective
dilution for disinfectant X was 1/450, then the phenol coefficient of X would
be 5. The higher the phenol coefficient value, the more effective the
disinfectant under these test conditions. A value greater than 1 means that the
disinfectant is more effective than phenol. The phenol coefficient test is a
useful initial screening procedure, but the phenol coefficient can be
misleading if taken as a direct indication of disinfectant potency during
normal use. This is because the phenol coefficient is determined under
carefully controlled conditions with pure bacterial cultures, whereas disinfectants
are normally used on complex populations in the presence of organic matter and
with significant variations in environmental factors such as pH, temperature,
and presence of salts. To more realistically estimate disinfectant
effectiveness, other tests are often used. The rates at which selected bacteria
are destroyed with various chemical agents may be experimentally determined and
compared. A use dilution test can also be carried out. Stainless steel cylinders
are contaminated with specific bacterial species under carefully controlled
conditions. The cylinders are dried briefly, immersed in the test disinfectants
for 10 minutes, transferred to culture media, and incubated for two days. The
disinfectant concentration that kills the organisms in the sample with a 95%
level of confidence under these conditions is determined. Disinfectants also can
be tested under conditions designed to simulate normal in-use situations.
In-use testing techniques allow a more accurate determination of the proper
disinfectant concentration for a particular situation.
BIOLOGICAL CONTROL OF MICROORGANISMS
The emerging field of biological control of microorganisms demonstrates
great promise. Scientists are learning to exploit natural control processes
such as predation of one microorganism on another, viral-mediated lysis, and
toxin-mediated killing. While these control mechanisms occur in nature, their
approval and use by humans is relatively new. Studies evaluating control of
Salmonella, Shigella, and E. coli by gram-negative predators such as B.dellovibrio suggest that poultry farms may be sprayed
with the predator to reduce potential contamination. The control of human
pathogens using bacteriophage is gaining wide support and appears to be
effective in the eradication of a number of bacterial species by lysing the
pathogenic host. This seems intuitive, knowing that the virus lyses its
specific bacterial host, yet unnerving when one thinks about maybe swallowing,
injecting, or applying a virus to the human body. The use of microbial toxins
(such as bacteriocins) to control susceptible a population suggests yet another
method for potential control of other microorganisms.
GENERAL CHARACTERISTICS OF ANTIMICROBIAL
DRUGS
As Ehrlich so clearly saw, to
be successful a chemotherapeutic agent
must have selective toxicity: it must kill or inhibit the microbial pathogen
while damaging the host as little as possible. The degree of selective toxicity
may be expressed in terms of (1) the therapeutic dose the drug level required
for clinical treatment of a particular infection and (2) the toxic dose the
drug level at which the agent becomes too toxic for the host. The therapeutic
index is the ratio of the toxic dose to the therapeutic dose. The larger the
therapeutic index, the better the chemotherapeutic agent (all other things
being equal). A drug that disrupts a
microbial function not found in host animal cells often has a greater selective
toxicity and a higher therapeutic index. For example, penicillin inhibits
bacterial cell wall peptidoglycan synthesis but has little effect on host cells
because they lack cell walls; therefore penicillin’s therapeutic index is high.
A drug may have a low therapeutic index because it inhibits the same process in
host cells or damages the host in other ways. The undesirable effects on the
host, or side effects, are of many kinds and may involve almost any organ
system. Because side effects can be
severe, chemotherapeutic agents should be administered with great care. Some bacteria and fungi are able to naturally
produce many of the commonly employed antibiotics. In contrast, several
important chemotherapeutic agents, such as sulfonamides, trimethoprim, ciprofloxacin,
isoniazid, and dapsone, are synthetic manufactured by chemical procedures
independent of microbial activity. Some antibiotics are semisynthetic natural
antibiotics that have been structurally modified by the addition of chemical
groups to make them less susceptible to inactivation by pathogens (e.g.,
ampicillin and methicillin). In addition, many semisynthetic drugs have a
broader spectrum of antibiotic activity than does their parent molecule. This
is particularly true of the semisynthetic penicillins (e.g., ampicillin,
amoxycillin) versus the naturally produced penicillin G and penicillin V. D
rugs vary considerably in their range of effectiveness. Many are narrow-spectrum drugs that are; they are
effective only against a limited variety of pathogens. Others are
broad-spectrum drugs that attack many different kinds of pathogens. Drugs may
also be classified based on the general microbial group they act against:
antibacterial, antifungal, anti- protozoan, and antiviral. A few agents can be
used against more than one group; for example, sulfonamides are active against
bacteria and some protozoa. Finally, chemotherapeutic agents can be either
cidal or static. Static agents reversibly inhibit growth; if the agent is
removed, the microorganisms will recover and grow again. Although a cidal agent
kills the target pathogen, it may be static at low levels. The effect of an
agent also varies with the target species: an agent may be cidal for one
species and static for another. Because static agents do not directly destroy
the pathogen, elimination of the infection depends on the host’s own immunity
mechanisms. A static agent may not be effective if the host is
immunosuppressed. Some idea of the
effectiveness of a chemotherapeutic agent against a pathogen can be obtained
from the minimal inhibitory concentration (MIC). The MIC is the lowest
concentration of a drug that prevents growth of a particular pathogen. On the
other hand, the minimal lethal concentration (MLC) is the lowest drug
concentration that kills the pathogen. A cidal drug generally kills pathogens
at levels only two to four times the MIC, whereas a static agent kills at much
higher concentrations, if at all.
DETERMINING THE LEVEL OF ANTIMICROBIAL
ACTIVITY
Determination of antimicrobial effectiveness against specific
pathogens is essential for proper therapy. Testing can show which agents are
most effective against a pathogen and give an estimate of the proper therapeutic
dose.
Dilution
Susceptibility Tests
Dilution susceptibility tests can be used to determine MIC and MLC
values. Antibiotic dilution tests can be done in both agar and broth. In the
broth dilution test, a series of broth tubes (usually Mueller-Hinton broth)
containing antibiotic concentrations in the range of 0.1 to 128 g/ml (two-fold
dilutions) is prepared and inoculated with a standard density of the test
organism. The lowest concentration of the antibiotic resulting in no growth
after 16 to 20 hours of incubation is the MIC. The MLC can be ascertained if
the tubes showing no growth are then cultured into fresh medium lacking
antibiotic. The lowest antibiotic concentration from which the microorganisms
do not grow when transferred to fresh medium is the MLC. The agar dilution test
is very similar to the broth dilution test. Plates containing Mueller-Hinton
agar and various amounts of antibiotic are inoculated and examined for growth.
Several automated systems for susceptibility testing and MIC determination with
broth or agar cultures have been developed.
Disk Diffusion Tests
If a rapidly growing microbe
such as Staphylococcus or Pseudomonas is
being tested, a disk diffusion technique may be used to save time and media.
The principle behind the assay technique is fairly simple. When an
antibiotic-impregnated disk is placed on agar previously inoculated with the
test bacterium, the antibiotic diffuses radially outward through the agar,
producing an antibiotic concentration gradient. The antibiotic is present at
high concentrations near the disk and affects even minimally susceptible
microorganisms (resistant organisms will grow up to the disk). As the distance
from the disk increases, the antibiotic concentration decreases and only more
susceptible pathogens are harmed. A clear zone or ring is present around an
antibiotic disk after incubation if the agent inhibits bacterial growth. The
wider the zone surrounding a disk, the more susceptible the pathogen is. Zone
width also is a function of the antibiotic’s initial con- centration, its
solubility, and its diffusion rate through agar. Thus zone width cannot be used
to compare directly the effectiveness of different antibiotics. Currently the disk diffusion test most often
used is the Kirby- Bauer method, which was developed in the early 1960s at the
University of Washington Medical School by William Kirby, A. W. Bauer, and
their colleagues. Freshly grown bacteria are used to inoculate the entire
surface of a Mueller-Hinton agar plate. After the agar surface has dried for
about 5 minutes, the appropriate antibiotic test disks are placed on it, either
with sterilized forceps or with a multiple applicator device. The plate is immediately placed at 35°C.
After 16 to 18 hours of incubation, the diameters of the zones of inhibition
are measured to the nearest millimeter.
Kirby-Bauer test results are interpreted using a table that relates zone
diameter to the degree of microbial resistance. A plot of MIC (on a logarithmic
scale) versus zone inhibition diameter (arithmetic scale) is prepared for each
antibiotic. These plots are then used to
find the zone diameters corresponding to the drug concentrations actually
reached in the body. If the zone diameter for the lowest level reached in the
body is smaller than that seen with the test pathogen, the pathogen should have
an MIC value low enough to be destroyed by the drug. A pathogen with too high and
MIC value (too small a zone diameter) is resistant to the agent at normal body
concentrations.
The E test
The E test from AB BIODISK may be used in sensitivity testing under
some conditions. It is particularly convenient for use with anaerobic
pathogens. Agar medium is streaked in three different directions with the test
organism and plastic E test strips are
placed on the surface so that they extend out radially from the center. Each strip contains a gradient of an antibiotic
and is labeled with a scale of MIC values. The lowest concentration in the
strip lies at the center of the plate. After 24 to 48 hours of incubation, an
elliptical zone of inhibition appears. MICs are determined from the point of
intersection between the inhibition zone and the strip’s scale of MIC
values.
ANTIBACTERIAL DRUGS
Since Fleming’s discovery of penicillin, many antibiotics have been
found that can damage pathogens in several ways. A few antibacterial drugs are
described here with emphasis on their mechanisms of action.
Inhibitors of Cell Wall
Synthesis
The most selective antibiotics
are those that interfere with bacterial cell wall synthesis. Drugs such as
penicillins, cephalosporins, vancomycin, and bacitracin have a high therapeutic
index because they target structures not found in eukaryotic cells.
Penicillins
Most penicillin (e.g.,
penicillin G or benzylpenicillin) are derivatives of 6-aminopenicillanic acid
and differ from one another with respect to the side chain attached to the
amino group. The most crucial feature of
the molecule is the -lactam ring,
which is essential for bioactivity. Many penicillin-resistant bacteria produce penicillinase
(also called lactamase), an enzyme that in- activates the antibiotic by
hydrolyzing a bond in the lactam ring. The structure of the penicillins
resembles the terminal d-alanyl-d-alanine found on the peptide side chain of
the peptidoglycan subunit. It is thought that this structural similarity blocks
the enzyme catalyzing the transpeptidation reaction that forms the
peptidoglycan cross-links. Thus formation of a complete cell wall is blocked,
leading to osmotic lysis. This mechanism is consistent with the observation
that penicillins act only on growing bacteria that are synthesizing new
peptidoglycan. However, the mechanism of penicillin action is actually more
complex. It has been discovered that penicillins bind to several periplasmic
proteins (penicillin-binding proteins, or PBPs) and may also destroy bacteria
by activating their own autolytic enzymes. However, penicillin may kill
bacteria even in the absence of autolysins or murein hydrolases. Lysis could occur
after bacterial viability has already been lost. Penicillin may stimulate
proteins called bacterial holins to form holes or lesions in the plasma membrane,
leading directly to membrane leakage and death. Murein hydrolases also could
move through the holes, disrupt the peptidoglycan, and lyse the cell. Penicillins
differ from each other in several ways. The two naturally occurring
penicillins, penicillin G and penicillin V, are narrow-spectrum drugs.
Penicillin G is effective against gonococci, meningococci, and several
gram-positive pathogens such as streptococci and staphylococci. However, it
must be administered by injection (parenterally) because it is destroyed by
stomach acid. Penicillin V is similar to penicillin G in spectrum of activity
but can be given orally because it is more resistant to acid. The semisynthetic
penicillins, on the other hand, have a broader spectrum of activity. Ampicillin
can be administered orally and is effective against gram-negative bacteria such
as Haemophilus, Salmonella, and Shigella.
Carbenicillin and ticarcillin are potent against Pseudomonas and
Proteus. An increasing number of bacteria have become resistant to natural
penicillins and many of the semisynthetic analogs. Physicians frequently employ
specific semisynthetic penicillins that are not destroyed by -lactamases to
combat antibiotic resistant pathogens. These include methicillin, nafcillin,
and oxacillin. However, this practice has been confounded by the emergence of
methicillin- resistant bacteria. Although penicillins are the least toxic of
the antibiotics, about 1 to 5% of the adults in the United States develop
allergies to them. Occasionally, a person will die of a violent allergic
response; therefore patients should be questioned about penicillin allergies
before treatment is begun.
Cephalosporins Cephalosporins
are a family of antibiotics originally isolated in 1948 from the fungus Cephalosporium.
They contain a lactam structure that is
very similar to that of the penicillins.
As might be expected from their structural similarities to penicillins,
cephalosporins also inhibit the transpeptidation reaction during peptidoglycan
synthesis. They are broad-spectrum drugs frequently given to patients with
penicillin allergies (although about 10% of patients allergic to penicillin are
also allergic to cephalosporins). Cephalosporins are broadly categorized into
four generations (groups of drugs that were sequentially developed) based on
their spectrum of activity. First-generation cephalosporins are more effective
against gram-positive pathogens than gram-negatives. Second-generation drugs,
developed after the first generation, have improved effects on gram-negative
bacteria with some anaerobe coverage. Third-generation drugs are particularly
effective against gram-negative pathogens, and some reach the central nervous
system. This is of particular note because many antimicrobial agents do not
cross the blood-brain barrier. Finally, fourth-generation cephalosporins are
broad spectrum with excellent gram-positive and gram-negative coverage and,
like their third-generation predecessors, inhibit the growth of the difficult
opportunistic pathogen Pseudomonas aeruginosa.
Vancomycin and Teicoplanin
Vancomycin is a glycopeptide antibiotic produced by Streptomyces orientalis. It
is a cup-shaped molecule composed of a peptide linked to a disaccharide. The
peptide portion blocks the transpeptidation reaction by binding specifically to
the d-alanine-d-alanine terminal sequence on the pentapeptide portion of
peptidoglycan. The antibiotic is bactericidal for Staphylococcus and some
members of the genera Clostridium, Bacillus, Streptococcus, and Enterococcus. It is given both orally and intravenously,
and has been particularly important in the treatment of antibiotic-resistant
staphylococcal and enterococcal infections. However, vancomycin-resistant
strains of Enterococcus have become widespread and cases of resistant
Staphylococcus aureus have appeared. In this case, resistance is conferred when
bacteria change the terminal d-alanine to either d-lactate or a d-serine
residue. Vancomycin resistance poses a serious public health threat: vancomycin
has been considered the “drug of last resort” in cases of antibiotic-resistant S.
aureus. Clearly newer drugs must be
developed. Teicoplanin, another glycopeptide antibiotic, is produced by the
actinomycete Actinoplanes teichomyceticus.
It is similar in structure and mechanism of action to vancomycin but has
fewer side effects. It is active against staphylococci, enterococci, streptococci,
clostridia, Listeria, and many gram-positive pathogens.
Protein Synthesis Inhibitors
Many antibiotics inhibit
protein synthesis by binding with the prokaryotic ribosome and other components
of protein synthesis. Because these drugs discriminate between prokaryotic and
eukaryotic ribosomes, their therapeutic index is fairly high but not as high as
that of cell wall inhibitors. Several different steps in protein synthesis can
be affected: aminoacyl-tRNA binding, peptide bond formation, mRNA reading, and
translocation.
Aminoglycosides although
considerable variation in structure occurs among several important
aminoglycoside antibiotics, all contain a cyclohexane ring and amino sugars.
Streptomycin, kanamycin, neomycin, and tobramycin are synthesized by different
species of the genus Streptomyces, whereas gentamicin comes from another
actinomycete, Micromonospora purpurea.
Aminoglycosides bind to the 30S (small) ribosomal subunit and interfere
with protein synthesis by directly inhibiting the synthesis process and causing
misreading of the mRNA. These
antibiotics are bactericidal and tend to be most effective against
gram-negative pathogens. Streptomycin’s usefulness has decreased greatly due to
widespread drug resistance but may still be effective when other
aminoglycosides are not tolerated or are contraindicated due to interactions
with other drugs (e.g., HIV protease inhibitors). Gentamicin is used to treat Proteus,
Escherichia, Klebsiella, and Serratia infections. Aminoglycosides can be quite
toxic, however, and can cause deafness, renal dam- age, loss of balance,
nausea, and allergic responses.
Tetracyclines the tetracyclines
are a family of antibiotics with a common four-ring structure to which a
variety of side chains are attached.
Oxytetracycline and chlortetracycline are produced naturally by Streptomyces
species, whereas others are semisynthetic drugs. These antibiotics are similar
to the aminoglycosides and combine with the 30S subunit of the ribosome. This
inhibits the binding of aminoacyl-tRNA molecules to the A site of the ribosome.
Their action is only bacteriostatic. Tetracyclines
are broad-spectrum antibiotics that are active against most bacteria, including
rickettsia, chlamydiae, and mycoplasmas. Although their use has declined in
recent years, they are still sometimes used to treat acne.
Macrolides The macrolide antibiotics
contain 12- to 22-carbon lac- tone rings linked to one or more sugars. Erythromycin binds to the 23S rRNA of the 50S
ribosomal subunit to inhibit peptide chain elongation during protein synthesis.
Erythromycin is a relatively broad-spectrum anti- biotic effective against
gram-positive bacteria, mycoplasmas, and a few gram-negative bacteria but is
usually only bacteriostatic. It is used with patients who are allergic to
penicillins and in the treatment of whooping cough, diphtheria, diarrhea caused
by Campylobacter, and pneumonia from Legionella or Mycoplasma infections.
Clindamycin is effective against a variety of bacteria, including staphylococci,
and anaerobes such as Bacteroides.
Azithromycin, which has surpassed erythromycin in use, is particularly
effective against many bacteria including Chlamydia trachomatis.
Chloramphenicol was first
produced from cultures of Streptomyces venezuelae but it is now synthesized
chemically. Like erythromycin, this antibiotic binds to 23S rRNA on the 50S
ribosomal subunit to inhibit the peptidyl transferase reaction. It has a very
broad spectrum of activity but, unfortunately, is quite toxic. The most common
side effect is depression of bone mar- row function, leading to aplastic anemia
and a decreased number of white blood cells. Consequently, this antibiotic is
used only in life-threatening situations when no other drug is adequate.
Metabolic Antagonists
Several valuable drugs act as antimetabolites:
they antagonize, or block, the functioning of metabolic pathways by
competitively inhibiting the use of metabolites by key enzymes. These drugs can
act as structural analogs, molecules that are structurally similar to naturally
occurring metabolic intermediates. These analogs compete with intermediates in
metabolic processes because of their similarity but are just different enough
that they prevent normal cellular metabolism. By preventing metabolism, they
are bacteriostatic but broad spectrum; their removal reestablishes the
metabolic activity.
Sulfonamides or Sulfa Drugs Sulfonamides , or sulfa drugs, are structurally related to
sulfanilamide, an analog of p-aminobenzoic acid, or PABA. PABA is used in the synthesis of the cofactor
folic acid (folate). When sulfanilamide or another sulfonamide enters a
bacterial cell, it competes with PABA for the active site of an enzyme involved
in folic acid synthesis, causing a decline in folate concentration. This
decline is detrimental to the bacterium because folic acid is a precursor of
purines and pyrimidines, the bases used in the construction of DNA, RNA, and
other important cell constituents. The resulting inhibition of purine and
pyrimidine synthesis leads to cessation of protein synthesis and DNA
replication. Sulfonamides are selectively toxic for many bacterial and
protozoan pathogens because these microbes manufacture their own folate and
cannot effectively take up this cofactor, whereas humans do not synthesize
folate (we must obtain it in our diet). Sulfonamides thus have a high therapeutic
index. The increasing resistance of many bacteria to sulfa drugs limits their
effectiveness. Furthermore, as many as 5% of the patients receiving sulfa drugs
experience adverse side effects, chiefly allergic responses such as fever,
hives, and rashes.
Trimethoprim is a synthetic antibiotic that also interferes with the
production of folic acid. It does so by binding to dihydrofolate reductase
(DHFR), the enzyme responsible for converting dihydrofolic acid to
tetrahydrofolic acid, competing against the dihydrofolic acid substrate. It is
a broad-spectrum antibiotic often used to treat respiratory and middle ear
infections, urinary tract infections, and traveler’s diarrhea. It can be
combined with sulfa drugs to increase efficacy of treatment by blocking two key
steps in the folic acid pathway. The
inhibition of two successive steps in a single biochemical pathway means that
less of each drug is needed in combination than when used alone. This is termed
a synergistic drug interaction.
Nucleic Acid Synthesis Inhibition
The antibacterial drugs that inhibit nucleic acid synthesis function
by inhibiting
(1) DNA polymerase and DNA helicase
(2) RNA polymerase to block replication or transcription,
respectively. These drugs are not as selectively toxic as other antibiotics
because procaryotes and eucaryotes do not differ greatly with respect to
nucleic acid synthesis.
Quinolones
The quinolones are synthetic drugs that contain the 4-quinolone ring.
The quinolones are important antimicrobial agents that inhibit nucleic acid
synthesis. They are increasingly used to treat a wide variety of infections.
The first quinolone, nalidixic acid was synthesized in 1962. Since that time,
generations of fluoroquinolones have been produced. Three of these
ciprofloxacin, norfloxacin, and ofloxacin are currently used in the United
States, and more fluoroquinolones are being synthesized and tested. Quinolones
act by inhibiting the bacterial DNA gyrase and topoisomerase II. DNA gyrase
introduces negative twist in DNA and helps separate its strands. Inhibition of
DNA gyrase disrupts DNA replication and repair, bacterial chromosome separation
during division, and other processes involving DNA.
Fluoroquinolones also inhibit topoisomerase II, another enzyme that
untangles DNA during replication. It is not surprising that quinolones are
bactericidal.
The quinolones are
broad-spectrum antibiotics. They are highly effective against enteric bacteria
such as Escherichia coli and Klebsiella pneumoniae. They can be used with Haemophilus, Neiserria,
P. aeruginosa, and other gram-negative pathogens. The quinolones also are
active against gram-positive bacteria such as S. aureus, Streptococcus pyogenes,
and Mycobacterium tuberculosis. Thus they are used in treating a wide range
of infections.
ANTIFUNGAL DRUGS
Treatment of fungal infections generally has been less successful than
that of bacterial infections largely because eukaryotic fun- gal cells are much
more similar to human cells than are bacteria. Many drugs that inhibit or kill
fungi are therefore quite toxic for humans and thus have a low therapeutic
index. In addition, most fungi have a detoxification system that modifies many
antifungal agents. Therefore antibiotics are fungistatic only as long as
repeated application maintains high levels of unmodified antibiotic.
Nonetheless, a few drugs are useful in treating many major fungal diseases.
Effective antifungal agents frequently either extract membrane sterols or
prevent their synthesis. Similarly, because fungi have cell walls made of
chitin, the enzyme chitin synthase is the target for antifungals such as polyoxin
D and nikkomycin. Fungal infections are
often subdivided into infections called superficial mycoses, subcutaneous
mycoses, and systemic mycoses. Treatment for these types of disease is very
different. Several drugs are used to treat superficial mycoses. Three drugs
containing imidazole miconazole, ketoconazole, and clotrimazole are
broad-spectrum agents available as creams and solutions for the treatment of
dermatophyte infections such as athlete’s foot, and oral and vaginal
candidiasis. They are thought to disrupt fungal membrane permeability and
inhibit sterol synthesis. Nystatin, a polyene anti- biotic from Streptomyces,
is used to control Candida infections of the skin, vagina, or alimentary tract.
It binds to sterols and damages the membrane, leading to fungal membrane
leakage. Griseofulvin, an antibiotic formed by Penicillium, is given orally to
treat chronic dermatophyte infections. It is thought to disrupt the mitotic
spindle and inhibit cell division; it also may inhibit protein and nucleic acid
synthesis. Side effects of griseofulvin include headaches, gastrointestinal upset
and allergic reactions. Ascomycota Systemic infections are very difficult to control
and can be fatal. Three drugs commonly used against systemic mycoses are
amphotericin B, 5-flucytosine, and fluconazole. Amphotericin B from Streptomyces
spp. binds to the sterols in fungal membranes, disrupting membrane permeability
and causing leakage of cell constituents. It is quite toxic to humans and used
only for serious, life-threatening infections. The synthetic oral antimycotic
agent 5-flucytosine (5-fluorocytosine) is effective against most systemic
fungi, although drug resistance often develops rapidly. The drug is converted
to 5-fluorouracil by the fungi, incorporated into RNA in place of uracil, and
disrupts RNA function. Its side effects include skin rashes, diarrhea, nausea,
aplastic anemia, and liver damage. Atovaquone and pentamidine are used to treat
Pneumocystis jiroveci (formerly called P.carinii). Some reports indicate that
pentamidine interferes with P. jiroveci metabolism, although the drug only
moderately inhibits glucose metabolism, protein synthesis, RNA synthesis, and
intracellular amino acid transport in vitro. Fluconazole is used in the treatment
of candidiasis, cryptococcal meningitis, and coccidioidal meningitis. Because
adverse effects of fluconazole are relatively uncommon, it is used
prophylactically to prevent life-threatening fungal infections in AIDS patients
and other individuals who are severely immunosuppressed. Subcutaneous mycoses,
such as mycetoma and sporotrichosis, are typically treated with combinations of
therapies that would be used for superficial and systemic mycoses. As with
systemic mycoses, strict attention to potential toxic side effects is
warranted.
ANTIVIRAL DRUGS
Because viruses enter host cells and make use of host cell enzymes and
constituents, it was long thought that a drug that blocked virus reproduction
would be toxic for the host. However, inhibitors of virus specific enzymes and
life cycle processes have been discovered, and several drugs are used
therapeutically. Most antiviral drugs
disrupt either critical stages in the virus life cycle or the synthesis of
virus-specific nucleic acids. Amantadine
and rimantadine can be used to prevent influenza an infections. When given early
in the infection (in the first 48 hours), they reduce the incidence of influenza
by 50 to 70% in an exposed population. Amantadine blocks the penetration and
uncoating of influenza virus particles. Adenine arabinoside or vidarabine
disrupts the activity of DNA polymerase and several other enzymes involved in DNA
and RNA synthesis and function. It is given intravenously or applied as an
ointment to treat herpes infections. A third drug, acyclovir, is also used in
the treatment of herpes infections. Upon phosphorylation, acyclovir resembles
deoxyGTP and inhibits the viral DNA polymerase. Unfortunately,
acyclovir-resistant strains of herpes have developed. Effective acyclovir derivatives
and relatives are now available. Valacyclovir is an orally administered prodrug
form of acyclovir. Prodrugs are inactive until metabolized. Ganciclovir,
penciclovir, and penciclovir’s oral form, famciclovir, are effective in
treatment of herpes viruses. Another kind of drug, foscarnet, inhibits the virus
DNA polymerase in a different way. Foscarnet is an organic analog of
pyrophosphate that binds to the polymerase active site and blocks the cleavage
of pyrophosphate from nucleoside triphosphate substrates. It is used in
treating herpes and cytomegalovirus infections. Several broad-spectrum anti-DNA
virus drugs have been developed. A good example is the drug HPMPC, or
cidofovir. It is effective against papovaviruses, adenoviruses, herpes viruses,
iridoviruses, and poxviruses. The drug acts on the viral DNA polymerase as a
competitive inhibitor and alternative substrate of dCTP. It has been used
primarily against cytomegalovirus but also against herpes simplex and human
papillomavirus infections. Research on anti-HIV drugs has been particularly
active. Many of the first drugs to be developed were reverse transcriptase
inhibitors such as azidothymidine (AZT) or zidovudine, lamivudine (3TC),
didanosine (ddI), zalcitabine (ddC), and stavudine (d4T). These interfere with
reverse transcriptase activity and therefore block HIV reproduction. More
recently HIV protease inhibitors have also been developed. Three of the most
used are saquinvir, indinavir, and ritonavir. Protease inhibitors are effective
because HIV, like many viruses, translates multiple proteins as a single
polypeptide. This polypeptide must then be cleaved into individual proteins
required for virus replication. Protease inhibitors mimic the pep- tide bond
that is normally attacked by the protease. The most successful HIV treatment
regimen has been a cocktail of agents given at high dosages to prevent the
development of drug resistance. For example, the combination of AZT, 3TC, and
ritonavir is very effective in reducing HIV plasma concentrations almost to
zero. However, the treatment does not eliminate proviral HIV DNA that still
resides in memory T cells and possibly elsewhere.
Probably the most publicized antiviral agent has been Tamiflu (generically,
oseltamivir phosphate). Tamiflu is a neuraminidase inhibitor that has received
much attention in light of predictions of a twenty-first-century influenza
pandemic, including avian influenza (“bird flu”). While Tamiflu is not a cure
for neurominidase expressing viruses, two clinical trials showed that patients
who took Tamiflu were relieved of flu symptoms 1.3 days faster than patients
who did not take Tamiflu. However, prophylactic use has resulted in viral
resistance to Tamiflu. Tamiflu is not a substitute for yearly flu vaccination
and frequent hand-washing.
ANTIPROTOZOAN DRUGS
The mechanism of action for most antiprotozoan drugs is not completely
understood. Drugs such as chloroquine, atovaquone, mefloquine, iodoquinol,
metronidazole, and nitazoxanide, for ex- ample, have potent antiprotozoan
action, but a clear mechanism by which protozoan growth is inhibited is
unknown. However, most antiprotozoan drugs appear to act on protozoan nucleic
acid or some metabolic event. Chloroquine is used to treat malaria. Several
mechanisms of action have been reported. It can raise the internal pH, clump
the plasmodial pigment, and intercalate into plasmodial DNA. Chloroquine also
inhibits heme polymerase, an enzyme that converts toxic heme into nontoxic hemazoin.
Inhibition of this enzyme leads to a buildup of toxic heme. Mefl oquine is also
used to treat malaria and has been found to swell the Plasmodium falciparum food
vacuoles, where it may act by forming toxic complexes that damage membranes and
other plasmodial components. Metronidazole is used to treat Entamoeba infections.
Anaerobic organisms readily reduce it to the active metabolite within the
cytoplasm. Aerobic organisms appear to reduce it using ferrodoxin (a protein of
the electron transport system). Reduced metronidazole interacts with DNA,
altering its helical structure and causing DNA fragmentation; it prevents
normal nucleic acid synthesis, resulting in cell death. A number of antibiotics
that inhibit bacterial protein synthesis are also used to treat protozoan infection.
These include the aminoglycosides clindamycin and paromomycin. Aminoglycosides
can be considered polycationic molecules that have a high affinity for nucleic
acids. Specifically, aminoglycosides possess high affinities for RNAs.
Different aminoglycoside antibiotics bind to different sites on RNAs. RNA
binding interferes with the normal expression and function of the RNA,
resulting in cell death. Interference
of eukaryotic electron transport is one common activity of some antiprotozoan
drugs. Atovaquone is used to treat Toxoplasma gondii. It is an analog of ubiquinone,
an integral component of the eukaryotic electron transport system. As an analog
of ubiquinone, atovaquone can act as a competitive inhibitor and thus suppress
electron transport. The ultimate metabolic effects of electron transport
blockade include inhibited or delayed synthesis of nucleic acids and ATP.
Another drug that interferes with electron transport is nitazoxanide, which is
used to treat cryptosporidiosis. It appears to exert its effect through interference
with the pyruvate: ferredoxin oxidoreductase. It has also been reported to form
toxic free radicals once the nitro group is reduced intracellularly. Pyrimethamine and dapsone, used to treat Toxoplasma
infections, appear to act in the same way as trimethoprim interfering with
folic acid synthesis by inhibition of dihydrofolate reductase. As with other antimicrobial therapies,
traditional drug development starts by identifying a unique target to which a
drug can bind and thus prevent some vital function. A second consideration is
often related to drug spectrum: how many different species have that target so
that the proposed drug can be used broadly as a chemotherapeutic agent? This is
also true for use of agents needed to remove protozoan parasites from their
hosts. However, because protozoa are eucaryotes, the potential for drug action
on host cells and tissues is greater than it is when targeting procaryotes.
Most of the drugs used to treat protozoan infection have signifcant side effects;
nonetheless, the side effects are usually acceptable when weighed against the
parasitic burden.
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even though it is a very long article, it is very clear and very much informative. almost i can prepare microbio notes for whole year. really happy about ur blog...
ReplyDeleteThis is very powerful I ve been diagnosed with HIV 3years ago and I've become in denial at first for a year but later I accepted it and I'm living with depression so I struggle to keep my cd4 up bit I'm very healthy and I just checked my cd4 was 460 and they advising me to being medication but I'm not ready I wanna take them when at least it 350 I'm only 24years old I have a 6year old healthy boy negative and I've been very stressed lately I slept with a guy a month ago without a condom I always protect my self but this time he took the condom out without me noticing I became so scared knowing my status and I can't live with my self knowing that I infected one another person I told him that he should take p.e.p anti virus he said no he is clean there is no need unless I know I'm not clean I was so scared not ready to tell him following day took me to his doctor for me to come he is clean he tested in front of me n I fogged my result sent him someone else's result with my name I regret it and I'm scared don't know how to tell him I'm scared can't live with my self knowing that I did what I did after protecting all the guys I've been with for so long it;s about a day for me to tell him that I HIV positive that i came across testimony of a lady been cured of HIV from Dr James herbal mix, for me been so desperate I picked up interest and contacted Dr James and told him my problems and he asked me some few questions and then said I should send him money some so he can send me his powerful herbal mixed medicine and I did,2 days later he courier the herbal mixed medicine to me through DHL speed post the medicine got to me in 5 days time and I used it morning and night as he prescribed for me for 3 weeks and I was cured, I couldn't believe it because it was like a magic to me
ReplyDeletethen I was bold enough to meet the guy and told him that I had HIV but lucky am cured now, I recommended him to Dr James who cured him and he was cured too today we have been in a good relationship and we have 2years old son now and he is HIV negative Thanks to Dr James herbal mix if you are HIV positive or suffering from
CANCER,
HERPES,
DIABETES,
HEPATITIS B,
PID Virus. contact Dr James on
Email drjamesherbalmix@gmail.com
What's app +2348152855846
This is very powerful I ve been diagnosed with HIV 3years ago and I've become in denial at first for a year but later I accepted it and I'm living with depression so I struggle to keep my cd4 up bit I'm very healthy and I just checked my cd4 was 460 and they advising me to being medication but I'm not ready I wanna take them when at least it 350 I'm only 24years old I have a 6year old healthy boy negative and I've been very stressed lately I slept with a guy a month ago without a condom I always protect my self but this time he took the condom out without me noticing I became so scared knowing my status and I can't live with my self knowing that I infected one another person I told him that he should take p.e.p anti virus he said no he is clean there is no need unless I know I'm not clean I was so scared not ready to tell him following day took me to his doctor for me to come he is clean he tested in front of me n I fogged my result sent him someone else's result with my name I regret it and I'm scared don't know how to tell him I'm scared can't live with my self knowing that I did what I did after protecting all the guys I've been with for so long it;s about a day for me to tell him that I HIV positive that i came across testimony of a lady been cured of HIV from Dr James herbal mix, for me been so desperate I picked up interest and contacted Dr James and told him my problems and he asked me some few questions and then said I should send him money some so he can send me his powerful herbal mixed medicine and I did,2 days later he courier the herbal mixed medicine to me through DHL speed post the medicine got to me in 5 days time and I used it morning and night as he prescribed for me for 3 weeks and I was cured, I couldn't believe it because it was like a magic to me
ReplyDeletethen I was bold enough to meet the guy and told him that I had HIV but lucky am cured now, I recommended him to Dr James who cured him and he was cured too today we have been in a good relationship and we have 2years old son now and he is HIV negative Thanks to Dr James herbal mix if you are HIV positive or suffering from
CANCER
HERPES
DIABETES
HEPATITIS B
PID Virus. contact Dr James on
Email drjamesherbalmix@gmail.com
What's app +2348152855846