Download Selective toxicity of antibiotics

Lecture 3
Influence of Environment factors on Microbes.
Effect of Physical Factors.
Effect of chemical factors.
Relation to temperature.
M.o. can withstand low temperatures fairly well.
(Cholera vibrio does not lose its viability at –32 C
Diphteria are able to withstand freezing for 3 months
Bacillus spores withstand at –253 for 3 days.)
The viruses are especially resistant to low temperature: -50_-70 for years.
Only certain species of pathogenic bacteria are very sensetive to low temperature
(meningococcus, gonococcus).
On the contrary - Most part of pathogenic microbes are very sensetive to high
temperature and many asporogenicfrom them perish at 60 C within 30-60 min.
Dry heating can kills the spores at 160 C, and steam pressure kills them at 120 C.
Acordingly the relation to temperature all microbes can be divided on some groups.
Figure 3.1.
Psychrophiles – optimum below 20 C, sometimes below 0 C; they are
commonly found in Arctic and Antarctic environments and occasionally in household
refrigirators, where they are important agents of food spoilage. The limit may simply
be the freezing temperature of the cell and its environment – liquid water is required
for metabolic function. In an Antarctic pond kept from freezing by its high CaCl2
content, active metabolism of microorganisms has been observed to about –10 C.
Mesophiles – have optimal growth temperatures between 20 and 45 C; all of
the normal resident bacteria of the human body; some bacteria are restricted to grow
near optimal temperature, others can grow over a range of temperatures; most human
pathogens are mesophiles and thus are able to grow rapidly and establish an infection
within the human body at 37 C.
Thermophiles – optimal growth temperatures above 40 C (Ex. Bacillus
stearothermophilus 55 to 70 C).
Extreme thermophiles ( hyperthermophiles) – above 80C (Ex. bacteria grow
within streaming effluents of hot springs, areas of volcanic activity etc. They have
caused the origin of life.
Relation to oxygen.
All microorganisms require elemental oxygen to build their biochemical
components, but not all of them require atmospheric oxygen. Most heterotrophic
bacteria obtain oxygen from the same molecule that serves as a carbon source
(CH2O).
Autotrophs obtain oxygen from CO2.
Most aerobic bacteria have an enzymes oxygenases that can add atmospheric
oxygen to organic molecules.
But oxygen itself is toxic for some bacteria (for example – destroyed
nitrogenase)
In addition many oxygen containing toxic compounds are produced inside the
cell during metabolism:
Hydrogen peroxide ( H2O2), superoxide (O2-), hydroxyl radical( OH).
All aerobes produce the enzyme superoxide dismutase, which destroys
superoxide. H2O2 can be destroyed by enzyme – catalase and peroxidase.
Bacteria that have such enzymes can survive in presence of free oxygen,
Bacteria which lack both of these enzymes quickly die in an oxygen- containing
environment. That is why, all microorganism can be divided accordingly their
relation to presence of oxygen.
Table.3.1.
Presence of
Microbial Class
Response to
Oxygen
Obligate aerobes Require
oxygen
Facultative
Can grow
anaerobes
with or
without
oxygen
Microaerophiles Grow best
with low
oxygen
Aerotolerant
Grow without
anaerobes
oxygen, but
not killed by it
Obligate
Killed by
Catalase
Present
Superoxide
Dismutase
Present
Example
Present
Present
Pseudomonas
aeruginosa
Escherichia coli
Present
Present
Campylobacter jejuni
Absent
Present
Streptococcus
pneumoniae
Absent
Absent
Methanococcus
anaerobes
oxygen
vannielii
Effect of light
Many pathogenic bacteria are very susceptible to different kinds of light.
UV rays(200-300nm), X-rays(0.005-2.0 nm), and can be used for sterilization.
Viruses are very quickly inactivated under uv-rays, but small doses of x-rays give the
multiplication .
Effect of desiccation
M.o. have a different resistance to desication.
Gonococci, meningococci, treponemas,leptospiras are very sensitive.
Cholera –2 days, dysentery – 7 days, diphtheria – 30, tuberculosis –90 days.
But in sputum tuberculosis bacteria survive for 10 month, and spores of anthrax more
than 10 years.
Effect of chemical factors.
Depending on the physicochemical composition of the medium, concentration,
the length of contact chemical substances have a different effect on microbes. In
small doses they act as stimulants. In bactericidal concentrations they paralyse the
dehydrogenase activity of bacteria.
According to their effect on bacteria, bactericidal chemical substances can be
subdivided into:
surface-active substances,
phenols and dyes,
salts of heavy metals,
oxidizing agents, and
the formaldehyde group.
Surface-active. Bacterial cells lose their negative charge and acquire a positive
charge which damages the normal function of the cytoplasmic membrane.
fatty acids and soaps
Phenols, (cresol). injure the cell wall and the cell proteins. Some substances of
this group inhibit the function of the coenzyme which participates in the
dehydrogenation of glucose and lactic acid.
Dyes are able to inhibit the growth of bacteria. Dyes with bactericidal
properties include brilliant green, rivanol, tripaflavine, acriflavine, etc.
Salts of heavy metals (lead, copper, zinc, silver, mercury)
cause coagulation of the cell proteins.
Some metals (silver, gold, copper, zinc, lead, etc.) have an oligodynamic action.
The mechanism: the positively charged metallic ions are adsorbed on the
negatively charged bacterial surface, and block the permeability of the cytoplasmic
membrane: the nutrition and reproduction of bacteria are disturbed.
Viruses also are sensitive to the salts of heavy metals – they become irreversibly
inactivated.
Oxidizing agents Chlorine, potassium permanganate, hydrogen peroxide
chloraminc act on the sulphohydryl groups of active proteins.
Oxidizing agents impairs dehydrogenases, hydrolases. amylases and proteinases
of bacteria used as disinfectants.
In medicine iodine is used successfully as an anti-microbial substance in the
form of iodine tincture which not only oxidizes the active groups of the proteins of
bacterial cytoplasm, but brings about their denaturation.
Many species of viruses are resistant to the action of ether, chloroform, ethyl
and methyl alcohol. Viruses are destroyed by sodium hydroxide, potassium
hydroxide, chloramine, chloride of lime, chlorine, and other oxidizing agents.
Formaldehyde is used as a 40 per cent solution known as formalin. It acts to the
amino groups of proteins which causes their denaturation.
Formaldehyde kills both the vegetative forms as well as the spores.
It is applied for decontaminating diphtheria and tetanus toxins as a result of
which they are transformed into anatoxins ( formol toxoids).
Some viruses inactivated by formalin can sometimes renew their infectivity.
Antiseptics is of great significance in medical practice.
Yet in 1865, Russian surgeon N. Pirogov pointed out the necessity of
destroying the source of intrahospital infection and tried chlorine water, silver nitrate,
iodine and other antiseptic substances in combating wound suppurations.
In 1867 J. Lister used phenol extensively as an antiseptic.
The science of antiseptics played a large role in the development of surgery. The
practical application of microbiology in surgery brought a decrease in the number of
postoperative complications, including gangrene, and considerably diminished the
death rate in surgical wards.
This trend received further development after E. Bergman and others who
introduced aseptics into surgical practice representing a whole system of measures
directed at preventing the access of microbes into wounds.
Aseptics is attained by desinfection of the air and equipment of the operating
room, by sterilization of surgical instruments and material, and by disinfecting the
hands of the surgeon and the skin on the operative field.
Film and plastic isolators are used in the clinic for protection against the
penetration of microorganisms. Soft surgical film isolators attached to the operative
field fully prevent bacteria from entering the surgical wound from the environment,
particularly from the upper respiratory passages of the personnel of the operating
room.
In decontaminating the environment from pathogenic microorganisms by
antagonism an important role is played by phages widespread in the soil and water
and by phytoncides, volatile substances of many plants.
The using of chemical substances for treating patients with infectious diseases ( and
in some cases for prophylaxis) is called chemiotherapy.
The basis of modern chemotherapy was founded by P. Ehrlich.
First drugs on the basis of Arsenicum:
Germanin (Ehrlich, 1990)
Salvarsan (Ehrlich, Bertheim, 1907)
Neosalvarsan (drug № 914) (Ehrlich, Bertheim, 1910)
Trypansamid (Jecobs, 1919)
Figure 3.2. P. Ehrlich.
Erlich developed main priciples of synthesis and application of chemical
preparations: methilene blue and salvarsan (arsenic derivates).
Main priciples are:
Chemopreparation should have:
- a specific action,
- a maximal therapeutic effect,
- minimal toxicity for the body.
Practical part
Isolation of anaerobic bacteria pure culture
Oxygen Requirements
Bacteria can be divided into several general groups on the basis of their oxygen
requirements. The first group, the strict aerobes, require free oxygen in order to grow.
Since our atmosphere is approximately 20 percent oxygen, growing them is no problem
as long as the bacteria are exposed to air.
The strict (obligate) anaerobes are organisms that not only will not grow in the
presence of free oxygen but may actually be killed by its presence. The mechanism of
this toxicity is discussed in Chapter 5. A number of techniques have been devised to
grow anaerobic bacteria. One common method is to add a reducing agent, such as
sodium thioglycolate, that will react with the free oxygen in the medium. In other cases,
special cultural equipment is employed to remove the oxygen mechanically and replace
the atmosphere with hydrogen and carbon dioxide (see Figure 3.3).
Figure 3.3 Petri dishes are placed inside the Gas-Pak jar ( Anaerostate) and
water is added to the GasPak envelope. This produces hydrogen and carbon dioxide.
The jar also contains palladium, which catalyzes the reaction of oxygen with hydrogen
to form water. The carbon dioxide generated is to support the growth of CO2-requiring
organisms.
A subgroup of the obligately anaerobic bacteria comprises those organisms that grow
best with reduced oxygen tension but not necessarily under obligately anaerobic
conditions. Such organisms are designated as microaerophilic, but no single
explanation for their oxygen requirement is available.
A large group of organisms is designated as facultative aerobes or facultative
anaerobes. These organisms can grow anaerobically, and under such conditions will
ferment carbohydrates to form stable fermentation products such as lactic acid, acetic
acid, and so forth. When they are grown in the presence of air, however, the facultative
organisms will change their metabolism to an aerobic one in which carbohydrates are
oxidized to water and carbon dioxide.
Finally, there is a group of bacteria referred to as aerotolerant because these
organisms will grow in the presence of air but do not possess an oxidative metabolism.
They do not use oxygen in their metabolism but carry out a fermentative degradation of
carbohydrates even in the presence of oxygen.
Lecture 4.
Influence of Environment factors on Microbes.
Effect of Biological Factors on microbes
In nature microorganisms constitute a component of the biocoenosis (a
community of plants and animals living in a part of the habitat with more or less
homogenous conditions of life - ecological niches).
Among various groups of microbes there are several types of relationships:
symbiosis, metabiosis, synergism, and antagonism.
Symbiosis represents an intimate mutually relation of organisms of different
species. They develop together better than separately. Sometimes the adaptation of
two organisms becomes so great, that they lose their ability to exist separately
(symbiosis of the fungus and blue-green algae, nitrogen-fixing bacteria and cellulosedecomposing bacteria, various fungi with the plants, yeast-like fungi and lamblias).
Metabiosis is that type of relationship in which one organism continues the
process caused by another organism (nitrifying and ammonizing bacteria).
Synergism is characterized by the increase in the physiological functions of the
microbial association (yeasts, lactobacilli, fusobacteria, and Borrelia).
During antagonistic relationships there is a struggle among microbes for
existence in associations (for oxygen, nutrients and a habitat). Certain species have
antagonistic properties in relation to other species
For example, lactic acid bacteria are antagonistic in relation to the causative
agents of dysentery, plague, etc.
Blue-pus bacteria inhibit the growth of dysentery, enteric fever microbes,
anthrax bacilli, cholera vibrio, causative agents of plague, and staphylococci,
meningococci. The normal inhabitants of the human body (e. g. colibacilli,
enterococci, lactobacilli, microflora of the skin and nasopharynx, etc.) have
especially potent antagonistic properties.
Biological factors have received widespread application in treating many
infectious diseases
Products of the life activities of bacteria, fungi, higher plants,
and animal tissues known as antibiotics.
In decontaminating the environment from pathogenic microorganisms by
antagonism an important role is played by phages widespread in the soil and water
and by phytoncides, volatile substances of many plants.
The using of chemical substances for treating patients with infectious diseases is
called chemiotherapy.
After discovery of antibiotics by A. Fleming (1929) they were included in definite
group of the chemiopreparations.
Figure 4.1. A. Fleming
In wide meaning chemipreparations can be subdevided as:
preparations which are produced by chemical synthesis: Arsenic, Bismuth, Mercury,
Acridine, Alcaloid and Sulphonamide preparations. Antimalarial and antiviral drugs.
And those which are obtained from biological objects - antibiotics.
Antibiotics
Antibiotics (Fr. anti against, bios life) are chemical substances excreted by some
microorganisms that inhibit the growth, or kill other microbes.
Antimicrobics include also agents, produced by animal and plant cells.
(in recent years some antibiotics have been obtained semisynthetically).
Ch. Darwin began scientific investigation into the problems of natural selection and
interspecies struggle. Antagonistic interrelations between microorganisms of various
species were first observed by L. Pasteur in 1887. He established that anthrax bacteria
die rapidly in mixed cultures with putrefying bacteria, and he characterized this
phenomenon as a struggle for existence.
The main causes for this antagonism may be competition for oxygen or nutrient
substances, the excretion into the cultural medium of acid or basic substances
inhibiting growth, and the accumulation of chemical substances with the help of
which some species of microbes inhibit the growth of others.
The essence of this phenomenon is that in the process of evolution of plant and
animal organisms the most varied and subtle adaptations were formed, which reflects
the general biological law of the struggle for existence. The latter, as E. Metchnikoff
pointed out, has a more universal nature, should be applied to microbes, and can be
used for treatment and prophylaxis of infectious diseases in animals and humans.
I. Metchnikoff is the founder of the study of antagonism in microbes and of the
practical use of this phenomenon. He employed lactic acid bacteria for inhibiting
harmful microflora of the intestine.
In 1871-72 V. Manassein and A. Polotebnov were the first to use the therapeutic
properties of fungi from the genus Penicillium. S. Vinogradsky observed the
phenomenon of antagonism in soil microbes. In 1904 M. Tartakovsky used a green
mould against the microbes which cause diseases in chickens.
In 1909 Lashchenkov and in 1922 A. Fleming isolated the enzyme lysozyme which
is capable of inhibiting a number of microorganisms.
However, the study of antibiotics began in 1929, when A. Fleming proved that the
filtrate of a broth culture of the fungus Penicillium notation has antibacterial
properties in relation to Gram-positive microorganisms. In 1940 E. Chain and H.
Florey obtained a relatively stable preparation of penicillin. In 1942 Z. Ermolyeva
prepared penicillin from Penicillium crustosum.
Further development of this problem is associated with the works of various
scientists: R. Dubos isolated gramicidin and tyrocidin from the cultural liquid of S.
brevis; S. Waksman and coworkers devised a method of producing streptomycin; B.
Tokin discovered antimicrobial substances from plants — phytoncides, and others
who enriched modern medical practice with numerous preparations widely used for
treating infectious diseases.
Antibiotics are obtained by special methods employed in the medical industry. For
the production of antibiotics strains of fungi, actinomycetes, and bacteria are used,
which are seeded in a nutrient substrate. After a definite growth period the antibiotic
is extracted, purified and concentrated, checked for innocuousness and potency of
action.
Classification
Produced by
1. Fungi (penicillin, cephalosporin, grizeofulvin)
2. Actinomycetes (tetracycline, streptomycin, kanamycin, neomycin, nystatin,
chloramphenicol, vancomycin, erythromycin, amphotericin
3. Bacteria (polymixin, gramicidin, bacitracin, gentamicin)
4. Animal ( lysozime, erithrin, eckmolin(from fishs)
5. Plant (phytoncides, hlorellin)
6. Semisynthetic or Synthetic
Chemical structure
1.Beta- lactamic ( penicillins, cephalosporines)
2. Glycopeptides ( vancomycine, teicoplanin)
3. Aminoglycosides ( streptomycine, gentamicine, kanamycin)
4.Tetracyclines ( tetracycline, doxycycline)
5.Macrolides ( erythromycine, clarithromycine)
Figure 4.2.
Mechanism of action
Mechanism of action
1.Cell wall synthesis inhibitors
penicillin, cephalosporin
2.Inhibitors membrane function
polymixin, nystatin, gramicidin
3.Protein synthesis inhibitors
chloramphenicol, neomycin, tetracyclines, erythromycin
4.DNA synthesis inhibitors
antitumour, rifampicin, quinolones:, ciprofloxacin, norfloxacin etc.
5.RNA synthesis inhibitors (actinomycin, kanamycin, neomycin)
6. Some other
Synthesis of antibiotics
Biological synthesis – producing strains and special nutrient media
1. Chemical synthesis
2. Combined synthesis
Spectrum of action
Figure 4.3.
Effects
- microbicidal
- microbiostatic
Selective toxicity of antibiotics
they must exhibit greater toxicity to the infecting pathogen than to the host organism
(Ex. -lactam effect on trans-peptidase)
Major side effects
1. Toxicity to mammalian cells
aminoglycosides (streptomycin)– deafness, vertigo, nephrotoxicity
amphotericin – nephrotoxicity
penicillin – myoclonic seizures
cephalosporin – vitamin K synthesis
chloramphenicol – anemia, cancerogenes
tetracyclines – hepatitis
2. Toxicity to normal microflora – dysbacteriosis
3. Allergical reactions – skin rash, itching, rhinitis, urticaria, seldom throat edema,
anaphylactic shock
4. Immunodepressive reactions – inhibition of antibody synthesis
5. Herz-Heiner phenomena - intoxication by microbial endotoxins
Resistance of microbes to antibiotics.
With the extensive use of antibiotics in medical practice, many species of
pathogenic micro-organisms became resistant to them.
Types of microbial antibiotics resistance
Innate
Acquired as a result :
1.
Mutations
2.
Recombinations
3.
Modifications
Mechanisms of microbial antibiotics resistance:
- decreased permeability of cell membrane to this drug
- structural changes in the target – decreased sensitivity
- production of enzymes that degrade antibiotics
- decreased transformation of the drug to its active form
Selection of antimicrobial agent
- sensitivity of pathogen to the particular antimicrobic
- side effects (toxicity to mammalian cell and normal microbiota)
- biotransformation of antimicrobic
- chemical properties of antimicrobic that determine its distribution within the body
Figure4.4.
Application of antibiotic
Treatment of human and animal diseases
1. Prophylactic
2. Microbiological diagnostic (isolation of pure culture)
3. Gene engineering, molecular cloning, biotechnology etc.
4. Agriculture (increase of cattle growth, conservation of products etc.)
Determination of antimicrobial susceptibility of a microorganisms.
Bauer-Kirby diffusion test (inhibition zones around antibiotic
impregnated disks on agar surface, inoculated with strain of interest)
Figure 4.5
- Minimum Inhibitory Concentration test (dilutions of antimicrobic in
liquid media to determine the lowest concentration (MIC) that is
effective in preventing the growth of the pathogen)
- Minimum Bactericidal Concentration test (MBC) (“no growth” tube
suspensions from MIC test are plated on agar to determine the presence
and number of survived this specific exposure to antibiotic
microorganisms)