1

THE CONTROL OF MICROBIAL GROWTH Introduction The control of microbial growth is necessary in many practical situations, and significant advances in agriculture, medicine, and food science have been made through study of this area of microbiology. "Control of growth", as used here, means to prevent growth of microorganisms. This control is effected in two basic ways: (1) by killing microorganisms or (2) by inhibiting the growth of microorganisms. Control of growth usually involves the use of physical or chemical agents which either kill or prevent the growth of microorganisms. Agents which kill cells are called cidal agents; agents which inhibit the growth of cells (without killing them) are referred to as static agents. Thus the term bactericidal refers to killing bacteria and bacteriostatic refers to inhibiting the growth of bacterial cells. A bactericide kills bacteria, a fungicide kills fungi, and so on. Sterilization is the complete destruction or elimination of all viable organisms (in or on an object being sterilized). There are no degrees of sterilization: an object is either sterile or not. Sterilization procedures involve the use of heat, radiation or chemicals, or physical removal of cells. Moist heat sterilization is the most efficient biocidal agent. In the pharmaceutical industry it is used for: Surgical dressings, Sheets, Surgical and diagnostic equipment, Containers, Closures, Aqueous injections, Ophthalmic preparations and Irrigation fluids etc. Dry heat sterilization can only be used for thermo stable, moisture sensitive or moisture impermeable pharmaceutical and medicinal. These include products

like; Dry powdered drugs, Suspensions of drug in non aqueous solvents, Oils, fats waxes, soft hard paraffin silicone, Oily injections, implants, ophthalmic ointments and ointment bases etc. Gaseous sterilization is used for sterilizing thermolabile substances like; hormones, proteins, various heat sensitive drugs etc. U.V light is perhaps the most lethal component in ordinary sunlight used in sanitation of garments or utensils. Gamma-rays from Cobalt 60 are used to sterilize antibiotic, hormones, sutures, plastics and catheters etc. Filtration sterilizations are used in the treatment of heat sensitive injections , biological products, air and other gases for supply to aseptic areas. They are also used in industry as part of the venting systems on fermentors, centrifuges, autoclaves and freeze driers. Membrane filters are used for sterility testing. Terms commonly used Survivor curves They are plots of the logarithm of the fraction of survivors (microorganisms which retain viability following a sterilization process) against the exposure time or dose. D-value D-value is indicative of the resistance of any organism to a sterilizing agent. For radiation and heat treatment, D-value is the time taken at a fixed temperature or the radiation dose required to achieve a 90% reduction in viable count. Z-value Z-value represents the increase in temperature needed to reduce the D-value of an organism by 90%. Methods of Sterilization

The various methods of sterilization are: 1. Physical Method a. Thermal (Heat) methods b. Radiation method c. Filtration method 2. Chemical Method 3. Gaseous method 1. Heat Sterilization Heat sterilization is the most widely used and reliable method of sterilization, involving destruction of enzymes and other essential cell constituents. The process is more effective in hydrated state where under conditions of high humidity, hydrolysis and denaturation occur, thus lower heat input is required. Under dry state, oxidative changes take place, and higher heat input is required. This method of sterilization can be applied only to the thermostable products, but it can be used for moisture-sensitive materials for which dry heat (160-1800C) sterilization, and for moisture-resistant materials for which moist heat (1211340C) sterilization is used. The efficiency with which heat is able to inactivate microorganisms is dependent upon the degree of heat, the exposure time and the presence of water. The action of heat will be due to induction of lethal chemical events mediated through the action of water and oxygen. In the presence of water much lower temperature time exposures are required to kill microbe than in the absence of water. In this processes both dry and moist heat are used for sterilization. a.Dry Heat Sterilization: Examples of Dry heat sterilization are: 1. Incineration 2. Red heat

3. Flaming 4. Hot air oven It employs higher temperatures in the range of 160-1800C and requires exposures time up to 2 hours, depending upon the temperature employed. The benefit of dry heat includes good penetrability and non-corrosive nature which makes it applicable for sterilizing glasswares and metal surgical instruments. It is also used for sterilizing non-aqueous thermostable liquids and thermostable powders. Dry heat destroys bacterial endotoxins (or pyrogens) which are difficult to eliminate by other means and this property makes it applicable for sterilizing glass bottles which are to be filled aseptically. Boiling: 100o for 30 minutes. Kills everything except some endospores (Actually, for the purposes of purifying drinking water 100o for five minutes is probably adequate though there have been some reports that Giardia cysts can survive this process). To kill endospores, and therefore sterilize the solution, very long or intermittent boiling is required. Hot-air oven Dry heat sterilization is usually carried out in a hot air oven, which consists of the following: i) An insulated chamber surrounded by an outer case containing electric heaters. ii) A fan iii) Shelves iv) Thermocouples v) Temperature sensor vi) Door locking controls.

Operation i) Articles to be sterilized are first wrapped or enclosed in containers of cardboard, paper or aluminum. ii) Then, the materials are arranged to ensure uninterrupted air flow. iii) Oven may be pre-heated for materials with poor heat conductivity. iv) The temperature is allowed to fall to 400C, prior to removal of sterilized material. b. Moist Heat Sterilization: Moist heat may be used in three forms to achieve microbial inactivation 1. Dry saturated steam Autoclaving 2. Boiling water/ steam at atmospheric pressure 3. Hot water below boiling point Moist heat sterilization involves the use of steam in the range of 121-1340C. Steam under pressure is used to generate high temperature needed for sterilization. Saturated steam (steam in thermal equilibrium with water from which it is derived) acts as an effective sterilizing agent. Steam for sterilization can be either wet saturated steam (containing entrained water droplets) or dry saturated steam (no entrained water droplets). Autoclaves use pressurized steam to destroy microorganisms, and are the most dependable systems available for the decontamination of laboratory waste and the sterilization of laboratory glassware, media, and reagents. For efficient heat transfer, steam must flush the air out of the autoclave chamber. Before using the autoclave, check the drain screen at the bottom of the chamber and clean if blocked. If the sieve is blocked with debris, a layer of air may form at the bottom of the autoclave, preventing efficient operation. Autoclaves should be tested periodically with biological indicators like cultures of Bacillus

stearothermophilus to ensure proper function. This method of sterilization works well for many metal and glass items but is not acceptable for rubber, plastics, and equipment that would be damaged by high temperatures Autoclaves, or steam sterilizers essentially consist of following:

i) A cylindrical or rectangular chamber, with capacities ranging from 400 to 800 liters. ii) Water heating system or steam generating system iii) Steam outlet and inlet valves iv) Single or double doors with locking mechanism. v) Thermometer or temperature gauge vi) Pressure gauges Operation

For porous loads (dressings) sterilizers are generally operated at a minimum temperature of 1340C, and for bottled fluid, sterilizers employing a minimum temperature of 1210C are used. Ensure that there should be sufficient water in the autoclave to produce the steam. The stages of operation of autoclaves include air removal, steam admission and sterilization cycle (includes heating up, holding/exposure, and cooling stages). Gaseous Sterilization The chemically reactive gases such as formaldehyde, methanol, and ethylene oxide (CH2)2O possess biocidal activity. Ethylene oxide is a colorless, odorless, and flammable gas. The mechanism of antimicrobial action of the two gases is assumed to be through alkylations of sulphydryl, amino, hydroxyl and carboxyl groups on proteins and amino groups of nucleic acids. The concentration ranges (weight of gas per unit chamber volume) are usually in range of 800-1200 mg/L for ethylene oxide and 15-100 mg/L for formaldehyde with operating temperatures of 45-63C and 7075C respectively. Both of these gases being alkylating agents are potentially mutagenic and carcinogenic. They also produce acute toxicity including irritation of the skin, conjunctiva and nasal mucosa. a. Ethylene oxide sterilizer: An ethylene oxide sterilizer consists of a chamber of 100-300-Litre capacity and surrounded by a water jacket. Air is removed from sterilizer by evacuation, humidification and conditioning of the load is done by passing sub-atmospheric pressure steam, then evacuation is done again and preheated vaporized ethylene oxide is passed. After treatment, the gases are evacuated either directly to the outside atmosphere or through a special exhaust system.

Ethylene oxide gas has been used widely to process heat-sensitive devices, but the aeration times needed at the end of the cycle to eliminate the gas made this method slow. b. Low temperature steam formaldehyde (LTSF) sterilizer: An LTSF sterilizer operates with sub atmospheric pressure steam. At first, air is removed by evacuation and steam is admitted to the chamber. Liquid Sterilization a. Peracetic Acid liquid sterilization: Peracetic acid was found to be sporicidal at low concentrations. It was also found to be water soluble, and left no residue after rinsing. It was also shown to have no harmful health or environmental effects. It disrupts bonds in proteins and enzymes and may also interfere with cell membrane transportation through the rupture of cell walls and may oxidize essential enzymes and impair vital biochemical pathways. In a low-temperature liquid chemical sterile processing system, several steps must be followed for effective sterilization: 1. Pre-cleaning of the devices is necessary 2. Leak testing is done to ensure there are no leaks that could allow fluid to enter/leak the ampoules/vials and cause damage. 3. The appropriate tray/container must then be selected, and if the device has lumens, the appropriate connector attached. 4. The sterilant concentrate is provided in a sealed single- use cup and requires no pre-mixing or dilution. The disadvantages of this method of sterilization are that the devices must be immersible, must fit in the appropriate tray, and must be able to withstand the 55C temperature the process uses. b. b. Hydrogen Peroxide Sterilization: This method disperses a hydrogen peroxide solution in a vacuum chambe. This agent sterilizes by oxidizing key

cellular components, which inactivates the microorganisms. The temperature of this sterilization method is maintained in the 40-50C range, which makes it particularly well-suited for use with heat-sensitive and moisture-sensitive medical devices. The instruments are wrapped prior to sterilization, and can either be stored or used immediately. Radiation Sterilization Many types of radiation are used for sterilization like electromagnetic radiation (e.g. gamma rays and UV light), particulate radiation (e.g. accelerated electrons).The major target for these radiation is microbial DNA. Gamma rays and electrons cause ionization and free radical production while UV light causes excitation. Radiation sterilization with high energy gamma rays or accelerated electrons has proven to be a useful method for the industrial sterilization of heat sensitive products. But some undesirable changes occur in irradiated products, an example is aqueous solution where radiolysis of water occurs. Radiation sterilization is generally applied to articles in the dry state; including surgical instruments, sutures, prostheses, unit dose ointments, plastic syringes and dry pharmaceutical products. UV light, with its much lower energy, and poor penetrability finds uses in the sterilization of air, for surface sterilization of aseptic work areas, for treatment of manufacturing grade water, but is not suitable for sterilization of pharmaceutical dosage forms. a. Gamma ray Sterilizer: Gamma rays for sterilization are usually derived from cobalt-60 source, the isotope is held as pellets packed in metal rods, each rod carefully arranged within the source and containing 20 KCi of activity. This source is housed within a reinforced concrete building with 2 m thick walls. Articles being sterilized are passed through the irradiation chamber on a conveyor belt and move around the raised source.

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Ultraviolet Irradiation: The optimum wavelength for UV sterilization is 260 nm. A mercury lamp giving peak emission at 254 nm is the suitable source of UV light in this region. Electron Accelerator A high energy electron beam is generated by accelerating electrons from a hot filament down an evacuated tube under high potential difference. Articles to be sterilized are arranged on a horizontal conveyor belt and are irradiated from one or both sides. Filtration Sterilization Filtration process does not destroy but removes the microorganisms. It is used for both the clarification and sterilization of liquids and gases as it is capable of preventing the passage of both viable and non viable particles. The major mechanisms of filtration are sieving, adsorption and trapping within the matrix of the filter material. Sterilizing grade filters are used in the treatment of heat sensitive injections and ophthalmic solutions, biological products and air and other gases for supply to aseptic areas. They are also used in industry as part of the venting systems on fermentors, centrifuges, autoclaves and freeze driers. Membrane filters are used for sterility testing. Application of filtration for sterilization of gases: HEPA (High efficiency particulate air) filters can remove up to 99.97% of particles >0.3 micrometer in diameter. Air is first passed through prefilters to remove larger particles and then passed through HEPA filters. The performance of HEPA filter is monitored by pressure differential and airflow rate measurements. There are two types of filters used in filtration sterilization (a) Depth filters: Consist of fibrous or granular materials so packed as to form twisted channels of minute dimensions. They are made of diatomaceous earth, unglazed porcelain filter, sintered glass or asbestos.

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(b) Membrane filters: These are porous membrane about 0.1 mm thick, made of cellulose acetate, cellulose nitrate, polycarbonate, and polyvinylidene fluoride, or some other synthetic material.The membranes are supported on a frame and held in special holders. Fluids are made to transverse membranes by positive or negative pressure or by centrifugation. Application of filtration for sterilization of liquids: Membrane filters of 0.22 micrometer nominal pore diameter are generally used, but sintered filters are used for corrosive liquids, viscous fluids and organic solvents. The factors which affects the performance of filter is the titre reduction value, which is the ratio of the number of organism challenging the filter under defined conditions to the number of organism penetrating it. The other factors are the depth of the membrane, its charge and the tortuosity of the channels. Pasteurization is the use of mild heat to reduce the number of microorganisms in a product or food. In the case of pasteurization of milk the time and temperature depend on killing potential pathogens that are transmitted in milk, i.e., staphylococci, streptococci, Brucella abortus and Mycobacterium tuberculosis. For pasteurzation of milk: batch nethod: 63o/30minutes; flash method: 71o/15 seconds. Low temperature (refrigeration and freezing): Most organisms grow very little or not at all at 0o. Store perishable foods at low temperatues to slow rate of growth and consequent spoilage (e.g. milk). Low temperatures are not bactericidal. Psychrotrophs, rather than true psychrophiles, are the usual cause of food spoilage in refrigerated foods. Drying (removal of H2O): Most microorganisms cannot grow at reduced water activity (Aw < 0.90). Often used to preserve foods (e.g. fruits, grains, etc.).

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Methods involve removal of water from product by heat, evaporation, freezedrying, addition of salt or sugar.

Table 1: Merits, Demerits and Applications of Different Methods of Sterilization Methods Heat Mechanism Merits Destroys endotoxins Most Demerits Can Applicatio ns be Dry heat is applicable

sterilization bacterial

widely used applied method

and reliable only to the for of thermostabl sterilizing glasswares and metal surgical instruments and most dependable method for decontamin ation laboratory waste the sterilization and of moist heat is the sterilization e products , involving destruction of enzymes and cell constituents . other essential

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of laboratory glassware, media, and Gaseous sterilization Alkylation Penetrating ability gases reagents. Gases being Ethylene oxide gas been process agents are has to

of alkylating potentially mutagenic and

used widely heat-

carcinogeni sensitive Radiation Ionization acids c devices. It is a useful Undesirable Radiation sterilization in is generally applied to in dry the industrial of occur irradiated

sterilization of

nucleic method for changes

sterilization products,an articles heat example is the aqueous solution where radiolysis of occurs. state; including surgical sensitive products.

instruments, prostheses, unit plastics dose ointments,

water sutures,

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Filtration

Does

not It is used Does

not This is Sterilizing and grade filters viable are used in the treatment of heat sensitive injections and ophthalmic solutions, biological products and air and other gases for to areas. supply aseptic

sterilization destroy but for both the differentiate method removes the clarification between microorgani and sms of viable liquids particles sterilization non and gases as it is capable of preventing the passage of viable non both and viable

particles.

. Recommended use of heat to control bacterial growth Treatment Incineration Boiling Temperature >500o 100
o

Effectiveness Vaporizes organic

material

on

nonflammable surfaces but may destroy many substances in the process 30 minutes of boiling kills microbial

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pathogens

and

vegetative

forms

of

bacteria but may not kill bacterial endospores Three 30-minute intervals of boiling, Intermittent boiling Autoclave pressure (steam pressure) Dry heat (hot air oven) and under at 15# pressure 100o followed by periods of cooling kills bacterial endospores kills all forms of life including bacterial The substance being sterilized must be maintained at the effective T for the full time For materials that must remain dry and 160o/2 hours which are not destroyed at T between 121o and 170o Good for glassware, metal, not plastic or rubber items Same as above. Note increasing T by 10 170o/1 hour degrees shortens the sterilizing time by 50 percent kills most vegetative bacterial cells 63 /30 minutes
o

cooker 121o/15 minutes endospores.

Dry heat (hot air oven)

Pasteurization (batch method)

including pathogens such as streptococci, staphylococci and Mycobacterium tuberculosis Effect on bacterial cells similar to batch

Pasteurization (flash method)

72 /15 seconds

method; for milk, this method is more conducive to industry and has fewer undesirable effects on quality or taste

Control of microbial growth by chemical agents Antimicrobial agents are chemicals that kill or inhibit the growth microorganisms. Antimicrobial agents include chemical preservatives and

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antiseptics, as well as drugs used in the treatment of infectious diseases of plants and animals. Antimicrobial agents may be of natural or synthetic origin, and they may have a static or cidal effect on microorganisms. Types of antimicrobial agents Antiseptics: microbicidal agents harmless enough to be applied to the skin and mucous membrane; should not be taken internaslly. Examples: mercurials, silver nitrate, iodine solution, alcohols, detergents. Disinfectants: Agents that kill microorganisms, but not necessarily their spores,not safe for application to living tissues; they are used on inanimate objects such as tables, floors, utensils, etc. Examples: chlorine, hypochlorites, chlorine compounds, lye, copper sulfate, quaternary ammonium compounds. Note: disinfectants and antiseptics are distinguished on the basis of whether they are safe for application to mucous membranes. Often, safety depends on the concentration of the compound. For example, sodium hypochlorite (chlorine), as added to water is safe for drinking, but "chlorox" (5% hypochlorite), an excellent disinfectant, is hardly safe to drink. Common antiseptics and disinfectants and their uses are summarized in Table 2. Table 2. Common antiseptics and disinfectants Chemical Ethanol (50-70%) Isopropanol (50-70%) Formaldehyde (8%) Action Denatures Uses proteins Antiseptic used on skin

and solubilizes lipids Denatures proteins

Antiseptic used on skin and solubilizes lipids Reacts with NH2, SH Disinfectant, kills

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and COOH groups Tincture of Iodine (2% I2 in 70% alcohol) Chlorine (Cl2) gas Inactivates proteins Forms hypochlorous

endospores Antiseptic used on skin Disinfect drinking water; general disinfectant General antiseptic and used

acid (HClO), a strong oxidizing agent

Silver nitrate (AgNO3)

Precipitates proteins

in the eyes of newborns Inactivates proteins by Disinfectant, although reacting with sulfide occasionally used as an antiseptic on skin cell Skin antiseptics

Mercuric chloride Detergents (e.g.

groups quaternary Disrupts

and

ammonium compounds) membranes disinfectants Phenolic compounds (e.g. Antiseptics at low Denature proteins and carboloic acid, lysol, concentrations; disrupt cell hexylresorcinol, disinfectants at high membranes hexachlorophene) concentrations Disinfectant used to Ethylene oxide gas Alkylating agent sterilize plastics Preservatives: static agents used to inhibit the growth of microorganisms, most often in foods. If eaten they should be nontoxic. Examples; calcium propionate, sodium benzoate, formaldehyde, nitrate, sulfur dioxide. Table 3 is a list of common preservative and their uses. Table 3. Common food preservatives and their uses Preservative Effective Uses heat-sensitive objects such as rubber and

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Concentration Propionic acid and propionates Sorbic acid sorbates Benzoic acid and and 0.32% 0.2% 0.1% 0.32% unknown dioxide, 200-300 ppm 200 ppm unknown unknown unknown Antifungal agent in breads, cake, Swiss cheeses Antifungal agent in cheeses, jellies, syrups, cakes Antifungal agent in margarine, cider, relishes, soft drinks Antifungal agent in breads Antimicrobial agent in

benzoates Sodium diacetate Lactic acid Sulfur

cheeses,

buttermilk, yogurt and pickled foods Antimicrobial agent in dried fruits, grapes, molasses Antibacterial agent in cured meats, fish Prevents microbial spoilage of meats, fish, etc. Prevents microbial spoilage of

sulfites Sodium nitrite Sodium chloride Sugar Wood smoke

preserves, jams, syrups, jellies, etc. Prevents microbial spoilage of meats, fish, etc.

Chemotherapeutic agents: antimicrobial agents of synthetic origin useful in the treatment of microbial or viral disease. Antibiotics: antimicrobial agents produced by microorganisms that kill or inhibit other microorganisms. This is the microbiologist's definition. A more broadened definition of an antibiotic includes any chemical of natural origin (from any type of cell) which has the effect to kill or inhibit the growth of other types cells. Since most clinically-useful antibiotics are produced by microorganisms and are used to kill or inhibit infectious Bacteria, we will follow the classic definition.

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Antibiotics are low molecular-weight (non-protein) molecules produced as secondary metabolites, mainly by microorganisms that live in the soil. Most of these microorganisms form some type of a spore or other dormant cell, and there is thought to be some relationship (besides temporal) between antibiotic production and the processes of sporulation. Among the molds, the notable antibiotic producers are Penicillium and Cephalosporium , which are the main source of the beta-lactam antibiotics (penicillin and its relatives). In the Bacteria, the Actinomycetes, notably Streptomyces species, produce a variety of types of antibiotics including the aminoglycosides (e.g. streptomycin), macrolides (e.g. erythromycin), and the tetracyclines. Endospore-forming Bacillus species produce polypeptide antibiotics such as polymyxin and bacitracin. The table below (Table 4) is a summary of the classes of antibiotics and their properties including their biological sources. Table 4. Classes of antibiotics and their properties Biological source Spectrum (effective against) Mode action Inhibits Beta-lactams (penicillins and cephalosporins) Penicillium Penicillin Cephalothin G, notatum species Ampicillin, Amoxycillin Cephalosporium bacteria in cell steps wall of

Chemical class Examples

and Gram-positive (peptidoglycan ) synthesis and murein assembly Gram-positive Inhibits steps and negative Gram- in cell wall (peptidoglycan

Semisynthetic penicillin

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) synthesis and bacteria Clavamox Clavulanic Acid plus amoxycillin is clavuligerus murein assembly Gram-positive Suicide and negative bacteria Gram- inhibitor betalactamases Inhibits steps cell wall Gram- (peptidoglycan ) synthesis and murein assembly Inhibits steps Gram-positive in Carboxypenems Imipenem Streptomyces cattleya and negative bacteria cell wall Gram- (peptidoglycan ) synthesis and murein of

clavulanic acid Streptomyces

Gram-positive in Monobactams Aztreonam Chromobacter and violaceum negative bacteria

assembly Gram-positive Inhibit Aminoglycoside s Streptomycin Streptomyces griseus and negative Gram- translation (protein

Gentamicin

bacteria synthesis) Gram-positive Inhibit and GramMicromonospor translation negative a species (protein bacteria esp. synthesis) Pseudomonas

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Gram-positive Glycopeptides Vancomycin Streptomyces orientales bacteria, s aureus Gram-positive and Lincomycins Clindamycin Streptomyces lincolnensis negative bacteria anaerobic Bacteroides Gram-positive bacteria, esp. Staphylococcu

Inhibits in

steps murein

(peptidoglycan ) biosynthesis and assembly

Gram- Inhibits translation esp. (protein synthesis)

Gram-negative Inhibits Macrolides Erythromycin Streptomyces erythreus bacteria enterics, Neisseria, Legionella, Mycoplasma Polypeptides Polymyxin Bacillus polymyxa Gram-negative bacteria Damages cytoplasmic membranes Inhibits steps in murein (peptidoglycan ) biosynthesis and assembly Inactivate not translation (protein synthesis)

Bacitracin

Bacillus subtilis

Gram-positive bacteria

Polyenes

Amphotericin

Streptomyces

Fungi

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membranes nodosus containing sterols Inactivate Nystatin Streptomyces noursei Fungi (Candida) Gram-positive and Rifamycins Rifampicin Streptomyces mediterranei negative bacteria, Mycobacteriu m tuberculosis Gram-positive Tetracyclines Tetracycline Streptomyces species and negative bacteria, Rickettsias Gram-positive and Semisynthetic tetracycline negative Doxycycline bacteria, Rickettsias Ehrlichia, Chloramphenico Chloramphenico Streptomyces l l venezuelae GramGramGrammembranes containing sterols Inhibits transcription (eubacterial RNA polymerase) Inhibit translation (protein synthesis)

Inhibit translation (protein synthesis)

Borellia Gram-positive Inhibits and negative Gram- translation (protein

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bacteria

synthesis)

Kinds of Antimicrobial Agents and their Primary Modes of Action 1. Cell wall synthesis inhibitors Cell wall synthesis inhibitors generally inhibit some step in the synthesis of bacterial peptidoglycan. Generally they exert their selective toxicity against eubacteria because human cells lack cell walls. Beta lactam antibiotics Chemically, these antibiotics contain a 4-membered beta lactam ring. They are the products of two groups of fungi, Penicillium and Cephalosporium molds, and are correspondingly represented by the penicillins and cephalosporins. The beta lactam antibiotics inhibit the last step in peptidoglycan synthesis, the final cross-linking between between peptide side chains, mediated by bacterial carboxypeptidase and transpeptidase enzymes . Beta lactam antibiotics are normally bactericidal and require that cells be actively growing in order to exert their toxicity. Natural penicillins, such as Penicillin G or Penicillin V, are produced by fermentation of Penicillium chrysogenum. They are effective against streptococcus, gonococcus and staphylococcus, except where resistance has developed. They are considered narrow spectrum since they are not effective against Gram-negative rods. Semisynthetic penicillins first appeared in 1959. A mold produces the main part oif the molecule (6-aminopenicillanic acid) which can be modified chemically by the addition of side shains. Many of these compounds have been developed to have distinct benefits or advantages over penicillin G, such as increased spectrum of activity (effectiveness against Gram-negative rods), resistance to penicillinase, effectiveness when administered orally, etc. Amoxycillin and Ampicillin have

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broadened spectra against Gram-negatives and are effective orally; Methicillin is penicillinase-resistant. Clavulanic acid is a chemical sometimes added to a semisynthetic penicillin preparation. Thus, amoxycillin plus clavulanate is clavamox or augmentin. The clavulanate is not an antimicrobial agent. It inhibits beta lactamase enzymes and has given extended life to penicillinase-sensitive beta lactams. Although nontoxic, penicillins occasionally cause death when administered to persons who are allergic to them. In the U.S. there are 300 - 500 deaths annually due to penicillin allergy. In allergic individuals the beta lactam molecule attaches to a serum protein which initiates an IgE-mediated inflammatory response. Cephalolsporins are beta lactam antibiotics with a similar mode of action to penicillins that are produced by species of Cephalosporium. The have a low toxicity and a somewhat broader spectrum than natural penicillins. They are often used as penicillin substitutes, against Gram-negative bacteria, and in surgical prophylaxis. They are subject to degradation by some bacterial betalactamases, but they tend to be resistant to beta-lactamases from S. aureus . Bacitracin is a polypeptide antibiotic produced by Bacillus species. It prevents cell wall growth by inhibiting the release of the muropeptide subunits of peptidoglycan from the lipid carrier molecule that carries the subunit to the outside of the membrane Teichoic acid synthesis, which requires the same carrier, is also inhibited. Bacitracin has a high toxicity which precludes its systemic use. It is present in many topical antibiotic preparations, and since it is not absorbed by the gut, it is given to "sterilize" the bowel prior to surgery. 2. Cell membrane inhibitors disorganize the structure or inhibit the function of bacterial membranes. The integrity of the cytoplasmic and outer membranes is

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vital to bacteria, and compounds that disorganize the membranes rapidly kill the cells. However, due to the similarities in phospholipids in eubacterial and eukaryotic membranes, this action is rarely specific enough to permit these compounds to be used systemically. The only antibacterial antibiotic of clinical importance that acts by this mechanism is Polymyxin, produced by Bacillus polymyxis. Polymyxin is effective mainly against Gram-negative bacteria and is usually limited to topical usage. Polymyxins bind to membrane phospholipids and thereby interfere with membrane function. Polymyxin is occasionally given for urinary tract infections caused by Pseudomonas that are gentamicin, carbenicillin and tobramycin resistant. The balance between effectiveness and damage to the kidney and other organs is dangerously close, and the drug should only be given under close supervision in the hospital. 3. Protein synthesis inhibitors Many therapeutically useful antibiotics owe their action to inhibition of some step in the complex process of translation. Their attack is always at one of the events occurring on the ribosome and rather than the stage of amino acid activation or attachment to a particular tRNA. Most have an affinity or specificity for 70S (as opposed to 80S) ribosomes, and they achieve their selective toxicity in this manner. The most important antibiotics with this mode of action are the tetracyclines, chloramphenicol, the macrolides (e.g. erythromycin) and the aminoglycosides (e.g. streptomycin). The aminoglycosides are products of Streptomyces species and are represented by streptomycin, kanamycin, tobramycin and gentamicin. These antibiotics exert their activity by binding to bacterial ribosomes and preventing the initiation of protein synthesis. Aminoglycosides have been used against a wide variety of bacterial infections caused by Gram-positive and Gram-negative bacteria. Streptomycin has been used extensively as a primary drug in the treatment of

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tuberculosis. Gentamicin is active against many strains of Gram-positive and Gram-negative bacteria, including some strains of Pseudomonas aeruginosa. Kanamycin (a complex of three antibiotics, A, B and C) is active at low concentrations against many Gram-positive bacteria, including penicillinresistant staphylococci. Gentamicin and Tobramycin are mainstays for treatment of Pseudomonas infections. An unfortunate side effect of aminoglycosides has tended to restrict their usage: prolonged use is known to impair kidney function and cause damage to the auditory nerves leading to deafness. The tetracyclines consist of eight related antibiotics which are all natural products of Streptomyces, although some can now be produced semisynthetically. Tetracycline, chlortetracycline and doxycycline are the best known. The tetracyclines are broad-spectrum antibiotics with a wide range of activity against both Gram-positive and Gram-negative bacteria. The tetracyclines act by blocking the binding of aminoacyl tRNA to the A site on the ribosome. Tetracyclines inhibit protein synthesis on isolated 70S or 80S (eukaryotic) ribosomes, and in both cases, their effect is on the small ribosomal subunit. However, most bacteria possess an active transport system for tetracycline that will allow intracellular accumulation of the antibiotic at concentrations 50 times as great as that in the medium. This greatly enhances its antibacterial effectiveness and accounts for its specificity of action, since an effective concentration cannot be accumulated in animal cells. Thus a blood level of tetracycline which is harmless to animal tissues can halt protein synthesis in invading bacteria. The tetracyclines have a remarkably low toxicity and minimal side effects when taken by animals. The combination of their broad spectrum and low toxicity has led to their overuse and misuse by the medical community and the wide-spread

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development of resistance has reduced their effectiveness. Nonetheless, tetracyclines still have some important uses, such as in the treatment of Lyme disease. 4. Effects on Nucleic Acids Some chemotherapeutic agents affect the synthesis of DNA or RNA, or can bind to DNA or RNA so that their messages cannot be read. Either case, of course, can block the growth of cells. The majority of these drugs are unselective, however, and affect animal cells and bacterial cells alike and therefore have no therapeutic application. Two nucleic acid synthesis inhibitors which have selective activity against procaryotes and some medical utility are nalidixic acid and rifamycins. 5. Competitive Inhibitors The competitive inhibitors are mostly all synthetic chemotherapeutic agents. Most are "growth factor analogs" which are structurally similar to a bacterial growth factor but which do not fulfill its metabolic function in the cell. Some are bacteriostatic and some are bactericidal.