《微生物生物学》课程教学资源（书籍文献）World of Microbiology and Immunology，Brigham Narins, Editor，Volumes 1 and 2 A-Z.pdf_P31-P35

Anthrax WORLD OF MICROBIOLOGY AND IMMUNOLOGY 20 • • with the bacterium. This can allow the organism to multiply to large numbers that overwhelm the immune system. The capsule also contains an antigen that has been designated a protective antigen. The antigen is protective, not to the host being infected, but to the bacterium. The protective antigen dissolves protein, which can allow the bacterium to “punch” through the membrane surrounding cells of the host, such as the epithelial cells that line the lung. One inside the cells, a bacterium is safe from the host’s immune defenses. A second toxic component, which is called lethal factor, destroys immune cells of the host. Finally, a third toxic factor is known as edema factor (named because it results in the accumulation of fluid at the site of infection). Edema factor disables a molecule in the host called calmodulin, which is used to regulate many chemical reactions in the body. The end result of the activity of the toxic factors of Bacillus anthracis is to quell the immune response and so, to allow the infection to spread. As the bacteria gain a foothold, toxins enter the bloodstream and circulate throughout the body causing destruction of blood cells and tissues. The damage can prove to be overwhelming to treatment efforts and death occurs. Anthrax infections are difficult to treat because the initial symptoms are similar to other, less serious infections, such as the flu. By the time the diagnosis is made, the infection can be too advanced to treat. A vaccine for anthrax does exist. But to date, only those at high risk for infection (soldiers, workers in meat processing plants, anthrax research scientists) have received the vaccine, due to the possible serious side effects that can occur. Work to establish a safer vaccine is underway. The edema factor may be a potential target of a vaccine. Another promising target is the protective antigen of the capsule. If the action of this antigen could be blocked, the bacteria would not be able to hide inside host cells, and so could be more effectively dealt with by the immune response and with antibiotics. See also Anthrax, terrorist use of as a biological weapon; Bioterrorism Light micrograph of Bacillus anthracis, showing the typical hair-like pattern of growth in a liquid. womi_A 5/6/03 1:06 PM Page 20

WORLD OF MICROBIOLOGY AND IMMUNOLOGY Anthrax, terrorist use as a biological weapon 21 • • ANTHRAX, FORENSIC IDENTIFICATION • see GENETIC IDENTIFICATION OF MICROORGANISMS ANTHRAX, TERRORIST USE AS A Anthrax, terrorist use as a biological weapon BIOLOGICAL WEAPON During the past two decades, the potential use of biological weapons by terrorists has received a great deal of attention, particularly in the United States. The existence of an anthrax bioweapons development campaign by the government of Iraq was revealed during the Persian Gulf War from 1990 to 1991. Then, in the aftermath of the September 11, 2001 terrorist attacks on the World Trade Center buildings in New York City and the Pentagon in Washington, DC., letters containing a powdered form of Bacillus anthracis, the bacteria that causes anthrax, were mailed to government representatives, members of the news media, and others in the United States. The anthrax-laced powder inside the letters was aerosolized (i.e., the spores became airborne) when the letters were opened, and in a few cases were inhaled. The death of a Florida man was the first case of an inhalational anthrax death in the United States since 1978 and as of June 2002, more than 20 cases and five deaths were attributed to the terrorist attack. Although a relatively new weapon in the hands of modern potential bioterrorists, the threat of death from the inhalation of anthrax has been part of human history since antiquity. Some scholars argue that it is the sooty “morain” in the Bible’s Book of Exodus, and is likely the “burning wind of plague” that begins Homer’s Iliad. As well, the use of microorganisms such as the anthrax bacteria as weapons is not new. In ancient military campaigns, diseased bodies (including those who died of anthrax) were used to poison wells and were catapulted into cities under siege. Research into the military use of anthrax was carried out during World War I by combatants on all sides of the conflict, and by World War II anthrax research was actively underway. For example, Allied efforts in Canada, the United States, and Britain to develop anthrax-based weapons included the production of five million anthrax “cakes,” designed to be dropped on Germany to infect wells and the food chain. The weapons were never used. Only within the past several decades, however, have biological weapons, including anthrax, been added to the arsenal of terrorists. For example, the Japanese cult Aum Shinrikyo (which released sarin gas into the Tokyo subway system in 1995, killing 12 people and hospitalizing 5,000) was developing anthrax-based weapons. Indeed, the group had released crude anthrax preparations in Tokyo on at least eight separate occasions in 1993. These incidents were the first time that anthrax was used as a weapon against a civilian population. In addition, state-sanctioned terrorism by the government of Iraq has also, purportedly, involved the production of anthrax bioweapons, and Western intelligence sources insist that Iraq—or terrorist groups operating with Iraq’s assistance—continues (despite United Nations’ efforts at inspection and destruction) to develop biological weapons, including anthrax-based weapons. Finally, during the terrorist attacks of the United States in the latter part of 2001 the use of anthrax by a terrorist or terrorists (as of June 2002, yet unidentified) pointed out how easily the lethal agent could be delivered. This ease of delivery of anthrax is one feature that has made the bacterium an attractive weapon for terrorists. Scenarios developed by United States government agencies have shown that even a small crop dusting plane carrying only a hundred kilograms of anthrax spores flying over a city could deliver a potentially fatal dose to up to three million people in only a few hours. Although variations in weather patterns and concentration variables would substantially reduce the number of expected actual deaths, such an attack could still result in the deaths of thousands of victims and result in a devastating attack on the medical and economic infrastructure of the city attacked. In a less sophisticated effort, spores could simply be released into air intake vents or left in places like a subway tunnel, to be dispersed in the air over a much small area. Another feature of anthrax that has led to its exploitation by terrorists is the physiology of the bacterium. Bacillus anthracis can live as a vegetative cell, growing and dividing in a rapid and cyclical fashion. The bacterium can also form a metabolically near-dormant form known as a spore. An individual spore is much smaller and lighter than the growing bacterium. Indeed, the spores can drift on air currents, to be inhaled into the lungs. Once in the lungs, the spores can resuscitate into an actively growing and dividing bacterium. The infections that are collectively termed anthrax can result. Although millions of spores can be released from a few grams (fractions of an ounce) of Bacillus anthracis, only about 5,000 to 8,000 spores are sufficient to cause the lung infection when they are inhaled. If left untreated or not promptly treated with the proper antibiotics (e.g., Cipro), the lung infection is almost always fatal. Non-inhalation contact with Bacillus anthracis can result in cutaneous anthrax—a condition more treatable with conventional antibiotic therapy. An often-overlooked aspect of the use of anthrax as a terrorist weapon is the economic hardship that the dispersal of a small amount of the spores would exact. A report from the Centers for Disease Control and Prevention, entitled The Economic Impact of a Bioterrorist Attack, estimated the costs of dealing with an anthrax incident at a minimum of US$26 billion per 100,000 people. In just a few months in 2001 alone, a flurry of anthrax incidents, most of which turned out to be hoaxes, cost the United States government millions of dollars. The choice of anthrax as a weapon by terrorists reflects the growing awareness of the power of biological research and biotechnology among the general community. The ability to grow and disperse infectious microorganisms was once restricted to specialists. However, the explosion of biotechnology in the 1980s and 1990s demonstrated that the many basic microbiological techniques are fairly simple and attainable. Experts in microbiology testifying before Congress, estimated that crude weapons could be developed with approximately $10,000 worth of equipment. A laboratory sufficient to grow and harvest the bacteria and to dry down the material to powdered form could fit into the average sized household basewomi_A 5/6/03 1:06 PM Page 21

Anti-adhesion methods WORLD OF MICROBIOLOGY AND IMMUNOLOGY 22 • • ment. The more highly trained the terrorist, the more effective weapons could be expected to be produced. Even though Bacillus anthracis could be grown in such a makeshift laboratory, the preparation of the spores and the drying of the spores into a powder is not a trivial task. As an example, even after a decade of dedicated effort, United Nations inspectors who toured Iraq bioweapons facilities after the Gulf War found that Iraq had only managed to develop crude anthrax preparations. Still, the Iraq bioweapons program managed to produce 8,500 liters of concentrated anthrax. Regardless, despite the technical challenges, the production of anthrax spores in quantities great enough to cause a huge loss of life is not beyond the capability of a small group of equipped and funded terrorists. This small size and nondescript nature of a bioweapons facility could make detection of such a lab very difficult. Accordingly, the terrorist potential of anthrax will remain a threat for the foreseeable future. See also Bacteria and bacterial infection; Biological warfare; Bioterrorism, protective measures; Bioterrorism; Epidemics and pandemics; Vaccine AAnti-adhesion methods NTI-ADHESION METHODS The adhesion of bacteria and other microorganisms to nonliving and living surfaces is a crucial part of the contamination and infection processes. In fact, the growth of microorganisms on surfaces is the preferred mode of existence. The ability to block adhesion would prevent surface growth. There are numerous examples of surface growth of microorganisms. Adherence and growth of bacteria such as Escherichia coli on urinary catheters (synthetic tubes that are inserted into the bladder to assist hospitalized patients in removing urine from the body) is a large problem in hospitals. The chance of a urinary tract infection increases by up to10% for each day of catheterization. Neiserria meningitidis, the agent that causes meningitis, relies upon adhesion with host cells. The adhesion of this and many other bacteria, including disease causing Escherichia coli, is mediated by a surface tube-like protein appendage called a pilus. Other bacterial proteins are involved in adhesion, typically by recognizing and biding to another protein on the surface of the host cell. Microorganism proteins that function in adhesion are generically known as adhesins. Workers in biohazard protective suits respond to an anthrax incident in Florida. womi_A 5/6/03 1:06 PM Page 22

WORLD OF MICROBIOLOGY AND IMMUNOLOGY Antibiotic resistance, tests for 23 • • Some strains of E. coli that infect intestinal cells do so by manufacturing and then releasing an adhesin, which is incorporated into the membrane of the host cell. Thus, the bacteria install their own receptor in the host tissue. Adhesion need not rely on the presence of adhesins. The chemistry of the surface can also drive adhesion. For example, the surface of the spores of bacillus and the capsule surrounding Pasteurella multocida are described as being hydrophobic; that is, they tend not to associate with water. This hydrophobicity will drive the spore or bacterium to associate with a surface of similar chemistry. In order to block adhesion that is the result of the above mechanisms, the molecular details of these mechanisms must be unraveled. This is an on-going process, but advances are being made through research. Adhesion of Escherichia coli can depend on the presence of an adhesin called FimH. Antibodies to FimH can block adhesion, presumable by binding to the FimH protein, preventing that protein from binding to the receptor on the surface of the host cell. Furthermore, the three-dimensional structure of this adhesion is similar to that of adhesins from other bacteria. A vaccine devised against FimH might then have some protective effect against the adhesion of other bacteria. In the case of the capsule-mediated adhesion, such as the example above, capsular antibodies may also thwart adhesion. The drawback with this approach is that capsular material is not a potent stimulator of the immune system. For microorganisms that secrete their own receptor, such as Escherichia coli, or which have receptor molecules protruding from their own surface (an example is the hemagglutinin protein on the surface of Bordetella pertussis), adhesion could be eliminated by blocking the manufacture or the release of the receptor molecule. In Canada, field trials began in the summer of 2001 on a vaccine to the adhesin target of Escherichia coli O157:H7. This pathogen, which can be permanently debilitating and even lethal to humans who ingest contaminated food or water, often lives in the intestinal tracts of cattle. By eliminating the adhesion of the bacteria, they could be “flushed” out of the cattle. Thus, a vital reservoir of infection would have been overcome. The vaccine could be ready for the market by as early as 2003. Another anti-adhesion strategy is to out-compete the target bacteria for the available spots on the surface. This approach has been successful in preventing bacterial vaginal infections. Suppositories loaded with bacteria called Lactobacillus are administered. Colonization of the vaginal wall by the Lactobacillus can retard or even prevent the subsequent colonization of the wall by a harmful type of bacteria. The same bacteria are present in yogurt. Indeed, consumption of yogurt may help prevent intestinal upset due to colonization of the gut by harmful organisms. Non-living surfaces, such as catheters and other implanted material, are colonized by, in particular, bacteria. In seeking to prevent adhesion, scientists have been experimenting with different implant materials, with the incorporation of antimicrobial compounds into the implant material, and with the “pre-coating” of the material. In the case of antimicrobial compounds, promising results have been obtained in laboratory studies using material that can slowly release antibiotics. The disadvantage of this approach is that the presence of residual antibiotic could encourage the formation of resistance. Pre-coating implant material with an antimicrobial compound that is permanently bonded has also been promising in lab studies. See also Biofilm formation and dynamic behavior; Infection and resistance; Probiotics AAntibiotic resistance, tests for NTIBIOTIC RESISTANCE, TESTS FOR Bacteria can sometimes adapt to the antibiotics used to kill them. This adaptation, which can involve structural changes or the production of enzymes that render the antibiotic useless, can make the particular bacterial species resistant to the particular antibiotic. Furthermore, a given bacterial species will usually display a spectrum of susceptibilities to antibiotics, with some antibiotics being very effective and others totally ineffective. For another bacterial species, the pattern of antibiotic sensitivity and resistance will be different. Thus, for diagnosis of an infection and for clinical decisions regarding the best treatment, tests of an organism’s response to antibiotics are essential. A standard method of testing for antibiotic resistance involves growth of the target bacteria in the presence of various concentrations of the antibiotic of interest. Typically, this test is performed in a specially designed plastic dish that can be filled with agar (a Petri plate). Contamination of the agar, which would spoil the test results, is guaranteed by the sterility of the plate and the lid that fits over the agar-containing dish. The type of agar used is essential for the validity of the tests results. Typically, Iso-Sensitest agar is used. The hardened agar surface receives a suspension of the test bacteria, which is then spread out evenly over the surface of the agar. The intention is to form a so-called lawn of organisms as growth occurs. Also on the agar surface are discs of an absorbent material. A plate is large enough to house six discs. Each disc has been soaked in a known and different concentration of the same or of different antibiotics. As growth of the bacteria occurs, antibiotic diffuses out from each disc into the agar. If the concentration of the antibiotic is lethal, no growth of the bacteria will occur. Finally, the diffusing antibiotic will be below lethal concentration, so that growth of bacteria can occur. The result is a ring of no growth around a disc. From comparison with known standards, the diameter of the growth inhibition ring will indicate whether the bacteria are resistant to the antibiotic. Automated plate readers are available that will scan the plates, measure the diameter of the growth inhibition zones and consult a standard database to indicate the antibiotic resistance or susceptibility of the sample bacteria. In the past 15 years, the use of fluorescent indicators has become popular. A myriad of compounds are available that will fluoresce under illumination of specific wavelengths. Among the uses for the fluorescent compounds is the viability womi_A 5/6/03 1:06 PM Page 23

Antibiotics WORLD OF MICROBIOLOGY AND IMMUNOLOGY 24 • • of a bacterium. For example, living bacteria will fluoresce in the presence of acridine orange, while dead bacteria will not. These probes combined with the optical technique of confocal laser microscopy, now enables populations of cells to be viewed without disrupting them to see if they fluoresce or not in the presence of an antibiotic of interest. The ability of living bacteria to fluoresce can also be exploited by another machine called a flow cytometer. This machine operates essentially by forcing a suspension of bacteria (or other cells) through an opening so that only one bacterium at a time passes by a sensor. The sensor monitors each passing bacterium and can sort these into categories, in this case, fluorescing (living) from non-fluorescing (dead). The entire process can be completely quickly. This provides an almost “real-time” assessment of the proportion of a population that has been killed by an antibiotic. If the proportion of dead bacteria is low, resistance is indicated. All the assessments of antibiotic effectiveness need to be done in a controlled manner. This necessitates the use of standard test types of bacteria (strains that are known to be resistant to the particular antibiotic as well as other strains that are known to be sensitive to the antibiotic). The concentration of the bacteria used is also important. Too many bacteria can “dilute” out the antibiotic, producing a false indication of resistance. Controls need to be included to verify that the experiment was not subject to contamination, otherwise the possibility that a finding of resistance was due to a contaminating bacteria could not be discounted. In clinical settings, a finding of resistance would prompt the search for another antibiotic. Often, identification of the bacteria will suggest, from previous documented tests of others, an antibiotic to which the organism will be susceptible. But, increasingly, formerly effective antibiotics are losing their potency as bacteria acquire resistance to them. Thus, tests of antibiotic resistance grow in importance. AAntibioticsNTIBIOTICS Antibiotics are natural or synthetic compounds that kill bacteria. There are a myriad of different antibiotics that act on different structural or biochemical components of bacteria. Antibiotics have no direct effect on virus. Prior to the discovery of the first antibiotic, penicillin, in the 1930s, there were few effective ways of combating bacterial infections. Illnesses such as pneumonia, tuberculosis, and typhoid fever were virtually untreatable, and minor bacterial Antibiotic susceptible and resistant strains of Stapylococcus. womi_A 5/6/03 1:07 PM Page 24