Bacteriology at UW-Madison

The Microbial World

Lectures in Microbiology by Kenneth Todar PhD    University of Wisconsin-Madison    Department of Bacteriology

THE CONTROL OF MICROBIAL GROWTH

Introduction

"Control of microbial growth", as used here, means to inhibit or prevent growth of microorganisms. This control is affected 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.

In microbiology, sterilization refers to the complete destruction or elimination of all viable organisms in or on a substance being sterilized. There are no degrees of sterilization: an object or substance is either sterile or not. Sterilization procedures involve the use of heat, radiation or chemicals, or physical removal of cells.

Methods of Sterilization

Heat: most important and widely used. For sterilization one must consider the type of heat, and most importantly, the time of application and temperature to ensure destruction of all microorganisms. Endospores of bacteria are considered the most thermoduric of all cells so their destruction guarantees sterility.

Incineration: burns organisms and physically destroys them. Used for needles, inoculating wires, glassware, etc. and objects not destroyed in the incineration process.

Boiling: 100^o for 30 minutes. Kills everything except some endospores. To kill endospores, and therefore sterilize a solution, very long (>6 hours) boiling, or intermittent boiling is required (See Table 1 below).

Autoclaving (steam under pressure or pressure cooker)
Autoclaving is the most effective and most efficient means of sterilization. All autoclaves operate on a time/temperature relationship. These two variables are extremely important. Higher temperatures ensure more rapid killing. The usual standard temperature/pressure employed is 121ºC/15 psi for 15 minutes. Longer times are needed for larger loads, large volumes of liquid, and more dense materials. Autoclaving is ideal for sterilizing biohazardous waste, surgical dressings, glassware, many types of microbiologic media, liquids, and many other things. However, certain items, such as plastics and certain medical instruments (e.g. fiber-optic endoscopes), cannot withstand autoclaving and should be sterilized with chemical or gas sterilants. When proper conditions and time are employed, no living organisms will survive a trip through an autoclave.

Schematic diagram of a laboratory autoclave in use to sterilize microbiological culture medium. Sterilization of microbiological culture media is is often carried out with the autoclave. When microbiological media are prepared, they must be sterilized and rendered free of microbial contamination from air, glassware, hands, etc.  The sterilization process is a 100% kill, and guarantees that the medium will stay sterile unless exposed to contaminants.

An autoclave for use in a laboratory or hospital setting.

Why is an autoclave such an effective sterilizer? The autoclave is a large pressure cooker; it operates by using steam under pressure as the sterilizing agent. High pressures enable steam to reach high temperatures, thus increasing its heat content and killing power. Most of the heating power of steam comes from its latent heat of vaporization. This is the amount of heat required to convert boiling water to steam. This amount of heat is large compared to that required to make water hot. For example, it takes 80 calories to make 1 liter of water boil, but 540 calories to convert that boiling water to steam. Therefore, steam at 100º C has almost seven times more heat than boiling water.

Moist heat is thought to kill microorganisms by causing denaturation of essential proteins. Death rate is directly proportional to the concentration of microorganisms at any given time. The time required to kill a known population of microorganisms in a specific suspension at a particular temperature is referred to as thermal death time (TDT). Increasing the temperature decreases TDT, and lowering the temperature increases TDT. Processes conducted at high temperatures for short periods of time are preferred over lower temperatures for longer times.

Environmental conditions also influence TDT. Increased heat causes increased toxicity of metabolic products and toxins. TDT decreases with pronounced acidic or basic pHs. However, fats and oils slow heat penetration and increase TDT. It must be remembered that thermal death times are not precise values; they measure the effectiveness and rapidity of a sterilization process. Autoclaving 121ºC/15 psi for 15 minutes exceeds the thermal death time for most organisms except some extraordinary sporeformers. 

Dry heat (hot air oven): basically the cooking oven. The rules of relating time and temperature apply, but dry heat is not as effective as moist heat (i.e., higher temperatures are needed for longer periods of time). For example 160^o/2hours or 170^o/1hour is necessary for sterilization. The dry heat oven is used for glassware, metal, and objects that won't melt.

Irradiation: usually destroys or distorts nucleic acids. Ultraviolet light is commonly used to sterilize the surfaces of objects,  although x-rays, gamma radiation and electron beam radiation are also used.

Ultraviolet lamps are used to sterilize workspaces and tools used in microbiology laboratories and health care facilities. UV light at germicidal wavelengths (two peaks, 185 nm and 265 nm) causes adjacent thymine molecules on DNA to dimerize, thereby inhibiting DNA replication (even though the organism may not be killed outright, it will not be able to reproduce). However, since microorganisms can be shielded from ultraviolet light in fissures, cracks and shaded areas, UV lamps should only be used as a supplement to other sterilization techniques.

An ultraviolet sterilization cabinet.

Gamma radiation and electron beam radiation are forms of ionizing radiation used primarily in the health care industry. Gamma rays, emitted from cobalt-60, are similar in many ways to microwaves and x-rays. Gamma rays delivered during sterilization break chemical bonds by interacting with the electrons of atomic constituents. Gamma rays are highly effective in killing microorganisms and do not leave residues or have sufficient energy to impart radioactivity.

Electron beam (e-beam) radiation, a form of ionizing energy, is generally characterized by low penetration and high-dose rates. E-beam irradiation is similar to gamma radiation in that it alters various chemical and molecular bonds on contact. Beams produced for e-beam sterilization are concentrated, highly-charged streams of electrons generated by the acceleration and conversion of electricity.

e-beam and gamma radiation are for sterilization of items ranging from syringes to cardiothoracic devices.

Filtration involves the physical removal (exclusion) of all cells in a liquid or gas. It is especially important for sterilization of solutions which would be denatured by heat (e.g. antibiotics, injectable drugs, amino acids, vitamins, etc.). Portable units can be used in the field for water purification and industrial units can be used to "pasteurize" beverages. Essentially, solutions or gases are passed through a filter of sufficient pore diameter (generally 0.22 micron) to remove the smallest known bacterial cells.

This water filter for hikers and backpackers is advertised to "eliminate Giardia, Cryptosporidium and most bacteria." The filter is made from 0.3 micron pleated glass fiber with a carbon core.

Chemical and gas

Chemicals used for sterilization include the gases ethylene oxide and formaldehyde, and liquids such as glutaraldehyde. Ozone, hydrogen peroxide and peracetic acid are also examples of chemical sterilization techniques are based on oxidative capabilities of the chemical.

Ethylene oxide (ETO) is the most commonly used form of chemical sterilization. Due to its low boiling point of 10.4ºC  at atmospheric pressure, EtO) behaves as a gas at room temperature. EtO chemically reacts with amino acids, proteins, and DNA to prevent microbial reproduction. The sterilization process is carried out in a specialized gas chamber. After sterilization, products are transferred to an aeration cell, where they remain until the gas disperses and the product is safe to handle.

ETO is used for cellulose and plastics irradiation, usually in hermetically sealed packages.  Ethylene oxide can be used with a wide range of plastics (e.g. petri dishes, pipettes, syringes, medical devices, etc.) and other materials without affecting their integrity.

An ethylene oxide sterilization gas chamber.

Ozone sterilization has been recently approved for use in the U.S. It uses oxygen that is subjected to an intense electrical field that separates oxygen molecules into atomic oxygen, which then combines with other oxygen molecules to form ozone.

Ozone is used as a disinfectant for water and food. It is used in both gas and liquid forms as an antimicrobial agent in the treatment, storage and processing of foods, including meat, poultry and eggs. Many municipalities use ozone technology to purify their water and sewage. Los Angeles has one of the largest municipal ozone water treatment plants in the world. Ozone is used to disinfect swimming pools, and some companies selling bottled water use ozonated water to sterilize containers.

An ozone fogger for sterilization of egg surfaces. The system reacts ozone with water vapors to create powerful oxidizing radicals. This system is totally chemical free and  is effective against bacteria, viruses and hazardous microorganisms which are deposited on egg shells.

An ozone sterilizer for use in the hospital or other medical environment.

Low Temperature Gas Plasma (LTGP) is used as an alternative to ethylene oxide. It uses a small amount of liquid hydrogen peroxide (H₂O₂), which is energized with radio frequency waves into gas plasma. This leads to the generation of free radicals and other chemical species, which destroy organisms.

An LTGP sterilizer that pumps vaporized H₂O₂ into the chamber.

Non Sterilizing Methods to Control Microbial Growth

Many physical and chemical technologies are employed by our civilization to control the growth of (certain) microbes, although sterility may not the desired end-point. Rather, preventing spoilage of food or curing infectious disease might be the desired outcome.

Applications of Heat

The lethal temperature varies in microorganisms. The time required to kill depends on the number of organisms, species, nature of the product being heated, pH, and temperature. Autoclaving, which kills all microorganisms with heat, is commonly employed in canning, bottling, and other sterile packaging procedures. This is an ultimate form of preservation against microbes. But, there are some other uses of heat to control growth of microbes although it may not kill all organisms present.

Boiling: 100^o for 30 minutes (more time at high altitude). Kills everything except some endospores. It also inactivates viruses.  For the purposes of purifying drinking water, 100^o for five minutes is a "standard" in the mountains" though there have been some reports that Giardia cysts can survive this process. Longer boiling might be recommended for Mississippi River water the closer to the Gulf.

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. But pasteurization kills many spoilage organisms, as well, and therefore increases the shelf life of milk especially at refrigeration temperatures (2°C).

Milk is usually pasteurized by heating, typically at 63°C for 30 minutes (batch method) or at 71°C for 15 seconds (flash method), to kill bacteria and extend the milk's usable life. The process kills pathogens but leaves relatively benign microorganisms that can sour improperly stored milk.

During the process of ultrapasteurization, also known as ultra high-temperature (UHT) pasteurization, milk is heated to temperatures of 140 °C. In the direct method, the milk is brought into contact with steam at 140°C for one or two seconds. A thin film of milk falls through a chamber of high-pressure steam, heating the milk instantaneously. The milk is flash cooled by application of a slight vacuum, which serves the dual purpose of removing excess water in the milk from condensing steam. In the indirect method of ultrapasteurization, milk is heated in a plate heat exchanger. It takes several seconds for the temperature of the milk to reach 140°C, and it is during this time that the milk is scalded, invariably leading to a burned taste. If ultrapasteurization is coupled with aseptic packaging, the result is a long shelf life and a product that does not need refrigeration.

A review of protocols and recommendations for the use of heat to control microbial growth is given in Table 1.

Low temperature (refrigeration and freezing): Most organisms grow very little or not at all at 0^oC. Perishable foods are stored 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. Although a few microbes will grow in supercooled solutions as low as minus 20^oC, most foods are preserved against microbial growth in the household freezer.

Drying (removal of H₂O): Most microorganisms cannot grow at reduced water activity (A_w < 0.90). Drying is often used to preserve foods (e.g. fruits, grains, etc.). Methods involve removal of water from product by heat, evaporation, freeze-drying, and addition of salt or sugar.

Irradiation (UV, x-ray, gamma radiation): destroys microorganisms as described under "sterilization". Many spoilage organisms are readily killed by irradiation.

In some parts of Europe, fruits and vegetables are irradiated to increase their shelf life up to 500 percent. The practice has not been accepted in the U.S.  UV light can be used to pasteurize fruit juices by flowing the juice over a high intensity ultraviolet light source. UV systems for water treatment are available for personal, residential and commercial applications and may be used to control bacteria, viruses and protozoan cysts.

The FDA has approved irradiation of poultry and pork to control pathogens, as well as foods such as fruits, vegetables, and grains to control insects, and spices, seasonings, and dry enzymes used in food processing to control microorganisms. Food products are treated by subjecting them to radiation from radioactive sources, which kills significant numbers of insects, pathogenic bacteria and parasites.

According to the FDA, irradiation does not make food radioactive, nor does it noticeably change taste, texture, or appearance.  Irradiation of food products to control food-borne disease in humans has been generally endorsed by the United Nation's World Health Organization and the American Medical Association. Two important Disease-causing bacteria that can be controlled by irradiation include Escherichia coli 0157:H7 and Salmonella species.

Control of microbial growth by chemical agents

Antimicrobial agents are chemicals that kill or inhibit the growth microorganisms. Antimicrobial agents include chemical preservatives and 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 internally. Examples include alcohols, mercurials, silver nitrate, iodine solution, alcohols, detergents.

Disinfectants: agents that kill microorganisms, but not necessarily their spores, but are not safe for application to living tissues; they are used on inanimate objects such as tables, floors, utensils, etc. Examples include, hypochlorites, chlorine compounds, lye, copper sulfate, quaternary ammonium compounds, formaldehyde and phenolic compounds.

Common antiseptics and disinfectants and their uses are summarized in Table 2. 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. 

Preservatives: static agents used to inhibit the growth of microorganisms, most often in foods. If eaten they should be nontoxic. Examples are calcium propionate, sodium benzoate, formaldehyde, nitrate and sulfur dioxide. Table 3a and 3b are lists of common preservative and their uses.

Chemotherapeutic agents (synthetic antibiotics): antimicrobial agents of synthetic origin useful in the treatment of microbial or viral disease. Examples are sulfonilamides, isoniazid, ethambutol, AZT, nalidixic acid and chloramphenicol. Note that the microbiologist's definition of a chemotherapeutic agent requires that the agent be used for antimicrobial purpose and excludes synthetic agents used for therapy against diseases that are not of microbial origin. Hence, pharmacology distinguishes the microbiologist's chemotherapeutic agent as a "synthetic antibiotic".

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. Note also (above), pharmacologists refer to any antimicrobial chemical used in the treatment of infectious disease as as antibiotic.

Three bacterial colonies growing on this plate secrete antibiotics that diffuse into the medium and inhibit the growth of a mold.

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.

Semisynthetic antibiotics are molecules produced my a microbe that are subsequently modified by an organic chemist to enhance their antimicrobial properties or to render them unique for a pharmaceutical patent.

Antimicrobial Agents Used in the Treatment of Infectious Disease

The modern era of antimicrobial chemotherapy began following Fleming's discovery in 1929 of the powerful bactericidal substance penicillin, and Domagk's discovery in 1935 of synthetic chemicals (sulfonamides) with broad antimicrobial activity. In the early 1940's, spurred partially by the need for antibacterial agents in WW II, penicillin was isolated, purified and injected into experimental animals, where it was found to not only cure infections but also to possess incredibly low toxicity for the animals. This fact ushered into being the age of antibiotic chemotherapy and an intense search for similar antimicrobial agents of low toxicity to animals that might prove useful in the treatment of infectious disease. The rapid isolation of streptomycin, chloramphenicol and tetracycline soon followed, and by the 1950's, these and several other antibiotics were in clinical usage.

The most important property of a clinically-useful antimicrobial agent, especially from the patient's point of view, is its selective toxicity, i.e., the agent acts in some way that inhibits or kills bacterial pathogens but has little or no toxic effect on the animal taking the drug This implies that the biochemical processes in the bacteria are in some way different from those in the animal cells, and that the advantage of this difference can be taken in chemotherapy.

Antibiotics may have a cidal (killing) effect or a static (inhibitory) effect on a range of microbes. The range of bacteria or other microorganisms that are affected by a certain antibiotic is expressed as its spectrum of action. Antibiotics effective against procaryotes which kill or inhibit a wide range of Gram-positive and Gram-negative bacteria are said to be broad spectrum . If effective mainly against Gram-positive or Gram-negative bacteria, they are narrow spectrum. If effective against a single organism or disease, they are referred to as limited spectrum.

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 of the molecule (6-aminopenicillanic acid) which can be modified chemically by the addition of side chains. Many of these compounds have been developed to have distinct benefits or advantages over penicillin G, such as increased spectrum of activity (e.g.  effectiveness against Gram-negative rods), resistance to penicillinase or  effectiveness when administered orally. Amoxycillin and Ampicillin have 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 beta-lactamases, but they tend to be resistant to beta-lactamases from S. aureus.

Chemical structure of some Beta Lactam antibiotics.

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 vital to bacteria, and compounds that disorganize the membranes rapidly kill the cells. However, due to the similarities in phospholipids in bacterial and eucaryotic 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 polymyxa. 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 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 tuberculosis. Gentamicin is active against many strains of Gram-positive and Gram-negative bacteria, including some strains of Pseudomonas aeruginosa. Kanamycin is active at low concentrations against many Gram-positive bacteria, including penicillin-resistant 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 damage to the auditory nerves leading to deafness.

The chemical structure of tobramycin.

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 (eucaryotic) 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 development of resistance has reduced their effectiveness. Nonetheless, tetracyclines still have some important uses, such as in the treatment of Lyme disease.

The chemical structure of tetracycline.

Chloramphenicol has a broad spectrum of activity that exerts a bacteriostatic effect. It is effective against intracellular parasites such as the rickettsiae. Unfortunately, aplastic anemia, which is dose related, develops in a small proportion (1/50,000) of patients. Chloramphenicol was originally discovered and purified from the fermentation of a Streptomyces, but currently it is produced entirely by chemical synthesis. Chloramphenicol inhibits the bacterial enzyme peptidyl transferase thereby preventing the growth of the polypeptide chain during protein synthesis.

Chloramphenicol is entirely selective for 70S ribosomes and does not affect 80S ribosomes. Its unfortunate toxicity towards the small proportion of patients who receive it is in no way related to its effect on bacterial protein synthesis. However, since mitochondria originated from procaryotic cells and have 70S ribosomes, they are subject to inhibition by some of the protein synthesis inhibitors including chloroamphenicol. This likely explains the toxicity of chloramphenicol. The eucaryotic cells most likely to be inhibited by chloramphenicol are those undergoing rapid multiplication, thereby rapidly synthesizing mitochondria. Such cells include the blood forming cells of the bone marrow, the inhibition of which could present as aplastic anemia. Chloramphenicol was once a highly prescribed antibiotic and a number of deaths from anemia occurred before its use was curtailed. Now it is seldom used in human medicine except in life-threatening situations (e.g. typhoid fever).

The chemical structure of chloroamphenicol.

The Macrolides is a family of antibiotics whose structures contain large lactone rings linked through glycoside bonds with amino sugars. The most important members of the group are erythromycin and azithromycin. Erythromycin is active against most Gram-positive bacteria, Neisseria, Legionella and Haemophilus, but not against the Enterobacteriaceae. Macrolides inhibit bacterial protein synthesis by binding to the 50S ribosomal subunit. Binding inhibits elongation of the protein by peptidyl transferase or prevents translocation of the ribosome or both. Macrolides are bacteriostatic for most bacteria but are cidal for a few Gram-positive bacteria.


The chemical structure of erythromycin.

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 classes of nucleic acid synthesis inhibitors which have selective activity against procaryotes and some medical utility are quinolones and rifamycins.

Quinolones are broad-spectrum agents that rapidly kill bacteria and are well absorbed after oral administration. Nalidixic acid and ciprofloxacin belong to this group. They act by inhibiting the activity of  bacterial DNA gyrase, preventing the normal functioning of DNA. Bacterial DNA exists in a supercoiled form and the enzyme DNA gyrase, a topoisomerase, is responsible for introducing negative supercoils into the structure. Humans possess DNA gyrase but it is structurally distinct from the bacterial enzyme and remains unaffected by the activity of quinolones. Overuse of these drugs in certain situations is selecting quinolone resistant mutants and these may threaten the long term use of such compounds.

The chemical structure of nalidixic acid.

Some quinolones penetrate macrophages and neutrophils better than most antibiotics and are thus useful in treatment of infections caused by intracellular parasites. However, the main use of nalidixic acid is in treatment of lower urinary tract infections (UTI). The compound is unusual in that it is effective against several types of Gram-negative bacteria such as E. coli, Enterobacter aerogenes, K. pneumoniae and  species which are common causes of UTI. It is not usually effective against Pseudomonas aeruginosa, and Gram-positive bacteria are resistant.  However, a fluoroquinolone, Ciprofloxacin (Cipro) was recently recommended as the drug of choice for prophylaxis and treatment of anthrax.

The chemical structure of ciprofloxacin.

The rifamycins are the products of Streptomyces. Rifampicin is a semisynthetic derivative of rifamycin that is active against Gram-positive bacteria (including Mycobacterium tuberculosis) and some Gram-negative bacteria. Rifampicin acts quite specifically on eubacterial RNA polymerase and is inactive towards RNA polymerase from animal cells or towards DNA polymerase. The antibiotic binds to the beta subunit of the polymerase and apparently blocks the entry of the first nucleotide which is necessary to activate the polymerase, thereby blocking mRNA synthesis. It has been found to have greater bactericidal effect against M.tuberculosis than other anti-tuberculosis drugs, and it has largely replaced isoniazid as one of the front-line drugs used to treat the disease, especially when isoniazid resistance is indicated. It is effective orally and penetrates well into the cerebrospinal fluid and is therefore useful for treatment of tuberculosis meningitis, as well as meningitis caused by Neisseria meningitidis.

The chemical structure of rifampicin.

Sulfonamides were introduced as chemotherapeutic agents by Domagk in 1935, who showed that one of these compounds (prontosil) had the effect of curing mice with infections caused by beta-hemolytic streptococci. Chemical modifications of the compound sulfanilamide gave compounds with even higher and broader antibacterial activity. The resulting sulfonamides have broadly similar antibacterial activity, but differ widely in their pharmacological actions. Bacteria which are almost always sensitive to the sulfonamides include Streptococcus pneumoniae, beta-hemolytic streptococci and E. coli. The sulfonamides have been extremely useful in the treatment of uncomplicated UTI caused by E. coli, and in the treatment of meningococcal meningitis (because they cross the blood-brain barrier). The most useful sulfonamides are sulfanilamide, Gantrisin and Trimethoprim.

The sulfonamides are inhibitors of the bacterial enzymes required for the synthesis of tetrahydrofolic acid (THF), the vitamin form of folic acid essential for 1-carbon transfer reactions. Sulfonamides are structurally similar to para aminobenzoic acid (PABA), the substrate for the first enzyme in the THF pathway, and they competitively inhibit that step. Trimethoprim is structurally similar to dihydrofolate (DHF) and competitively inhibits the second step in THF synthesis mediated by the DHF reductase. Animal cells do not synthesize their own folic acid but obtain it in a preformed fashion as a vitamin. Since animals do not make folic acid, they are not affected by these drugs, which achieve their selective toxicity for bacteria on this basis.

Sulfanilamide is similar in structure to para-aminobenzoic acid (PABA), an intermediate in the biosynthetic pathway for folic acid. Sulfanilamide can competitively inhibit the enzyme that has PABA as it's normal substrate by competitively occupying the active site of the enzyme.

Three additional synthetic chemotherapeutic agents have been used in the treatment of tuberculosis: isoniazid (INH), para-aminosalicylic acid (PAS), and ethambutol. The usual strategy in the treatment of tuberculosis has been to administer a single antibiotic (historically streptomycin, but now, most commonly, rifampicin is given) in conjunction with INH and ethambutol. Since the tubercle bacillus rapidly develops resistance to the antibiotic, ethambutol and INH are given to prevent outgrowth of a resistant strain. It must also be pointed out that the tubercle bacillus rapidly develops resistance to ethambutol and INH if either drug is used alone. Ethambutol inhibits incorporation of mycolic acids into the mycobacterial cell wall. Isoniazid has been reported to inhibit mycolic acid synthesis in mycobacteria and since it is an analog of pyridoxine (Vitamin B₆) it may inhibit pyridoxine catalyzed reactions as well. Isoniazid is activated by a mycobacterial peroxidase enzyme and destroys several targets in the cell. PAS is an anti-folate. PAS was once a primary anti-tuberculosis drug, but now it is a secondary agent, having been largely replaced by ethambutol.

 
The chemical structure of isoniazid.

Bacterial resistance to antibiotics

Penicillin became generally available for treatment of bacterial infections, especially those caused by staphylococci and streptococci, about 1946. Initially, the antibiotic was effective against all sorts of infections caused by these two Gram-positive bacteria. Resistance to penicillin in some strains of staphylococci was recognized almost immediately. (Resistance to penicillin today occurs in as many as 80% of all strains of Staphylococcus aureus). Surprisingly, Streptococcus pyogenes (Group A strep) have not fully developed resistance to penicillin and it remains a reasonable drug of choice for many types of streptococcal infections. Natural penicillins have never been effective against most Gram-negative pathogens (e.g. Salmonella, Shigella, Bordetella pertussis, Yersinia pestis, Pseudomonas) with the notable exception of Neisseria gonorrhoeae. Gram-negative bacteria are inherently resistant because their vulnerable cell wall is protected by an outer membrane that prevents permeation of the penicillin molecule.

The period of the late 1940s and early 1950s saw the discovery and introduction of streptomycin, chloramphenicol, and tetracycline, and the age of antibiotic chemotherapy came into full being. These antibiotics were effective against the full array of bacterial pathogens including Gram-positive and Gram-negative bacteria, intracellular parasites, and the tuberculosis bacillus. However, by 1953, during a Shigella outbreak in Japan, a strain of the dysentery bacillus was isolated which was multiple drug resistant, exhibiting resistance to chloramphenicol, tetracycline, streptomycin, and the sulfanilamides. There was also evidence mounting that bacteria could pass genes for multiple drug resistance between strains and even between species. It was also apparent that Mycobacterium tuberculosis was capable of rapid development of resistance to streptomycin which had become a mainstay in tuberculosis therapy.

By the 1960's it became apparent that some bacterial pathogens were developing resistance to antibiotic-after-antibiotic, at a rate faster than new antibiotics could be brought to market. A more conservative approach to the use of antibiotics has not been fully accepted by the medical and agricultural communities, and the problems of emerging multiple-drug resistant pathogens still loom. The most important pathogens to emerge in multiple drug resistant forms so far have been Mycobacterium tuberculosis and Staphylococcus aureus.
 

The basis of bacterial resistance to antibiotics

An antibiotic sensitivity test performed on an agar plate. The discs are seeded with antibiotics planted on the agar surface. Interpretation of the size of the bacterial "zones of inhibition" relates to the possible use of the antibiotic in a clinical setting. The organism is resistant to the antibiotics planted on the plate at 5 o'clock and 9 o'clock.

Bacterial resistance to an antimicrobial agent may be due to some innate property of the organism or it may due to acquisition of some genetic trait as described below.

Inherent (Natural) Resistance - Bacteria may be inherently resistant to an antibiotic. For example, a streptomycete may have some natural gene that is responsible for resistance to its own antibiotic; or a Gram-negative bacterium has an outer membrane that establishes a permeability barrier against the antibiotic; or an organism lacks a transport system for the antibiotic; or it lacks the target or reaction that is hit by the antibiotic.

Acquired Resistance - Bacteria can develop resistance to antibiotics, e.g. bacterial populations previously-sensitive to antibiotics become resistant. This type of resistance results from changes in the bacterial genome. Acquired resistance is driven by two genetic processes in bacteria: (1) mutation and selection (sometimes referred to as vertical evolution); (2) exchange of genes between strains and species (sometimes called horizontal evolution or horizontal gene transmission).

Vertical evolution is strictly a matter of Darwinian evolution driven by principles of natural selection: a spontaneous mutation in the bacterial chromosome imparts resistance to a member of the bacterial population. In the selective environment of the antibiotic, the wild type (non mutants) are killed and the resistant mutant is allowed to grow and flourish. The mutation rate for most bacterial genes is approximately 10^-8. This means that if a bacterial population doubles from 10⁸ cells to 2 x 10⁸ cells, there is likely to be a mutant present for any given gene. Since bacteria grow to reach population densities far in excess of 10⁹ cells, such a mutant could develop from a single generation during 15 minutes of growth.

Horizontal gene transmission (HGT) is the acquisition of genes for resistance from another organism. For example, a streptomycete has a gene for resistance to streptomycin (its own antibiotic), but somehow that gene escapes and gets into E. coli or Shigella. Or, more likely, some bacterium develops genetic resistance through the process of mutation and selection and then donates these genes to some other bacterium through one of several processes for genetic exchange that exist in bacteria.

Bacteria are able to exchange genes in nature by three processes: conjugation, transduction and transformation. Conjugation involves cell-to-cell contact as DNA crosses a sex pilus from donor to recipient. During transduction, a virus transfers the genes between mating bacteria. In transformation, DNA is acquired directly from the environment, having been released from another cell. Genetic recombination can follow the transfer of DNA from one cell to another leading to the emergence of a new genotype (recombinant). It is common for DNA to be transferred as plasmids between mating bacteria. Since bacteria usually develop their genes for drug resistance on plasmids (called resistance factors [R-factors] or resistance transfer factors [RTFs]), these genetic elements play heavily in the of spread drug resistance to other strains and species during genetic exchange processes.

The combined effects of fast growth rates, high populations of cells, genetic processes of mutation and selection, and the ability to exchange genes, account for the extraordinary rates of adaptation and evolution that can be observed in the bacteria. For these reasons bacterial adaptation (resistance) to the antibiotic environment seems to take place very rapidly in evolutionary time: bacteria evolve fast!
 

The medical problem of bacterial drug resistance

Obviously, if a bacterial pathogen is able to develop or acquire resistance to an antibiotic, then that substance becomes useless in the treatment of infectious disease caused by that pathogen (unless the resistance can somehow be overcome with secondary measures). So as pathogens develop resistance, we must find new (different) antibiotics to fill the place of the old ones in treatment regimes. Hence, natural penicillins have become useless against staphylococci and must be replaced by other antibiotics; tetracycline, having been so widely used and misused for decades, has become worthless for many of the infections where it once worked as a "wonder drug".

Not only is there a problem in finding new antibiotics to fight old diseases (because resistant strains of bacteria have emerged), there is a parallel problem to find new antibiotics to fight new diseases. In the past two decades, many "new" bacterial diseases have been discovered (Legionnaire's disease, gastric ulcers, Lyme disease, toxic shock syndrome, "skin-eating" streptococci). We are only now able to examine patterns of susceptibility and resistance to antibiotics among new pathogens that cause these diseases. Broad patterns of resistance exist in these pathogens, and it seems likely that we will soon need new antibiotics to replace the handful that are effective now against these bacteria, especially as resistance begins to emerge among them in the selective environment antibiotic chemotherapy.

Alternatives to Antibiotics

Phage therapy is the therapeutic use of lytic bacteriophages to treat pathogenic bacterial infections. Phage therapy is an alternative to antibiotics being developed for clinical use by research groups in Eastern Europe and the U.S. After having been extensively used and developed mainly in former Soviet Union countries for about 90 years, phage therapies for a variety of bacterial and poly microbial infections are now becoming available on an experimental basis in other countries, including the U.S. The principles of phage therapy have potential applications not only in human medicine, but also in dentistry, veterinary science, food science and agriculture.