General Concepts

Introduction

The broad classification of bacteria as anaerobic, aerobic, or facultative is based on the types of reactions they employ to generate energy for growth and other activities. In their metabolism of energy-containing compounds, aerobes require molecular oxygen as a terminal electron acceptor and cannot grow in its absence (see Chapter 4). Anaerobes, on the other hand, cannot grow in the presence of oxygen. Oxygen is toxic for them, and they must therefore depend on other substances as electron acceptors. Their metabolism frequently is a fermentative type in which they reduce available organic compounds to various end products such as organic acids and alcohols. The facultative organisms are the most versatile. They preferentially utilize oxygen as a terminal electron acceptor, but also can metabolize in the absence of oxygen by reducing other compounds. Much more usable energy, in the form of high-energy phosphate, is obtained when a molecule of glucose is completely catabolized to carbon dioxide and water in the presence of oxygen (38 molecules of ATP) than when it is only partially catabolized by a fermentative process in the absence of oxygen (2 molecules of ATP). The ability to utilize oxygen as a terminal electron acceptor provides organisms with an extremely efficient mechanism for generating energy. Understanding the general characteristics of anaerobiosis provides insight into how anaerobic bacteria can proliferate in damaged tissue and why special care is needed in processing clinical specimens that may contain them.

Oxygen Toxicity

Several studies indicate that aerobes can survive in the presence of oxygen only by virtue of an elaborate system of defenses. Without these defenses, key enzyme systems in the organisms fail to function and the organisms die. Obligate anaerobes, which live only in the absence of oxygen, do not possess the defenses that make aerobic life possible and therefore cannot survive in air.

During growth and metabolism, oxygen reduction products are generated within microorganisms and secreted into the surrounding medium. The superoxide anion, one oxygen reduction product, is produced by univalent reduction of oxygen:

O₂^e- → O₂^–

It is generated during the interaction of molecular oxygen with various cellular constituents, including reduced flavins, flavoproteins, quinones, thiols, and iron-sulfur proteins. The exact process by which it causes intracellular damage is not known; however, it is capable of participating in a number of destructive reactions potentially lethal to the cell. Moreover, products of secondary reactions may amplify toxicity. For example, one hypothesis holds that the superoxide anion reacts with hydrogen peroxide in the cell:

O₂^– + H₂O₂ → OH^– + OH^. + O₂

This reaction, known as the Haber-Weiss reaction, generates a free hydroxyl radical (OH·), which is the most potent biologic oxidant known. It can attack virtually any organic substance in the cell. A subsequent reaction between the superoxide anion and the hydroxyl radical produces singlet oxygen (O₂* ), which is also damaging to the cell:

O₂^– + OH → OH + O₂*

The excited singlet oxygen molecule is very reactive. Therefore, superoxide must be removed for the cells to survive in the presence of oxygen.

Most facultative and aerobic organisms contain a high concentration of an enzyme called superoxide dismutase. This enzyme converts the superoxide anion into ground-state oxygen and hydrogen peroxide, thus ridding the cell of destructive superoxide anions:

2O₂^– + 2H^{+Superoxide Dismutase} O₂ + H₂ O₂

The hydrogen peroxide generated in this reaction is an oxidizing agent, but it does not damage the cell as much as the superoxide anion and tends to diffuse out of the cell. Many organisms possess catalase or peroxidase or both to eliminate the H₂O₂. Catalase uses H₂O₂ as an oxidant (electron acceptor) and a reductant (electron donor) to convert peroxide into water and ground-state oxygen:

H₂O₂ + H₂O₂^Catalase 2H₂O + O₂

Peroxidase uses a reductant other than H₂O₂:

H₂O₂ + H₂R ^Peroxidase 2H₂O + R

One study showed that facultative and aerobic organisms lacking superoxide dismutase possess high levels of catalase or peroxidase. High concentrations of these enzymes may alleviate the need for superoxide dismutase, because they effectively scavenge H₂ O₂ before it can react with the superoxide anion to form the more active hydroxyl radical. However, most organisms show a positive correlation between the activity of superoxide dismutase and resistance to the toxic effects of oxygen.

In another study, facultative and aerobic organisms demonstrated high levels of superoxide dismutase. The enzyme was present, generally at lower levels, in some of the anaerobes studied, but was totally absent in others. The most oxygen-sensitive anaerobes as a rule contained little or no superoxide dismutase. In addition to the activity of superoxide dismutase, the rate at which an organism takes up and reduces oxygen was determined to be a factor in oxygen tolerance. Very sensitive anaerobes, which reduced relatively large quantities of oxygen and exhibited no superoxide dismutase activity, were killed after short exposure to oxygen. More tolerant organisms reduced very little oxygen or else demonstrated high levels of superoxide dismutase activity.

The continuous spectrum of oxygen tolerance among bacteria appears to be due partly to the activities of superoxide dismutase, catalase, and peroxidase in the cell and partly to the rate at which the cell takes up oxygen (Fig. 17-1). Clearly, other factors influence tolerance: the location of protective enzymes in the cell (surface versus cytoplasm), the rate at which cells form toxic oxygen products (e.g., the hydroxyl radical or singlet oxygen), and the sensitivities of key cellular components to the toxic oxygen products.

Figure 17-1

Effects of oxygen on aerobic, anaerobic, and facultative anaerobic bacteria.

Pathogenic Anaerobes

Anaerobic bacteria are widely distributed in nature in oxygen-free habitats. Many members of the indigenous human flora are anaerobic bacteria, including spirochetes and Gram-positive and Gram-negative cocci and rods. For example, the human colon, where oxygen tension is low, contains large populations of anaerobic bacteria, exceeding 10¹¹ organisms/g of colon content. Anaerobes in this region frequently outnumber facultative organisms by a factor of at least 100. Oxygen-sensitive organisms also are numerous in other areas of the body, such as the gingival crevices, tonsillar crypts, nasal folds, hair follicles, the urethra and vagina, and tooth surfaces.

Anaerobic indigenous flora components are potentially pathogenic if displaced from their normal habitat. Most anaerobic infections are endogenously acquired from members of the microflora, although Clostridium, found principally in the soil, also produces infections in humans. Proliferation of anaerobic bacteria in tissue depends on the absence of oxygen. Oxygen is excluded from the tissue when the local blood supply is impaired by trauma, obstruction, or surgical manipulation. Anaerobes multiply well in dead tissue. Multiplication of aerobic or facultative organisms in association with anaerobes in infected tissue also diminishes oxygen concentration and develops a habitat that supports growth of anaerobic bacteria.

Infections produced by anaerobic bacteria occur in all parts of the human body (Fig. 17-2). The infected tissues usually contain a mixture of several kinds of anaerobes and frequently also contain aerobic and facultative bacteria. The types of infections commonly produced by anaerobic bacteria are as follows:

Figure 17-2

Types of infection commonly produced by anaerobic bacteria.

Intra-abdominal infections.

Abscesses, postoperative wound infections, and generalized peritonitis produced by anaerobes occur as a consequence of bowel perforation during surgery or injury.

Pulmonary infections.

Anaerobic lung infections may originate in the bronchi or the blood. Aspirations from the upper respiratory tract, which contain large numbers of anaerobic bacteria, are responsible for initiating infection in the bronchi.

Pelvic infections.

Anaerobic infections of the vagina and uterus sometimes occur after gynecologic surgery or in association with malignancy of pelvic organs.

Brain abscesses.

Anaerobes infrequently produce meningitis, but are a common cause of brain abscesses. The infecting organisms usually originate in the upper respiratory tract.

Skin and soft tissue infections.

Combinations of anaerobes, aerobes, and facultative organisms often act synergistically to produce these infections.

Oral and dental infections.

These local infections frequently extend to the face and neck and sometimes to other areas of the body such as the brain.

Bacteremia and endocarditis.

Anaerobic bacteremia may follow disturbance in an area of the body where an established flora or an infection exists. Endocarditis, an inflammation of the endothelial lining of the heart cavities, is occasionally caused by anaerobic bacteria, especially anaerobic streptococci.

With the exception of the clostridia, which have been studied extensively, the mechanisms by which anaerobes cause infections in humans are not well understood. Clostridium species produce various toxins that destroy tissue cells, and two species, C botulinum and C tetani, release the neurotoxins responsible for botulism and tetanus, respectively. Enzymes excreted by other anaerobic bacteria, including proteases, lipases, hyaluronidase, chondroitin sulfatase, and neuraminidase, may play a role in infection by causing tissue cell destruction, and ß-lactamase may act as a virulence factor by inactivating antibiotics that possess a ß-lactam ring, such as the penicillins and cephalosporins. In addition, the capsules surrounding some anaerobic bacteria probably interfere with phagocytosis and act as a barrier against penetration by antimicrobial agents.

Processing of Clinical Specimens

When collecting specimens from patients for isolation and identification of anaerobic bacteria associated with infections, precautions must be taken to exclude air (Fig. 17-3). Materials for anaerobic culture are best obtained with a needle and syringe. Unless the specimen can be sent to the laboratory immediately, it is placed in an anaerobic transport tube containing oxygen-free carbon dioxide or nitrogen. The specimen is injected through the rubber stopper in the transport tube and remains in the anaerobic environment of the tube until processed in the bacteriology laboratory. If the specimen is collected with a swab, only a special commercially available anaerobic swab transport system is used.

Figure 17-3

Isolation and identification of anaerobes.

Specimens should be free of contaminating bacteria. Material from sites that are normally sterile, such as blood, spinal fluid, or pleural fluid, poses no problem provided the usual precautions are taken to decontaminate the skin properly before puncturing it to obtain the specimen. Fecal specimens, sputum specimens, or vaginal secretions cannot be cultured routinely for pathogenic anaerobes because they normally contain other anaerobic organisms. Aspirates from abscesses or the specific sites of infections must be obtained in these cases to avoid undue contamination with indigenous flora components.

Although several techniques are available for maintaining an oxygen-free environment during the processing of specimens for anaerobic culture, the anaerobic jar is the most common. It is a medium-sized glass or plastic jar with a tightly fitting lid containing palladium-coated alumina particles, which serve as a catalyst. It can be set up by two methods. The easiest uses a commercially available hydrogen and carbon dioxide generator envelope (GasPak) that is placed in the jar along with the culture plates. The generator is activated with water. Oxygen within the jar and the hydrogen that is generated are converted to water in the presence of the catalyst, thus producing anaerobic conditions. Carbon dioxide, which is also generated, is required for growth by some anaerobes and stimulates the growth of others. An alternative method for achieving anaerobiosis in the jar consists of evacuation and replacement. Air is evacuated from the sealed jar containing the culture plates and is replaced with an oxygen-free mixture of 80 percent nitrogen, 10 percent hydrogen, and 10 percent carbon dioxide.

More sophisticated procedures are used to isolate extremely oxygen-sensitive microorganisms that cannot be recovered by using the anaerobic jar. One, the roll tube method, consists of a stoppered test tube containing oxygen-free gas and a thin layer of prereduced agar medium on its inside surface. The medium in the tube is inoculated with a loop while the tube is rotated. This produces a spiral track on the agar surface. The tube is flushed with a stream of carbon dioxide to prevent entry of air while it is open during inoculation.

The anaerobic glove box isolator is another innovation developed for isolating anaerobic bacteria. It is essentially a large clear-vinyl chamber, with attached gloves, containing a mixture of 80 percent nitrogen, 10 percent hydrogen, and 10 percent carbon dioxide. A lock at one end of the chamber is fitted with two hatches, one leading to the outside and the other to the inside of the chamber. Specimens are placed in the lock, the outside hatch is closed, and the air in the lock is evacuated and replaced with the gas mixture. The inside hatch is then opened to introduce the specimen into the chamber. Conventional bacteriologic procedures are employed to process the specimen in the oxygen-free atmosphere.

Although these complex systems are needed to isolate anaerobic flora components, studies have shown that the anaerobic jar is adequate to recover clinically significant anaerobes. The extremely oxygen-sensitive bacteria of the microflora apparently are not associated with infectious processes.

Procedures for cultivation and identification of anaerobic bacteria are well established (Fig. 17-3). A variety of selective and nonselective media is available for cultivation of anaerobes. A reliable, nonselective medium consists of Brucella agar supplemented with sheep blood, hemin, cysteine, sodium carbonate, and menadione. Usual bacteriologic procedures are used to identify anaerobes. These are based on Gram-staining reactions, cellular and colony morphology, antibiotic sensitivity patterns, carbohydrate fermentation reactions, and other biochemical tests. Analysis of metabolic end products, especially organic acids, provides additional information useful in classifying these organisms.

References

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