Chapter 20  Antimicrobial Drugs 575 Other Antifungal Drugs Griseofulvin is an antibiotic produced by a species of Penicillium It has the interesting property of being active against superficial dermatophytic fungal infections of the hair (tinea capitis, or ringworm) and nails, even though its route of administration is oral The drug apparently binds selectively to the keratin found in the skin, hair follicles, and nails Its mode of action is primarily to block microtubule assembly, which interferes with mitosis and thereby inhibits fungal reproduction Tolnaftate is a common alternative to miconazole as a topical agent for the treatment of athlete’s foot Its mechanism of action is not known Undecylenic acid is a fatty acid that has antifungal activity against athlete’s foot, although it is not as effective as tolnaftate or the imidazoles Pentamidine is used in treating Pneumocystis pneumonia, a frequent complication of AIDS It also is useful in treating several protozoan-caused tropical diseases The drug’s mode of action is unknown, but it appears to bind DNA CHECK YOUR UNDERSTANDING ✓ What sterol in the cell membrane of fungi is the most common target for antifungal action? 20-13 Antiviral Drugs In developed parts of the world, it is estimated that at least 60% of infectious illnesses are caused by viruses, and about 15% by bacteria Every year, at least 90% of the U.S population suffers from a viral disease Even so, compared to the number of antibiotics available for treating bacterial diseases, there are relatively few antiviral drugs Many of the recently developed antiviral drugs are directed against HIV, the pathogen responsible for the pandemic of AIDS Therefore, as a practical matter the discussion of antivirals is often separated into agents that are directed at chemotherapy of HIV (see page 542) and those with more general (non-HIV) applications (see Table 20.5) Because viruses replicate within the host’s cells, very often using the genetic and metabolic mechanisms of the host’s own cells, it is relatively difficult to target the virus without damaging the host’s cellular machinery Many of the antivirals in use today are analogs of components of viral DNA or RNA However, as more becomes known about the reproduction of viruses, more targets suggest themselves for antiviral action Nucleoside and Nucleotide Analogs An early, obvious target for antiviral drugs was the reverse transcriptase step found in RNA viruses (page 253) and not used in human DNA This family of drugs has consisted mostly of nucleoside and nucleotide analogs (page 47) Among the nucleoside analogs, acyclovir is the one more widely used (Figure 20.16) Although best known for treating genital herpes, it is generally useful for most herpes virus infections, especially in immunosuppressed individuals The antiviral drugs famciclovir, which can be taken orally, and ganciclovir are derivatives of acyclovir and have a similar mode of action Ribavirin resembles the nucleoside guanine and accelerates the already high mutation rate of RNA viruses until the accumulation of errors reaches a crisis point, killing the virus The nucleoside analog lamivudine is used to treat hepatitis B More recently, a nucleotide analog, adefovir dipivoxil (Hepsera), has been introduced for patients resistant to the nucleoside lamivudine A nucleoside analog, cidofovir, is currently used for treating cytomegalovirus infections of the eye, but this drug is especially interesting because it shows promise as a possible treatment of smallpox Other Enzyme Inhibitors Two inhibitors of the enzyme neuraminidase (page 699) have been introduced for treatment of influenza These are zanamivir (Relenza) and oseltamivir (Tamiflu) Interferons Cells infected by a virus often produce interferon, which inhibits further spread of the infection Interferons are classified as cytokines, discussed in Chapter 17 Alpha interferon (see Chapter 16, page 471) is currently a drug of choice for viral hepatitis infections The production of interferons can be stimulated by a recently introduced antiviral, imiquimod This drug is often prescribed to treat genital warts CHECK YOUR UNDERSTANDING ✓ One of the most widely used antivirals, acyclovir, inhibits the synthesis of DNA Humans also synthesize DNA, so why is the drug still useful in treating viral infections? 20-14 Antivirals for Treating HIV/AIDS The interest in effective treatments for the pandemic of HIV infections requires a separate discussion of the many antiviral drugs developed for this HIV is an RNA virus, and its reproduction depends on the enzyme reverse transcriptase, which controls the synthesis of RNA from DNA (see page 388) In fact, the term antiretroviral currently implies that a drug is used to treat HIV infections (see the discussion of HAART on page 553) A well-known example of a nucleoside analog is zidovudine An example of a nucleotide analog is tenofovir In consideration of the large number of drugs required to treat HIV, especially to minimize development of resistant strains, combinations of drugs have been developed An example is Atripla, which combines tenofovir, emtricitabine, and efavirenz Not all drugs that inhibit reverse transcriptase are nucleoside or nucleotide analogs For example, a few non-nucleoside agents, such as nevirapine, block RNA synthesis by other mechanisms As the reproduction of HIV became better understood, other approaches to its control became available When the host cell (at the direction of the infecting HIV) makes a new virus, it must begin by cutting up large proteins with protease enzymes 576 Part Three  Interaction between Microbe and Host O O Guanine HN H2N C HOCH2 H C N C N C N HN CH N2N C C N C N C N CH HOCH2 O O H H HO H CH2 H CH2 Acyclovir Deoxyguanosine (a) Acyclovir structurally resembles the nucleoside deoxyguanosine Phosphate Normal thymidine kinase Guanine nucleotide DNA polymerase Incorporated into DNA Nucleoside (b) The enzyme thymidine kinase combines phosphates with nucleosides to form nucleotides, which are then incorporated into DNA Phosphate Thymidine kinase in virus-infected cell Acyclovir (resembles nucleoside) False nucleotide (acyclovir triphosphate) DNA polymerase blocked by false nucleotide Assembly of DNA stops (c) Acyclovir has no effect on a cell not infected by a virus, that is, with normal thymidine kinase In a virally infected cell, the thymidine kinase is altered and converts the acyclovir (which resembles the nucleoside deoxyguanosine) to a false nucleotide, which blocks DNA synthesis by DNA polymerase Figure 20.16  The structure and function of the antiviral drug acyclovir Q Why are viral infections generally difficult to treat with chemotherapeutic agents? The resulting fragments are then used to assemble new viruses Analogs of amino acid sequences in the large proteins can serve as inhibitors of these proteases by competitively interfering with their activity The protease inhibitors atazanavir, indinavir, and saquinavir have proved especially effective when combined with inhibitors or reverse transcriptase Drugs that use new targets of HIV reproduction are being considered, and several are undergoing clinical tests Among these are integrase inhibitors, which inhibit an enzyme that integrates viral DNA into the DNA of the infected cell The first of this new class of HIV antivirals to be approved is raltegravir Viral infection obviously requires entry into the cell Entry inhibitors include antivirals that target the receptors that HIV uses to bind to the cell before entry, such as CCR5 (see Figure 19.13, page 546) The first of a c1ass of drugs that target this infection step is maraviroc Entry of HIV into the cell can also be blocked by fusion inhibitors such as enfuvirtide This is a synthetic peptide that blocks cell fusion and entry by mimicking a region of Chapter 20  Antimicrobial Drugs 577 the gp41 HIV-l envelope (again, see Figure 19.13) It is, however, dauntingly expensive and must be injected twice daily Antiprotozoan and Antihelminthic Drugs For hundreds of years, quinine from the bark of the Peruvian cinchona tree was the only drug known to be effective for treating a parasitic infection (malaria) Peruvian natives had observed that quinine, which is an effective muscle relaxant, controlled the shivering symptomatic of malarial fever Actually, this characteristic is unrelated to quinine’s toxicity to the protozoan that causes malaria It was first introduced into Europe in the early 1600s and was known as “Jesuit’s powder.” There are now many antiprotozoan and antihelminthic drugs, although many of them are still considered experimental This does not preclude their use, however, by qualified physicians The Centers for Disease Control and Prevention (CDC) provides several of them on request when they are not available commercially Antiprotozoan Drugs Quinine is still used to control the protozoan disease malaria, but synthetic derivatives, such as chloroquine, have largely replaced it For preventing malaria in areas where the disease has developed resistance to chloroquine, the new drug mefloquine (Lariam) is often recommended, although serious psychiatric side effects have been reported As resistance to the most widely used and cheapest drug, chloroquine, becomes almost universal, the products of a Chinese shrub, artemisinin and artemisinin-based combination therapies (ACTs), have become the principal treatment of malaria Artemisinin was a traditional Chinese medicine long used for controlling fevers: Chinese scientists, following this lead, identified its antimalarial properties in 1971 ACTs act by killing the asexua1 stages of Plasmodium spp in the blood (Figure 12.18 on page 352), and they also affect the sexual stages that transmit the infection by mosquitoes Compared to choroquinine, ACTs are expensive—a problem in malaria-prone areas This has led to widespread distribution of low-cost, but ineffective, counterfeit ACTs Some of these contain enough of the genuine drug to evade simple tests, but these low dosages are accelerating development of resistance Quinacrine is the drug of choice for treating the protozoan disease giardiasis Diiodohydroxyquin (iodoquinol) is an important drug prescribed for several intestinal amebic diseases, but its dosage must be carefully controlled to avoid optic nerve damage Metronidazole (Flagyl) is one of the most widely used antiprotozoan drugs It is unique in that it acts not only against parasitic protozoa but also against obligately anaerobic bacteria For example, as an antiprotozoan agent, it is the drug of choice for vaginitis caused by Trichomonas vaginalis It is also used in treating giardiasis and amoebic dysentery The mode of action is to interfere with anaerobic metabolism, which incidentally these protozoans share with certain obligately anaerobic bacteria, such as Clostridium Tinidazole, a drug similar to metronidazole, is effective in treating giardiasis, amebiasis, and trichomoniasis Another antiprotozoan agent, and the first to be approved for the chemotheraphy of diarrhea caused by Cryptosporidium hominis, is nitazoxanide It is active in treating giardiasis and amebiasis Interestingly, it is also effective in treating several helminthic diseases, as well as having activity against some anaerobic bacteria Antihelminthic Drugs With the increased popularity of sushi, a Japanese specialty often made with raw fish, the CDC began to notice an increased incidence of tapeworm infections To estimate the incidence, the CDC documents requests for niclosamide, which is the usual first choice in treatment The drug is effective because it inhibits ATP production under aerobic conditions Praziquantel is about equally effective for the treatment of tapeworms; it kills worms by altering the permeability of their plasma membranes Praziquantel has a broad spectrum of activity and is highly recommended for treating several fluke-caused diseases, especially schistosomiasis It causes the helminths to undergo muscular spasms and also makes them susceptible to attack by the immune system Apparently, its action exposes surface antigens, which antibodies can then reach Mebendazole and albendazole are broad-spectrum antihelminthics that have few side effects and have become the drugs of choice for treating many intestinal helminthic infections The mode of action of both drugs is to inhibit the formation of microtubules in the cytoplasm, which interferes with the absorption of nutrients by the parasite These drugs are also widely used in the livestock industry; for veterinary applications they are relatively more effective in ruminant animals Ivermectin is a drug with a wide range of applications It is known to be produced by only one species of organism, Streptomyces avermectinius, which was isolated from the soil near a Japanese golf course It is effective against many nematodes (roundworms) and several mites (such as scabies), ticks, and insects (such as head lice) (Some mites and insects happen to share certain similar metabolic channels with affected helminths.) Its primary use has been in the livestock industry as a broad-spectrum antihelminthic Its exact mode of action is uncertain, but the final result is paralysis and death of the helminth without affecting mammalian hosts CHECK YOUR UNDERSTANDING ✓ What was the first drug available for use against parasitic infections? 20-15 Tests to Guide Chemotherapy LEARNING OBJECTIVE 20-16 Describe two tests for microbial susceptibility to chemo­ therapeutic agents Different microbial species and strains have different degrees of susceptibility to different chemotherapeutic agents Moreover, 578 Part Three  Interaction between Microbe and Host MIC MIC Figure 20.18  The E test (for epsilometer), a gradient diffusion method that determines antibiotic sensitivity and estimates minimal inhibitory concentration (MIC).  The plastic strip, which is placed on an agar surface inoculated with test bacteria, contains an increasing gradient of the antibiotic The MIC in μg/ml is clearly shown Figure 20.17  The disk-diffusion method for determining the activity Q Q dardized incubation The diameter of the zone can be measured; in general, the larger the zone, the more sensitive the microbe is to the antibiotic The zone diameter is compared to a standard table for that drug and concentration, and the organism is reported as sensitive, intermediate, or resistant For a drug with poor solubility, however, the zone of inhibition indicating that the microbe is sensitive will be smaller than for another drug that is more soluble and has diffused more widely Results obtained by the disk-diffusion method are often inadequate for many clinical purposes However, the test is simple and inexpensive and is most often used when more sophisticated laboratory facilities are not available A more advanced diffusion method, the E test, enables a lab technician to estimate the minimal inhibitory concentration (MIC), the lowest antibiotic concentration that prevents visible bacterial growth A plastic-coated strip contains a gradient of antibiotic concentrations, and the MIC can be read from a scale printed on the strip (Figure 20.18) of antimicrobials. Each disk contains a different chemo­therapeutic agent, which diffuses into the surrounding agar The clear zones indicate inhibition of growth of the microorganism swabbed onto the agar surface Which agent is the most effective against the bacterium being tested? the susceptibility of a microorganism can change with time, even during therapy with a specific drug Thus, a physician must know the sensitivities of the pathogen before treatment can be started However, physicians often cannot wait for sensitivity tests and must begin treatment based on their “best guess” estimation of the most likely pathogen causing the illness Several tests can be used to indicate which chemotherapeutic agent is most likely to combat a specific pathogen However, if the organisms have been identified—for example, Pseudomonas aeruginosa, beta-hemolytic streptococci, or gonococci—certain drugs can be selected without specific testing for susceptibility Tests are necessary only when susceptibility is not predictable or when antibiotic resistance problems develop The Diffusion Methods Probably the most widely used, although not necessarily the best, method of testing is the disk-diffusion method, also known as the Kirby-Bauer test (Figure 20.17) A Petri plate containing an agar medium is inoculated (“seeded”) uniformly over its entire surface with a standardized amount of a test organism Next, filter paper disks impregnated with known concentrations of chemotherapeutic agents are placed on the solidified agar surface During incubation, the chemotherapeutic agents diffuse from the disks into the agar The farther the agent diffuses from the disk, the lower its concentration If the chemotherapeutic agent is effective, a zone of inhibition forms around the disk after a stan- What is the MIC of the E test on the left? Broth Dilution Tests A weakness of the diffusion method is that it does not determine whether a drug is bactericidal and not just bacteriostatic A broth dilution test is often useful in determining the MIC and the minimal bactericidal concentration (MBC) of an antimicrobial drug The MIC is determined by making a sequence of decreasing concentrations of the drug in a broth, which is then inoculated with the test bacteria (Figure 20.19) The wells that not show growth (higher concentration than the MIC) can be cultured in broth or on agar plates free of the drug If growth occurs in this Chapter 20  Antimicrobial Drugs 579 Figure 20.19  A microdilution, or microtiter, plate used for testing for minimal inhibitory concentration (MIC) of antibiotics.  Such plates contain as many as 96 shallow wells that contain measured concentrations of antibiotics They are usually purchased frozen or freeze dried (page 168) The test microbe is added simultaneously, with a special dispenser, to all the wells in a row of test antibiotics A button of growth appears if the antibiotic has no effect on the microbe; the microbe is recorded as not sensitive If there is no growth in a well, the microbe is sensitive to the antibiotic at that concentration To ensure that the microbe is capable of growth in the absence of the antibiotic, wells that contain no antibiotic are also inoculated (positive control) To ensure against contamination by unwanted microbes, wells that contain nutrient broth but no antibiotics or inoculum are included (negative control) Q Doxycycline (Growth in all wells, resistant) Sulfamethoxazole (Trailing end point; usually read where there is an estimated 80% reduction in growth) Streptomycin (No growth in any well; sensitive at all concentrations) Ethambutol (Growth in fourth wells; equally sensitive to ethambutol and kanamycin) What is MIC? Kanamycin Decreasing concentration of drug broth, the drug was not bactericidal, and the MBC can be determined Determining the MIC and MBC is important because it avoids the excessive or erroneous use of expensive antibiotics and minimizes the chance of toxic reactions that larger-than-necessary doses might cause Dilution tests are often highly automated The drugs are purchased already diluted into broth in wells formed in a plastic tray A suspension of the test organism is prepared and inoculated into all the wells simultaneously by a special inoculating device After incubation, the turbidity may be read visually, although clinical laboratories with high workloads may read the trays with special scanners that enter the data into a computer that provides a printout of the MIC Other tests are also useful for the clinician; a determination of the microbe’s ability to produce β-lactamase is one example One popular, rapid method makes use of a cephalosporin that changes color when its β-lactam ring is opened In addition, a measurement of the serum concentration of an antimicrobial is especially important when toxic drugs are used These assays tend to vary with the drug and may not always be suitable for smaller laboratories The hospital personnel responsible for infection control prepare periodic reports called antibiograms that record the susceptibility of organisms encountered clinically These reports are especially useful for detecting the emergence of strains of pathogens resistant to the antibiotics in use in the institution CHECK YOUR UNDERSTANDING ✓ In the disk-diffusion (Kirby-Bauer) test, the zone of inhibition indicating sensitivity around the disk varies with the antibiotic Why? 20-16 Clinical Case Dr Singh sends her sample of P aeruginosa to the CDC for analysis (The ophthalmologist in the other P aeruginosa case also sends a sample.) Using a broth dilution assay, the MIC against these bacteria is 100 μg/ml The decimal reduction time (DRT) of gentamicin against this bacterium at 4°C was determined to be days and at 23°C, 20 How much time would be required to kill 200 cells at each temperature? (Hint: See Chapter 7.) 559 570 579 581 584 585 ▲ Resistance to Antimicrobial Drugs LEARNING OBJECTIVE 20-17 Describe the mechanisms of drug resistance One of the triumphs of modern medicine has been the development of antibiotics and other antimicrobials But the development of resistance to them by the target microbes is an increasing concern To illustrate this concept, human populations often have a relative resistance to diseases to which they have been exposed for many generations For example, when Europeans first colonized tropical climes, they proved highly susceptible to diseases to which they had never been exposed, although the local populations were relatively resistant Antibiotics represent, in a sense, a disease for bacteria When first exposed to a new antibiotic, the susceptibility of microbes tends to be high, and their mortality rate is also high; there may be only a handful of survivors from a FOUNDATION FIGURE 20.20 Bacterial Resistance to Antibiotics Blocking entry Antibiotic Inactivation by enzymes Antibiotic KEYCONCEPTS • There are only a few mechanisms of microbial resistance to antimicrobial agents: blocking the drug’s entry into the cell, inactivation of the drug by enzymes, alteration of the drug’s target site, efflux of the drug from the cell, or alteration of the metabolic pathways of the host • The mechanisms of bacterial resistance to antibiotics are limited Knowledge of these mechanisms is critical for understanding the limitations of antibiotic use Antibiotic Altered target molecule Enzymatic action Alteration of target molecule Inactivated antibiotic Efflux of antibiotic population of billions The surviving microbes usually have some genetic characteristic that accounts for their survival, and their progeny are similarly resistant Such genetic differences arise from random mutations These mutational differences can be spread horizontally among bacteria by processes such as conjugation (page 282) or transduction (page 234) Drug resistance is often carried by plasmids or by small segments of DNA called transposons, which can jump from one piece of DNA to another (Chapter 8, page 237) Some plasmids, including those called resistance (R) factors, can be transferred between bacterial cells in a population and between different but closely related bacterial populations (see Figure 8.28a, page 236) R factors often contain genes for resistance to several antibiotics Once acquired, however, the mutation is transmitted by normal reproduction, and the progeny carry the genetic characteristics of the parent microbe Because of the rapid reproductive rate of bacteria, only a short time elapses before practically the entire population is resistant to the new antibiotic Bacteria that are resistant to large numbers of antibiotics are popularly designated as superbugs Although the most publicized of superbugs is MRSA (page 568), superbug status has also been assigned to a range of bacteria, both gram-positive and gramnegative Often cited among these are Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, 580 Pseudomonas aeruginosa, and species of Enterobacter Faced with infections by such pathogens, medical science has only limited treatment options Mechanisms of Resistance There are only a few major mechanisms by which bacteria become resistant to chemotherapeutic agents See Figure 20.20 At least one clinically troublesome bacterium, Acinetobacter baumanii, has developed resistance by means of all five of the major target sites illustrated in Figure 20.20 Enzymatic Destruction or Inactivation of the Drug Destruction or inactivation by enzymes mainly affects antibiotics that are natural products, such as the penicillins and cephalosporins Totally synthetic chemical groups of antibiotics such as the fluoroquinolones are less likely to be affected in this manner, although they can be neutralized in other ways This may simply reflect the fact that the microbes have had fewer years to adapt to these unfamiliar chemical structures The penicillin/ cephalosporin antibiotics, and also the carbapenems, share a structure, the β-lactam ring, which is the target for β-lactamase enzymes that selectively hydrolyze it Nearly 200 variations of these enzymes are now known, each effective against minor variations in the β-lactam ring structure When this problem first appeared, the basic penicillin molecule was modified The first of these Chapter 20  Antimicrobial Drugs 581 penicillinase-resistant drugs was methicillin (see page 568), but resistance to methicillin soon appeared The best-known of these resistant bacteria is the widely publicized pathogen MRSA, which is resistant to practically all antibiotics, not just methicillin (see the box on page 423) In a recent year, the CDC ascribed 19,000 deaths to this pathogen In hospital patients, invasive infections with MRSA can cause as much as 20% mortality Also, S aureus is not the only bacterium of concern; other important pathogens, such as Streptococcus pneumoniae, have also developed resistance to β-lactam antibiotics Furthermore, MRSA has continued to develop resistance against a succession of new drugs such as vancomycin (the “antibiotic of last resort”), even though this antibiotic has a mode of action against cell wall synthesis that is totally different from that of the penicillins These highly adaptable bacteria have even developed resistance against antibiotic combinations that include clavulanic acid, specifically developed as an inhibitor of β-lactamases (see page 568) At first, MRSA was almost exclusively a problem in hospitals and similar health-related settings, accounting for about 20% of bloodstream infections there However, it is now the cause of frequent outbreaks in the general community, is more virulent, and affects otherwise healthy individuals These strains produce a toxin, a leukocidin, that destroys neutrophils, a primary innate defense against infection In consequence, the descriptive terminology now differentiates community-associated MRSA from health care–associated MRSA There is an obvious need for rapid tests to detect MRSA bacteria (generally from nasal swabs) so that infections can be isolated and transmission reduced The most promising of these are based on PCR technology and yield good results within or hours Prevention of Penetration to the Target Site within the Microbe Gram-negative bacteria are relatively more resistant to antibiotics because of the nature of their cell wall, which restricts absorption of many molecules to movements through openings called porins (see page 86) Some bacterial mutants modify the porin opening so that antibiotics are unable to enter the periplasmic space Perhaps even more important, when β-lactamases are present in the periplasmic space, the antibiotic remains outside the cell, where the enzyme, which is too large to enter even through an unmodified porin, can reach and inactivate it Alteration of the Drug’s Target Site The synthesis of proteins involves the movement of a ribosome along a strand of messenger RNA, as shown in Figure 20.4 Several antibiotics, especially those of the aminoglycoside, tetracycline, and macrolide groups, utilize a mode of action that inhibits protein synthesis at this site Minor modifications at this site can neutralize the effects of antibiotics without significantly affecting cellular function Interestingly, the main mechanism by which MRSA gained ascendancy over methicillin was not by a new inactivating enzyme, but by modifying the penicillin-binding protein (PBP) on the cell’s membrane β-Lactam antibiotics act by binding with the PBP, which is required to initiate the cross-linking of peptidoglycan and form the cell wall MRSA strains become resistant because they have an additional, modified, PBP The antibiotics continue to inhibit the activity of the normal PBPs, preventing their participation in forming the cell wall But the additional PBP present on the mutants, although it binds weakly with the antibiotic, still allows synthesis of cell walls that is adequate for survival of MRSA strains Clinical Case It would take 12 days to kill 200 cells at 4°C and 60 minutes at 23°C The gentamicin is more effective at the warmer temperature, but the tissues will deteriorate too quickly at this temperature Hence, the corneas are stored at 4°C to preserve the tissue even though gentamicin is less effective at 4°C How might storage of the corneas in gentamicin have contributed to these infections 559 570 579 581 584 585 ▲ Rapid Efflux (Ejection) of the Antibiotic Certain proteins in the plasma membranes of gram-negative bacteria act as pumps that expel antibiotics, preventing them from reaching an effective concentration This mechanism was originally observed with tetracycline antibiotics, but it confers resistance among practically all major classes of antibiotics Bacteria normally have many such efflux pumps to eliminate toxic substances Variations of Mechanisms of Resistance Variations on these mechanisms also occur For example, a microbe could become resistant to trimethoprim by synthesizing very large amounts of the enzyme against which the drug is targeted Conversely, polyene antibiotics can become less effective when resistant organisms produce smaller amounts of the sterols against which the drug is effective Of particular concern is the possibility that such resistant mutants will increasingly replace the susceptible normal populations Figure 20.21 shows how rapidly bacterial numbers increase as resistance develops Antibiotic Misuse Antibiotics have been much misused, nowhere more so than in the less-developed areas of the world Well-trained personnel are scarce, especially in rural areas, which is perhaps one reason why 582 Part Three  Interaction between Microbe and Host Bacteria (number/ml) 108 50 107 40 106 30 Bacteria count 105 20 104 10 103 10 Antibiotic resistance (mg/ml) Antibiotic resistance of bacterial population measured by amount of antibiotic needed to control growth Initiation of antibiotic therapy 11 Days Figure 20.21  The development of an antibiotic-resistant mutant during antibiotic therapy.  The patient, suffering from a chronic kidney infection caused by a gram-negative bacterium, was treated with streptomycin The red line records the antibiotic resistance of the bacterial population Until about the fourth day, essentially all of the bacterial population is sensitive to the antibiotic At this time, resistant mutants that require 50,000 μg/ml of antibiotic (a very high amount) to control them appear, and their numbers increase rapidly The black line records the bacterial population in the patient After antibiotic therapy is begun, the population declines until the fourth day At this time, mutants in the population that are resistant to streptomycin appear The bacterial population in the patient rises as these resistant mutants replace the sensitive population Q This test used streptomycin and a gram-negative bacterium What would the lines have looked like if penicillin G had been the antibiotic? antibiotics can almost universally be purchased without prescriptions in these countries A survey in rural Bangladesh, for example, showed that only 8% of antibiotics had been prescribed by a physician In much of the world, antibiotics are sold to treat headaches and for other inappropriate uses (Figure 20.22) Even when the use of antibiotics is appropriate, dose regimens are usually shorter than needed to eradicate the infection, thereby encouraging the survival of resistant strains of bacteria Outdated, adulterated (impure), and even counterfeit antibiotics are common The developed world is also contributing to the rise of antibiotic resistance The CDC estimates that in the United States, 30% of the antibiotic prescriptions for ear infections, 100% of the prescriptions for the common cold, and 50% of prescriptions for sore throats were unnecessary or inappropriate to treat the problem pathogen At least half of the more than 100,000 tons of antibiotics consumed in the United States each year are not used to treat disease but are used in animal feeds to promote growth— a practice that many people feel should be controlled (see the box on the facing page) Figure 20.22  Antibiotics have been sold without prescriptions for many decades in much of the world Q How does this practice lead to development of resistant strains of pathogens? Cost and Prevention of Resistance Antibiotic resistance is costly in many ways beyond those that are apparent in higher rates of disease and mortality Developing new drugs to replace those that have lost effectiveness is costly Almost all of these drugs will be more expensive, sometimes priced in a range that makes them difficult to afford even in highly developed countries In less-developed parts of the world, the costs are simply unaffordable There are many strategies that patients and health care workers can adopt to prevent the development of resistance Even if they feel they have recovered, patients should always finish the full regimen of their antibiotic prescriptions to discourage the survival and proliferation of the antibiotic-resistant microbes Patients should never use leftover antibiotics to treat new illnesses or use antibiotics that were prescribed to someone else Health care workers should avoid unnecessary prescriptions and ensure that the choice and dosages of antimicrobials are appropriate to the situation Prescribing the most specific antibiotic possible, instead of broad-spectrum antimicrobials, also decreases the chances that the antibiotic will inadvertently cause resistance among the patient’s normal flora Strains of bacteria that are resistant to antibiotics are particularly common among hospital workers, where antibiotics are in constant use When antibiotics are injected, as many are, the syringe must first be held vertically and cleared of air bubbles, a practice that causes aerosols of the antibiotic solution to form When the nurse or physician inhales these aerosols, the microbial inhabitants of the nostrils are exposed to the drug Inserting the needle into sterile cotton can prevent aerosols from forming Many hospitals have special monitoring committees to review the use of antibiotics for effectiveness and cost Clinical Focus 583 Livestock growers use antibiotics in the feed of closely penned animals because the drugs reduce the number of bacterial infections and accelerate the animals’ growth Today, more than half the antibiotics used worldwide are given to farm animals Meat and milk that reach the consumer’s table are not heavily laden with anti- biotics, so what is the risk of using antibiotics in animal feed? The constant presence of antibiotics in these animals is an example of “survival of the fittest.” Antibiotics kill some bacteria, but other bacteria have properties that help them survive How bacteria acquire resistance genes? Resistance to antimicrobial drugs in bacteria results from mutations These mutations can be transmitted to other bacteria via horizontal gene transfer (Figure A) What evidence would show that veterinary use of antibiotics promotes resistance? Vancomycin-resistant Enterococcus spp (VRE) were first isolated in France in 1986 and were found in the United States in 1989 Vancomycin and another glycopeptide, avoparcin, were widely used in animal feed in Europe In 1996, veterinary use of avoparcin was banned in Germany After the ban, VRE-positive samples decreased from 100% to 25%, and the human carrier rate dropped from 12% to 3% Campylobacter jejuni is a commensal in the intestines of poultry What human disease does C jejuni cause? Annually in the United States, Campylo­ bacter causes over million foodborne What FQs are used to treat human infections? (Hint: See Table 20.3.) S enterica after conjugation E coli infections Fluoroquinolone (FQ)-resistant C jejuni in humans emerged in the 1990s (Figure B) The emergence corresponds with the presence of FQ-resistant C jejuni in grocery store-purchased chicken meat FQ-resistant C jejuni could be selected for in patients who had previously taken an FQ However, a study of Campylobacter isolates from patients between 1997 and 2001 showed that patients infected with FQ-resistant C jejuni had not taken an FQ prior to their illness and had not traveled out of the United States Suggest a way to decrease emergence of FQ resistance The use of FQ in chicken feed was banned Resistance plasmid Figure A  Cephalosporin-resistance in E coli transferred by conjugation to Salmonella enterica in the intestinal tracts of turkeys of meat during processing at the slaughterhouse, and (3) use proper storage and cooking methods in 2005, in hope of reducing FQ resistance A variety of approaches may be necessary to reduce the possibility of illness: (1) prevent colonization in the animals at the farm, (2) reduce fecal contamination Data sources: CDC and National Microbial Resistance Monitoring System 30 FQ for humans Percent FQ-resistant Campylobacter As you read through this box, you will encounter a series of questions that microbiol­ ogists ask as they combat antibiotic resistance Try to answer each question before going on to the next one S enterica Antibiotics in Animal Feed Linked to Human Disease FQ for poultry FQ for poultry discontinued 25 20 15 10 1986 1988 1990 1992 1994 1996 1998 Year 2000 2002 2004 2006 2008 Figure B  Flouroquinolone-resistant Campylobacter jejuni in the United States, 1986–2008 584 Part Three  Interaction between Microbe and Host Area of synergistic inhibition, clear Disk with antibiotic amoxicillin-clavulanic acid Area of growth, cloudy Disk with antibiotic aztreonam (Figure 20.23) This phenomenon, called synergism, was introduced earlier For example, in the treatment of bacterial endocarditis, penicillin and streptomycin are much more effective when taken together than when either drug is taken alone Damage to bacterial cell walls by penicillin makes it easier for streptomycin to enter Other combinations of drugs can show antagonism For example, the simultaneous use of penicillin and tetracycline is often less effective than when either drug is used alone By stopping the growth of the bacteria, the bacteriostatic drug tetracycline interferes with the action of penicillin, which requires bacterial growth CHECK YOUR UNDERSTANDING Figure 20.23  An example of synergism between two different antibiotics. The photograph shows the surface of a Petri plate seeded with bacteria The paper disk at the left contains the antibiotic amoxicillin plus clavulanic acid The disk on the right contains the antibiotic aztreonam The dashed circles drawn over the photo show the clear areas surrounding each disk where bacterial growth would have been inhibited if there had been no synergy The additional clear area between these two areas and outside the drawn circles illustrates inhibition of bacterial growth through the effects of synergy Q What would the plate look like if the two antibiotics had been antagonistic? ✓ Tetracycline sometimes interferes with the activity of penicillin How? 20-18 Clinical Case Gentamicin is used in commercial storage medium for corneas because it has been reported to be more effective than penicillin or cephalothin in reducing the colony counts of staphylococci and gram-negative rods in a buffered storage medium Adding gentamicin is intended to preserve the medium before use, not to sterilize corneal tissue CHECK YOUR UNDERSTANDING ✓ What is the most common mechanism that a bacterium uses to resist the effects of penicillin? 20-17 Antibiotic Safety In our discussions of antibiotics, we have occasionally mentioned side effects These may be potentially serious, such as liver or kidney damage or hearing impairment Administering almost any drug involves assessing risks against benefits; this is called the therapeutic index Sometimes, the use of another drug can cause toxic effects that not occur when the drug is taken alone One drug may also neutralize the intended effects of the other For example, a few antibiotics have been reported to neutralize the effectiveness of contraceptive pills Also, some individuals may have hypersensitivity reactions, for example, to penicillins (see the box on page 537) A pregnant woman should take only those antibiotics that are classified by the U.S Food and Drug Administration as presenting no evidence of risk to the fetus Effects of Combinations of Drugs LEARNING OBJECTIVE 20-18 Compare and contrast synergism and antagonism The chemotherapeutic effect of two drugs given simultaneously is sometimes greater than the effect of either given alone Storage in an antibiotic could select for antibiotic-resistant bacteria What antimicrobial drug would work best to treat P aeruginosa? 559 570 579 581 584 585 ▲ The Future of Chemotherapeutic Agents LEARNING OBJECTIVE 20-19 Identify three areas of research on new chemotherapeutic agents As pathogens develop resistance to current chemotherapeutic agents, the need for new agents becomes more pressing However, developing new antimicrobial agents is not especially profitable Like vaccines, antimicrobials are used only on infrequent occasions for limited periods of time Pharmaceutical companies are understandably more interested in developing drugs that treat chronic conditions, such as high blood pressure or diabetes, for which a patient requires years of regular medication This has led to something of a “perfect storm”—increasing drug resistance combined with a decline in the development of new antibiotics Existing antibiotics continue to encounter problems with resistance in large part because their developers have relied on a limited range of targets (see Figure 20.2) A truly new ... penicillinase-resistant drugs was methicillin (see page 568), but resistance to methicillin soon appeared The best-known of these resistant bacteria is the widely publicized pathogen MRSA, which is resistant... the fourth day At this time, mutants in the population that are resistant to streptomycin appear The bacterial population in the patient rises as these resistant mutants replace the sensitive population... resistance, 17, 451 See also immunity to antibiotic drugs, 12, 237 See also antibiotic resistance to drought, modified into crop plants, 264 resistance factors in bacteria See R factors resistance