CHAPTER 38 Principles of Antibiotic Therapy*
In 1967 the U.S. Surgeon General declared, “The time has come to close the book on infectious diseases.” In 1993, 17 million people died of infectious diseases throughout the world, with 11.4 million deaths (mostly of children) caused by bacterial diarrhea and pneumonia. In the same year, 15.6 million died of cardiovascular disease and cancer combined.69 The four primary disease killers caused by infection are the same as in 1900: diarrhea, pneumonia, tuberculosis, and malaria.19 One third of the world population has tuberculosis, and Africa accounts for 90% of the 300 to 500 million new cases of malaria annually, with 1.5 to 2.7 million deaths per year. In World War II, 55 million people were killed; by 2010, 65 million will have died of acquired immunodeficiency syndrome (AIDS).
The Surgeon General was echoing the prevailing wisdom of the 1960s era of optimism regarding antibiotics. In the late 1950s the medical community became alarmed at the extent and rapidity of Staphylococcus aureus resistance to the penicillins, erythromycin, and tetracyclines and the discovery that bacteria could transfer the genes for antibiotic resistance among themselves. In the early 1960s, a plethora of new antibiotics became available: cephalosporins, β-lactamase–resistant penicillins, lincosamides, and new aminoglycosides. The belief that humankind would always stay several steps ahead of the microbes because they could not possibly match human intelligence was widely accepted. Assumptions are the genesis of most disasters, and, as one of “Murphy’s laws” states, “Optimism indicates that the situation is not clearly understood.”
The U.S. Centers for Disease Control and Prevention estimates that 65,000 to 90,000 deaths annually in U.S. hospitals result from nosocomial (hospital-acquired) infections. This figure may be a significant underestimate and the number may be closer to 200,000 to 300,000 because infectious disease deaths may be misclassified as cardiac arrest or respiratory or renal failure instead of their underlying microbial causes. In 1977, 100,000 gram-negative nosocomial bacteremic deaths were estimated annually in the United States57; bloodstream infections (septicemia and bacteremia) alone, among all nosocomial infections, may now be the eighth leading cause of death in the United States.114,115
Hospitals are currently plagued by vancomycin-resistant enterococci, vancomycin-resistant or glycopeptide-intermediate–resistant S. aureus, coagulase-negative staphylococci (CoNS), and other microorganisms resistant to multiple antibiotics, particularly Streptococcus pneumoniae and extended β-lactamase–producing enteric bacilli. The community is now beset by methicillin-resistant S. aureus (MRSA), which was previously thought to be a problem only in hospitals; penicillin-resistant and macrolide-resistant S. pneumoniae and viridans group streptococci (VGS); β-lactamase–producing Haemophilus influenzae and Moraxella catarrhalis; and widespread fluoroquinolone resistance. The oral cavity is home to β-lactam–resistant VGS and β-lactamase–producing Prevotella and Porphyromonas.
Mechanisms for resistance to antibiotics have likely always existed in some form to allow microbes to ensure their survival against competing microorganisms and find a niche in their environment to survive and thrive. Our current problems are of human origin, however—we disturbed the delicate microbial ecology for our own benefit, never realizing how formidable microbial retaliation would be. We are approaching the loss of one of our greatest gifts.
The importance of two medical discoveries that have essentially doubled the human life span in first-world countries since the 1850s—anesthesia and the control of infectious diseases—cannot be overestimated. Without the ability to operate internally within the human body free of excruciating pain, the gains of medical and dental surgery would be void. In the United States in 1776, the average life span was less than 40 years of age. In England in 1853, infectious disease was responsible for 37% of all deaths. At the beginning of the twentieth century in the United States, the infant mortality rate was 100 per 1000; now it is less that 10 per 1000.69 A child in 1900 had a 10% chance of death between ages 1 and 4 years from pneumonia or diarrhea.69 Many adults, infants, and children died of typhus, typhoid, diphtheria, whooping cough, yellow fever, malaria, influenza, measles, smallpox, and streptococcal and staphylococcal infections.
Even before the advent of the modern germ theory of disease in the 1870s, many individuals surmised that filth had a substantial bearing on disease. The “sanitary movement” began in Great Britain in the 1850s and the United States in the 1870s with improvement in wages, housing, education, and personal hygiene. Civil engineers cleaned the streets, water, and air, and cities removed refuse and their attendant rodent vectors of disease. Waste disposal, clean water, and hand hygiene by public health engineering have reduced the transmission of 35 to 40 infectious diseases.31
The modern era of infectious disease began with the first visualization of microbes by Anton van Leeuwenhoek in 1683, the “animicules” of dental plaque scraped from his upper gingiva and killed with salt (the first periodontal chemotherapy).47 In 1776, Edward Jenner administered the first smallpox vaccination. In 1848, Ignaz Semmelweiss introduced clean surgical operating technique (“gentlemen, wash your hands”). In 1854, John Snow showed the link between cholera and drinking water.47
In the 1860s, Louis Pasteur first used the word germ for living entities that produced disease, and Joseph Lister used carbolic acid to disinfect wounds. In the 1870s, Robert Koch proved the bacterial causation of anthrax and tuberculosis, and in the 1880s, Pasteur developed anthrax and rabies vaccines. In 1891, Paul Ehrlich showed that antibodies were responsible for immunity. In 1897, Ivanowski and Beiternick discovered viruses. The mosquito vector for yellow fever was shown in 1900, Treponema pallidum was found to be the cause of syphilis in 1905, human immunodeficiency virus (HIV) was identified in 1983, Helicobacter pylori was discovered as a cause of peptic ulcer in 1984, and the West Nile virus was identified in 1999.47
In the early 1900s, Paul Ehrlich used the term magic bullet for his predicted chemical that would affect only microbial cells and have no effect on mammalian cells. He later used fuchsin and mercury (Salvarsan) to treat syphilis. In 1928, Alexander Fleming serendipitously discovered that a mold, Penicillium chrysogenum, lysed staphylococci; this was later developed to its full potential by the isolation of penicillin from Penicillium notatum by Florey and colleagues at Oxford in the late 1930s and early 1940s. The first use of penicillin was in 1941 on an English police constable with streptococcal and staphylococcal skin abscesses. In the United States, penicillin was first used in 1942 on Anne Miller, who had streptococcal toxemia of pregnancy. All of these firsts have possibly overshadowed arguably the greatest of all medical advances: the demonstration in 1935 by Gerhard Domagk that sulfanilamide could be safely used systemically to treat infectious disease. The “dreaded disease of summer” (poliomyelitis) declined from 57,879 cases in the United States in 1953 to 72 cases in 1965 with the advent of the polio vaccine.71 By 1977, smallpox was eradicated from the world as a contagious disease. During 1900-1997, the American life span increased by 60% to a median age of 76.19
In the developing world, a different story has unfolded. In 1998, the World Health Organization determined that infectious disease caused 25% (13 million) of the 54 million deaths in the world that year, with pneumonia (3.5 million), AIDS (2.3 million), diarrhea (2.2 million), tuberculosis (1.5 million), malaria (1.1 million), and measles (1 million) the top killers.19 The incidence of emerging infections (defined by the Institute of Medicine as new, re-emerging, or drug-resistant infections whose incidence has increased in the last 2 decades or whose incidence threatens to increase) has increased.19 Now included in this category are legionnaires disease, toxic shock syndrome, respiratory syncytial virus, Lyme disease, Nipah virus, Hantavirus, hemorrhagic viral diseases (dengue, Ebola, Marburg), Escherichia coli O157:H7, malaria, yellow fever, cholera, and multidrug-resistant tuberculosis. All of these infections and more are potentially transmitted by 500,000 world refugees and 1.6 billion annual airline passengers, 500 million of which cross borders each year.69
All of the media attention to these potential pathogens has led to a second “germ panic” with the revival of the focal infection theory of disease,73 which alleges that many or most current diseases are caused by various microbes, including cardiovascular disease; various forms of emotional disorders such as obsessive-compulsive disorder, Tourette’s syndrome, autism, and schizophrenia; preterm births; chronic fatigue syndrome; and multiple sclerosis. The first germ panic of 1900-1940 was fostered by the focal infection theory as espoused by Hunter and colleagues, in which a localized infection in one area of the body could move and occur elsewhere in the body and cause various pathologic conditions, such as arthritis, neuritis, myalgia, osteomyelitis, endocarditis, brain abscess, skin abscess, pneumonia, anemia, indigestion, gastritis, pancreatitis, colitis, diabetes, emphysema, goiter, thyroiditis, Hodgkin’s disease, “obscure fever,” nervous diseases, headache, mental apathy, and mental incompetence.73 All of these were disorders for which medicine at the time (and many currently) had no explanations and no answers.
These foci of infection were conveniently located in areas of the body readily accessible to surgery (particularly in the wealthy): teeth, tonsils, and the facial sinuses, leading to an excessive number of dental extractions, tonsillectomies, and other surgeries in the first half of the twentieth century.42,73,117,118 The resurrection of the foci of infection concept today is based on limited scientific evidence and questionable studies that lack attention to sound epidemiologic methods.
Very rarely, microbes leave the oral cavity and metastasize to other areas of the body to initiate a nonspecific inflammatory infectious process manifested as liver, splenic, or brain abscesses or bacterial endocarditis. These microorganisms are almost always VGS and almost never pathogens associated with periodontal disease. That these metastatic infections are so rare is truly remarkable and speaks well for our immune defense mechanisms, particularly in the oral cavity and blood, and the reticence of microorganisms to leave their ecologic niches for foreign environments. Currently, little evidence suggests that the oral cavity is the source of significant systemic disease.72,73
Antibiotics are the most widely abused prescribed drugs on the basis of inappropriate indications, dosages, and duration of use. Approximately half of all antibiotics used in hospitals are given to patients without signs or symptoms of infection, in many cases to “prevent” infections and to ensure that “everything was done” to avoid later criticism. Antibiotics are often used as “drugs of fear”45 to cover for potential errors of omission or commission and prevent a claim of negligence. The abuse of negligence (tort) law has been a major contributing factor to the massive overuse of antibiotics and the attendant mortality rate associated with highly antibiotic-resistant microorganisms.
In hospitals, one third of antibiotics are used empirically, one third for prophylaxis, and one third with appropriate culture and sensitivity tests.69 Because hospitals save money by not using culture and sensitivity tests, the demand has been for broader spectrum antibiotics, which has created a vicious cycle by disturbing the hospital microbial ecology further and fostering even greater microbial resistance.98
Outpatient antibiotic use is characterized by the “80 : 80 rule”: 80% of all antibiotics are used in the community, and 80% are used for respiratory infections—most of which are viral in cause and not amenable to antibiotic therapy.69 Of the 50% of people with acute respiratory illness who seek medical treatment, 50% to 80% may receive an antibiotic, but pneumonia (the only respiratory tract disorder requiring an antibiotic) may account for only 2% of these cases.
The prescribing of antibiotics can vary 15-fold among physicians. Physicians who tend to prescribe many drugs also prescribe many antibiotics. Antibiotic prescriptions are a quick way to end an office visit and reduce return visits.90
Dentists prescribe 7% to 11% of all common antibiotics (β-lactams, macrolides, tetracycline, metronidazole, clindamycin), and abuse of such antibiotics can be substantial.18 In England, 33% to 87% of various antibiotics were judged to be inappropriately prescribed by dentists according to the Dental Practitioners Formulary.74 Experts in England are in agreement that antibiotics are used too long for the management of orofacial infections and that shorter durations are more appropriate and reduce the selection of drug-resistant microbes.56
In a survey of 505 Canadian dentists, the average length of antibiotic therapy was 6.92 days (range 1 to 21 days), and 17.5% did not use the 1997 American Heart Association (AHA) guidelines for endocarditis prophylaxis.27 Two thirds of the dentists used antibiotic prophylaxis for patients with rheumatic fever without rheumatic heart disease; 25%, for patients with HIV/AIDS; 70%, for prosthetic joints; and two thirds, for restorative dentistry not associated with significant bleeding even though not advocated by the AHA.
AHA prophylaxis for patients with cardiac valve prostheses was not used by 20% of dental specialists. The study concluded that antibiotics are underused for symptomatic infections, overused for surgical prophylaxis, and commonly used at suboptimal dosing with prolonged dosing schedules and often not according to antibiotic prophylaxis guidelines.27
In a survey of antibiotic use by 1606 members of the American Association of Endodontists, 12.5% used antibiotics as an analgesic for post-treatment pain; 37.3%, as antibiotic prophylaxis after surgery; 44.8%, after incision and drainage without systemic involvement or patient immunosuppression; and 12% to 54%, for situations in which they are not effective, such as the following: (1) irreversible pulpitis with moderate-severe symptoms with or without apical periodontitis; (2) asymptomatic necrotic pulps with chronic apical periodontitis but no swelling; (3) necrotic pulps with acute apical periodontitis, no swelling, and moderate-severe symptoms; and (4) asymptomatic necrotic pulps with chronic periapical periodontitis with or without a sinus tract.119 The authors concluded that not much had changed in the past 25 years.
Inappropriate antibiotic use in dentistry includes the following situations: (1) antibiotic therapy initiated after surgery to prevent an infection unlikely to occur and not documented effective for this purpose by clinical trials; (2) failure to use prophylactic antibiotics according to the principles established for such use; (3) use of antibiotics as analgesics in endodontics; (4) overuse in situations in which patients are not at risk for metastatic infections; (5) treatment of chronic periodontitis almost totally amenable to mechanical therapy; (6) administration of antibiotics instead of mechanical therapy for periodontitis; (7) long-term administration in the management of periodontal diseases; (8) antibiotic therapy instead of incision and drainage; (9) administration of antibiotics to avoid claims of negligence; and (10) administration in improper situations, dosage, and duration of therapy.69
To appreciate how microbes defend themselves against chemicals in their environment, one must first determine how antimicrobial agents kill microbes or prevent their replication. Antibiotics are chemicals most often, but not always, derived from microorganisms (commonly yeasts and fungi) that are intended in nature to perform as part of the system that maintains the ecologic balance in the microbial world. This system is composed of various entities, including bacteriophages (bacterial viruses); cationic peptides; antibiotics; and the quorum-sensing system that conveys chemical messages to microbes regarding metabolic activities, surface adhesion, colony formation, virulence, and the presence of chemicals intended to do harm. Virtually all clinically useful antibiotics are derived from naturally occurring entities, with only three synthetically produced: sulfonamides, fluoroquinolones, and oxazolidinones.
Antimicrobials affect the viability of microorganisms by five known processes: (1) inhibition of cell wall synthesis, (2) alteration of cell membrane integrity, (3) inhibition of ribosomal protein synthesis, (4) suppression of deoxyribonucleic acid (DNA) synthesis, and (5) inhibition of folic acid synthesis (Table 38-1, Figure 38-1). Microbial cell wall synthesis inhibition and membrane effects are extracytoplasmic, and inhibition of nucleic acid, protein, and folic acid synthesis is intracytoplasmic. Drugs that affect bacterial cell wall or membrane integrity and DNA synthesis are usually, but not always, bactericidal (inducing cell death), and protein and folic acid synthesis inhibitors are usually bacteriostatic (preventing cell growth or replication).
Whether an antimicrobial agent is bactericidal (cidal) or bacteriostatic (static) can also depend on its concentration at the infected site and the particular offending organism because some static drugs become cidal at high concentrations. The previous preference for cidal drugs over static antibiotics (cidal drugs allegedly do not rely on host defenses) has become less distinct because of the appreciation of the long postantibiotic effects (continued antibiotic activity when the drug blood levels have declined) of bacteriostatic drugs.
The principal cell wall inhibitors are β-lactam antibiotics and glycopeptides. Bacterial cell walls are rigid and composed of alternating peptidoglycan (murein) units of N-acetyl-d-glucosamine and N-acetylmuramic acid (NAM). These are cross-linked via short peptides by amide linkages to a d-alanyl group on NAM. Various bacterial enzymes (transglycosylases, transpeptidases, carboxypeptidases, endopeptidases), termed penicillin-sensitive enzymes or penicillin-binding proteins (PBPs), catalyze the formation of the rigid cell wall by incorporating new peptidoglycan into existing peptidoglycan by attaching a free amino group on the NAM-pentapeptide to a terminus opened by displacement of d-alanine. β-Lactam antibiotics competitively inhibit this final transpeptidation reaction to prevent three-dimensional rigid cell wall formation. The internal osmotic pressure of the bacterium causes lysis of the bacterial cell because the wall is no longer an effective barrier.
In addition, in some organisms, the β-lactams inhibit the inhibitor (derepression) of an endogenous bacterial autolysin (N-acetyl-muramyl-l-alanine amidase), which, when activated, causes the lysis of the bacterial cell wall, initiating bacterial suicide. Microbes that lose this autolysin system can become tolerant to antibiotics, with the antibiotic becoming bacteriostatic instead of bactericidal. Glycopeptides inhibit gram-positive bacterial cell wall synthesis by complexing with the d-alanyl-d-alanine portion of the muramyl peptide precursors to inhibit the action of transglycosylase and transpeptidase at a stage just before that of the β-lactams.
Polymyxin B disrupts the integrity of the cell membrane by displacing Ca++ and Mg++ from membrane lipid phosphate groups. Cationic antimicrobial peptides are part of humans’ natural skin and mucosal defense system and act by disrupting cell wall or membrane integrity by an effect on the gram-negative lipopolysaccharide component that literally puts holes in the wall or membrane.
The macrolides bind to the P site of the 50S ribosomal subunit to inhibit RNA-dependent protein synthesis by inhibiting peptidyl transferase or by increasing the dissociation of peptidyl tRNA from the ribosome. Clindamycin similarly attaches to the same 50S subunit and can compete with the macrolides for this site. Cross-resistance between these two disparate antibiotics is common. Tetracyclines attach to the 30S ribosomal subunit to block ribosomal protein synthesis by inhibiting the binding of tRNA to mRNA on the ribosome. Aminoglycosides attach to the 30S subunit to inhibit ribosomal protein synthesis, but may also induce the formation of abnormal bactericidal proteins. Streptogramins (quinupristin-dalfopristin) bind to two different sites on the 50S subunit of the 70S ribosome to prevent newly synthesized peptide chains from extruding from the ribosome. The oxazolidinone, linezolid, attaches to the 50S ribosome near the interface with the 30S subunit to prevent the initiation complex required for bacterial translation.
The 5′ nitro group of metronidazole is reduced in sensitive obligate anaerobes by nitro reductase to cell toxic nitro, nitroso, and hydroxylamine compounds that damage DNA or inhibit its synthesis. Fluoroquinolones inhibit topoisomerase IV and DNA gyrase that control the supercoiling of DNA and DNA replication, recombination, and repair. Fluoroquinolones may also induce the SOS response, which constitutes a repair system of DNA (the bacterial response to DNA damage) that normally functions to inhibit cell division to prevent the replication of damaged DNA. When the SOS repair system is affected by fluoroquinolones, unbalanced growth, vacuoles, filamentation, and cell lysis occur.
Sulfonamides and trimethoprim are antimetabolites that inhibit sequential steps in the bacterial synthesis of folic acid essential for one-carbon transfers in nucleic acid synthesis. Mammalian cells do not synthesize folic acid but acquire it from the environment. Sulfonamides are structural analogues of p-aminobenzoic acid (PABA) and block the conversion of PABA to dihydrofolic acid by inhibiting tetrahydropteroic acid synthetase, which has greater affinity for sulfonamides than PABA. Trimethoprim blocks the next step in folic acid synthesis by inhibiting dihydrofolate reductase, which catalyzes the conversion of dihydrofolic acid to tetrahydrofolic acid.
Microbial resistance to antibiotics has become a major factor in determining when and which antibiotic is used and dosages and length of administration. It also has spurred renewed interest in antibiotic pharmacokinetics and pharmacodynamics.
Procedures designed to reduce antibiotic-resistant pathogenic microorganisms have been developed, including education of health care providers and the general public, improved handwashing techniques, better hospital infection control, isolation of patients with highly resistant bacteria, control of antibiotic use in hospitals through formularies and pharmacist oversight, and the removal of antibiotics for growth promotion in agricultural animals. Many of these programs have had little effect to date.
All microbial resistance is local; the patterns and extent of this resistance are determined by the use of antibiotics in a particular community. What is true in Florida may not be true in Los Angeles or in Paris, London, Rome, or New Delhi. If tetracyclines are used widely in the community for acne or Lyme disease, a high resistance level to the drug is likely to be present in that locale. If not, the microbial resistance level is likely to be low. If an antibiotic or its analogue has been used widely in agriculture, this may strongly influence resistance patterns—to the point of rendering a new antibiotic far less useful. In Taiwan, virginiamycin (a streptogramin) has been used for more than 2 decades as a growth promoter in food animals. When quinupristin-dalfopristin, a new streptogramin, was tested on human bacterial isolates before its clinical introduction, more than 50% of some pathogens were already resistant to the drug. Antibiotics are truly societal drugs that cumulatively affect the individual receiving the drug and many others as well.50
Microorganisms have developed seven known mechanisms to evade the bactericidal or bacteriostatic actions of antimicrobials, as follows: (1) enzymatic inactivation, (2) modification/protection of the target site, (3) limited access of antibiotic (altered cell membrane permeability), (4) active drug efflux, (5) failure to activate the antibiotic, (6) use of alternative growth requirements, and (7) overproduction of target sites (Table 38-2).69,88
Enzymatic inactivation is one of the more common methods and is typified by β-lactamase hydrolysis of penicillins and cephalosporins and acetyltransferases that inactivate chloramphenicol, aminoglycosides, and tetracyclines. Altered target sites include ribosomal point mutations for tetracyclines, macrolides, and clindamycin; altered DNA gyrase and topoisomerase for fluoroquinolones; and modified PBPs for VGS and pneumococci. Most microorganisms have developed ways to alter their cell wall or membrane permeability to limit access of the antibiotic to its receptor by deleting outer membrane proteins or closing membrane pore channels. Altering antibiotic access to the cell interior usually does not confer a high level of resistance on the organism and must be combined with another mechanism for significant resistance potential. Several hundred efflux proteins are available that extrude waste products from the microbial cell but that now have been adapted over time to eliminate antibiotics specifically from the cell interior virtually as fast as they can enter. Enterococci can evade destruction by developing alternative metabolic growth requirements (auxotrophs). Sulfonamide resistance may occur from the overproduction of PABA, and some enteric organisms evade β-lactam antibiotics by overproducing β-lactamase (hyper–β-lactamase producers).
Antibiotic tolerance occurs when the antibiotic no longer kills the microorganism, but merely inhibits its growth or multiplication. Tolerant microorganisms start to grow after the antibiotic is removed, whereas resistant microorganisms multiply in the presence of the antibiotic. Tolerance is usually caused by the loss of autolysin activity through a failure to create or mobilize the autolytic enzymes. Vancomycin tolerance in S. pneumoniae is unique; a mutation in the sensor-response system controls the bactericidal autolysin activity.
Most experts agree that the major factor in the development and maintenance of antibiotic resistance in microbes is their ability to eliminate sensitive microorganisms and allow resistant ones to multiply and dominate. Although this selection process is crucial, other factors also contribute. Lengthy antibiotic regimens are commonly advocated to kill all the resistant strains or prevent stepped resistance (the development of resistance by a sequence of mutations occurring over several generations of microbial multiplication). Theoretically, if the antibiotic is given long enough, all these mutants are exposed to the antibiotic and killed at cell division. This is the rationale for taking the entire prescribed antibiotic rather than stopping the antibiotic when the patient is well. This concept is false for three reasons: (1) microbial mutations rarely occur during antibiotic treatment; (2) stepped resistance occurs even with prolonged antibiotic use4; and (3) most antibiotic resistance is gained by the transfer of genetic material between microbes, which is greatly enhanced by low-dose, prolonged antibiotic therapy.50 Combination antibiotic therapy against the stepped resistance seen with Mycobacterium tuberculosis is unique for this organism, but should not be extrapolated to all microbes. Also, a directive to “take all the antibiotic” assumes that the prescriber knows the exact duration of the infection, which is impossible.
Microbial resistance is most likely to occur when subtherapeutic antibiotic doses are used—doses that do not kill or inhibit the microorganism, but rather allow it to perceive the chemical as a threat to its survival and to react by mutation to resistance, acquisition, or transfer of resistance genes/virulence factors or induction (expression) of latent resistance genes.32,50 The gastrointestinal tract is a massive reservoir for resistance genes readily transferred within and between enteric microbial species,93 a process greatly enhanced by antibiotics that readily induce the expression or transfer of resistance genes, such as tetracyclines, imipenem, cefoxitin, and clavulanic acid.82
Bacteria-carrying resistance genes may have a reduction in “fitness” (a biologic cost) that results in slower growth rates, loss of virulence, and an increased biologic burden (synthesis of nucleic acids). Studies indicate, however, that many bacteria can adapt to this new genetic burden or even require resistance genes for survival. If this situation becomes common, removal of the antibiotic from the environment would have little effect on reducing resistance in the hospital or community, a point that may already have been reached with some microbes.
The most important acquired mechanism for β-lactam resistance, particularly in gram-negative microorganisms, is the production of various β-lactamases that hydrolyze the β-lactam ring to form a linear metabolite incapable of binding to PBPs. In 1984, 19 plasmid-mediated β-lactamases were known; the number now has increased to more than 340 chromosomally and plasmid-mediated β-lactamases—70 of the TEM-1 and TEM-2 and 20 of the SHV-1 types alone.11
β-Lactamases have been variously classified by Richmond-Sykes (I to V), Ambler (A to D), and Bush (1 to 4).11 β-Lactamase enzymes can be chromosomally mediated or easily transferred by transposable elements. Many are of the TEM type (from a patient named Temoniera in Greece, in whom a β-lactamase was isolated in the early 1960s) or the SHV type (sulfhydryl variable).11 The most pressing difficulties with β-lactamases are their widespread dissemination throughout the microbial environment, ability to move between widely disparate organisms, tendency to inhibit new antibiotic agents rapidly, and increasing resistance to β-lactamase inhibitors (clavulanic acid, sulbactam, and tazobactam). β-Lactamases have been observed in numerous gram-positive and gram-negative pathogens. The cohabitation of staphylococci and enterococci on human skin in hospitals has likely led to the incorporation of β-lactamase genes into enterococci after the latter organisms had successfully avoided this transfer for billions of years.
Point mutations have appeared more recently in TEM and SHV β-lactamases, resulting in extended-spectrum β-lactamases in Klebsiella pneumoniae that hydrolyze the latest cephalosporins (cefotaxime, ceftazidime, cefepime) and aztreonam. Certain enteric microorganisms (Escherichia coli, Citrobacter freundii, K. pneumoniae, Proteus mirabilis) can produce massive amounts of TEM-1 β-lactamase (hyper–β-lactamase producers) that can overwhelm β-lactamase inhibitors. Metallo-β-lactamases possess the broadest spectrum of inhibitory activity and hydrolyze all β-lactam antibiotics except monobactams (aztreonam) and are not inhibited by any of the β-lactamase inhibitors currently available.
The first plasmid encoded β-lactamases with the ability to hydrolyze cephalosporins were termed the extended-spectrum β-lactamases (ESBLs).13 These ESBLs microbes were also resistant to aminoglycosides, tetracyclines, and trimethoprim/sulfonamides.13 ESBLs cause resistance to third-generation cephalosporins (cefotaxime, ceftriaxone, ceftazidime) and monobactams (aztreonam) but are sensitive to cefamycins (cefoxitin, cefotetan) and carbapenems (imipenem, meropenem, ertapenem). These ESBLs are horizontally transmitted by mobile genetic elements from food, animals, or family members and induce greater mortality than enteric bacilli without these ESBLs.77
A mechanism by which bacteria move an antibiotic out of the cell as soon as it enters was first detected in E. coli by Levy in 1978; the first gene (qacA) encoding a multidrug efflux protein was subsequently detected in an isolate of S. aureus.69 Currently, more than 50 such systems have been described, and these cytoplasmic membrane transport proteins (multidrug efflux pumps) have likely evolved to protect the cell from foreign chemical invasion and allow its secretion of cell metabolic products.110 Efflux pumps operate in E. coli, Pseudomonas aeruginosa, staphylococci, Streptococcus pyogenes and S. pneumoniae, Bacillus subtilis, Pasteurella multocida, Neisseria gonorrhoeae, mycobacteria, and enterococci.110 For tetracyclines, these efflux pumps are the major mechanism for resistance and are becoming increasingly so for the fluoroquinolones.110 Efflux pumps are classified into five main groups: (1) the major facilitator family; (2) the small/staphylococcal multidrug resistance family; (3) the resistance, nodulation, and cell division family; (4) the adenosine 5′-triphosphate binding cassette superfamily; and (5) the multidrug and toxic compound extrusion family.110
These chromosomal and plasmid-mediated efflux transporter proteins may be quite specific for antibiotics and metabolic product substrates and are regulated by numerous genes and gene products. Repressors are also present and are highly regulated to prevent the accidental overproduction of efflux pumps. Tetracyclines derepress this system, leading to an overproduction of efflux proteins and increasing resistance to themselves and any other antibiotics carried by these proteins.81
Microorganisms possess three mechanisms for genetic variation: (1) local nucleotide changes in the genome, (2) rearrangement of genomic sequences, and (3) horizontal acquisition of DNA from other microorganisms. Such genetic alterations have allowed for their evolution and survival for 3.5 billion years. The rearrangement of genes and particularly the acquisition of new genetic information are commonplace and are now the major mechanism controlling microbial resistance to antibiotics.
In the 1950s, McClintock described genetic controlling elements that did not follow the Mendelian Laws of Genetics and acquired an independent existence (selfish genes, jumping genes). In the early 1970s, Hedges and Jacob first used the term transposon for a mobile genetic element conveying resistance to ampicillin. Microbes acquire new genetic information by three mechanisms—transformation, transduction, and conjugation—and use numerous transposable elements, such as bacteriophages, transposons, integrons, and plasmids.
During transformation, bacteria acquire “naked” DNA from their environment to incorporate into their genome. Such genetic transformations are uncommon and require unique circumstances involving genes, binding, uptake, and integration. At least 50 bacteria are sufficiently competent to acquire environmental genes from their fellow microbes, plants, yeasts, and animals. VGS and S. pneumoniae have the DNA recognition sites and a quorum-sensing peptide (competence-stimulating peptide) that allows for the acquisition of each other’s genes when released into the environment on their death. Because they coinhabit the oropharynx, and penicillin resistance occurs in a stepwise manner with gradual amino acid mutations in at least four PBPs for high penicillin resistance, this resistance likely evolved over many years and indicates that transformation is a slow but ultimately efficient mechanism for genetic change.
Transduction is the movement of DNA from one bacterium to another by a bacteriophage (bacterial virus) intermediary. Conjugation is the self-transfer of genetic information by plasmids or transposons to other microorganisms, generally by physical contact with a sex pilus in gram-negative organisms and stimulated by various pheromones (small peptides). Mobile elements commonly require site-specific combination sites but not DNA segment identity, allowing for broad DNA movement. Mobile elements of various types include bacteriophages, transposons, plasmids, integrons, and shufflons.
Transposons are DNA segments that cannot self-replicate, but can self-transfer between plasmids, bacteriophages, and chromosomes. Transposons can recruit as many genes as required for their purpose, and the mechanisms that control this process are essentially unknown. That we know so little about a system with so much potential for genetic change is worrisome. Between 30% and 40% of the human genome is composed of transposable element sequences or gene sequences directly derived from them.23
Plasmids may be conjugative (self-transmissible) or nonconjugative (unable to effect their own transfer) and may be narrow range (replication in only one or a few hosts) or broad range (replication in many hosts). Too great a concentration of plasmids in one microorganism is usually intolerable because of the high energy costs to maintain it; plasmids have an autoregulatory system (iterons) that allows them to determine their own rate of replication.
Plasmids may also be constitutive (ongoing formation) or inductive (formed only when stimulated or induced by a foreign chemical). Plasmids carry resistance genes and virulence genes or pathogenicity islands that carry all the components necessary to damage the host directly or initiate host responses, such as inflammation, that harm the host. Plasmids are common in oral and gastrointestinal Bacteroides, Porphyromonas, and Prevotella isolates.
Researchers previously hoped that resistance genes and their transporters would pose such a fitness problem for bacteria (requiring so much energy) that bacteria no longer exposed to the antibiotic would lose their resistance genes. Such genes may become so important for bacterial functions, however, that they become permanent. The tetracycline efflux pumps can become necessary for bacterial survival by functioning in Na+-K+ exchange across the bacterial membrane.6 The problem is compounded when the resistance gene for a particular antibiotic becomes part of an integron-containing multiple antibiotic resistance gene array. Eliminating the one antibiotic does nothing; all the antibiotics must be eliminated from the environment for the integron to be lost.
Antibiotic resistance has been enhanced further by the discovery of the integron, a genetic element that captures and disseminates genes by site-specific integration of DNA (gene cassettes) that can mediate resistance, virulence, and biochemical functions.79 Integrons have three distinct genes encoding for an integrase enzyme, a recombination site, and a promoter element.79 Integrons resemble bundled products with a computer operating system; they package resistance determinants to allow for widespread gene dissemination.
Each gene is a cassette, and five genes may commonly be present in one integron.79 Superintegrons have been isolated in Vibrio cholerae that contain hundreds of gene cassettes that encode many bacterial functions beyond those of resistance and virulence. Gene cassettes have been identified for all antibiotics except fluoroquinolones, and they exist for quaternary ammonium compounds. Integrons cannot promote self-transfer because they lack transporter genes, but they are commonly associated with transposons and conjugative plasmids.
Horizontal gene transfer (HGT), also known as lateral gene transfer, has been a major impetus for the exceptional diversity and survival of the microbial world. Ancient integrons, bacteriophages, plasmids, transposons, and now insertion sequence common regions have changed the otherwise clonal mode of prokaryotic life95 and allowed for gene capture and dissemination from the global gene pool.8 These mobile bacterial elements move from one bacterial cell to another via transduction and conjugation as circular, usually double-stranded DNA and often via a sex pilus as an extension from the donor to the recipient cell. Technically, a third form of DNA transfer is via transformation whereby “naked” DNA from disrupted cells is taken up from the extracellular fluid by certain “competent” cells.
These mobile genetic elements include plasmids, transopsons, bacteriophages, integrons, and insertion sequence common regions that move from bacterial cell to bacterial cell or, when translocated, from various DNA sites within the bacterial cell.8 These DNA elements carry genes for antibiotic resistance, heavy metal toxins, and virulence determinants and can induce or repair DNA damage.8
HGT encompasses two processes that move genetic material from one bacterium to another to enable unique phenotypic characteristics or to translocate genes from one location (plasmids) to another (chromosomes).59 The combination of integrons, transposons, and insertion elements results in antimicrobial resistance islands.59 Insertion sequences are mobile genetic elements that promote and translocate genes and are inserted into transposons to mobilize DNA resistance genes for β-lactamase of the CTX-M type.59
HGT facilitates genome rearrangement, deletions, and insertions to allow adaptation to changing environments and is induced by antimicrobials, metals, and organic contaminants (environmental stress).7 This facilitation of gene transfer occurs in four steps: (1) packaging of nucleic acids for transfer via excision and circularization of the transposons, (2) transfer of the DNA via contact with the recipient cell (conjugation), (3) entrance into the cell and integration with host chromosomal DNA, and (4) transfer of chromosomal DNA or the replicating elements into the daughter cells and subsequent generations.7
Microbial resistance to antibiotics in S. pneumoniae is most serious because the organism is responsible for 3000 cases of meningitis, 50,000 cases of bacteremia, 500,000 cases of pneumonia, and 2 million cases of otitis media in the United States annually and 3 to 5 million deaths annually worldwide.69 Resistance to sulfonamides was first detected in 1943 and to penicillin in the late 1960s in Australia and New Guinea. The mechanism of penicillin resistance is a single point mutation in PBP2x or PBP2b, with an altered PBP2a requiring mutation also in pBP2x (the organism has six PBPs).
High penicillin resistance (usually a plasma concentration of ≥2 µg/mL) is seen in 14% of U.S., 6.8% of Canadian, 10.4% of European, and 17.8% of Asian-Pacific isolates.38 Resistance of the pneumococcus to penicillin can vary significantly with geographic area: 38.8% in Tennessee, 15.3% in Maryland, 65.3% in Japan, 60.8% in Vietnam, 15.6% to 48.2% in Latin America, and 79.7% in Korea.69
Tolerance to vancomycin has been detected in an isolate responsible for meningitis and high-level resistance to quinupristin-dalfopristin and cefotaxime. Tetracycline resistance in S. pneumoniae is currently low but is increasing, which may pose a significant problem because doxycycline has become an important drug for community and nosocomial-acquired pneumonia caused by S. pneumoniae, Mycoplasma pneumoniae, Legionella pneumophila, and Chlamydia pneumoniae.
In a 1999 review of more than 10,000 bloodstream infections at 49 U.S. hospitals, S. aureus accounted for 16% and CoNS for 32% of all isolates, with most of the CoNS being methicillin-resistant.69 Some isolates are susceptible only to vancomycin; others are susceptible to linezolid and quinupristin-dalfopristin; and still others are susceptible to older agents such as macrolides, tetracyclines, aminoglycosides, rifampin, clindamycin, sulfonamides, and fluoroquinolones. The mechanism of methicillin resistance is an altered PBP2 (PBP2a or PBP2′) conferred through a mecA gene that results in a much lower binding affinity of methicillin for PBP2a. This mode of resistance requires the cooperation of PBP2 and PBP2a sites and two enzymes, one natural and one environmentally acquired.
The first MRSA isolate was detected in the United Kingdom in 1961, and MRSA remained rare in the United States until 1976. MRSA spread throughout the hospital by nasal secretions, hands, clothing, bedding, air currents, fomites, and skin boils (furuncles). The anterior nares is the prime carrier site of S. aureus in humans, with 80% of people either persistent or intermittent carriers and possibly 25% of healthy individuals colonized with CoNS. High concentrations of staphylococci are also found in the throat, axilla, and perineum (groin and upper thighs).
Of the 17 species of enterococci found in the human oral cavity and gastrointestinal and genitourinary tracts, Enterococcus faecalis accounts for 90% of human infections, and E. faecium accounts for approximately 10%.69 Enterococcal infections are classic examples of a relatively harmless commensal organism becoming a serious pathogen by the acquisition of multiple resistance genes.
Enterococci are intrinsically resistant to cephalosporins and have varying degrees of resistance to aminoglycosides, macrolides, tetracyclines, and clindamycin. Vancomycin resistance, particularly in E. faecium, has been of major concern since the late 1980s. Enterococci cause 800,000 nosocomial infections annually in the United States, with more than 50% caused by vancomycin-resistant E. faecium; resistance is more than 90% in E. faecium bacteremias. Currently, 17% of enterococcal strains in the United States are resistant to vancomycin.52
Vancomycin-resistant enterococcus (VRE) infections, particularly of the bloodstream type, are becoming extremely difficult to treat. Doxycycline has been enlisted more recently in the treatment of VRE.52 Enterococcal resistance is complicated further by the observations that (1) streptococci, staphylococci, and enterococci often share the same resistance genes; (2) β-lactamase in enterococci is identical to that in staphylococci, indicating sharing of genetic information; (3) enterococci can transfer resistance genes, particularly for vancomycin to staphylococci and other organisms, in vitro and in animal models; (4) staphylococci and enterococci coinhabit the skin; and (5) the possibility exists that vancomycin resistance may one day appear in many VGS.69
Chronic gastritis, peptic ulcer, and gastric cancer have been linked to H. pylori. Depending on the geographic area and the prevalence of antibiotic use, alarming reports have appeared of resistance to all antibiotic agents used in its management, including metronidazole, clarithromycin, tetracycline, and amoxicillin. Resistance to metronidazole acquired by a decreased ability to reduce its nitro group ranges from 10% to 50% in developed countries and up to 100% in developing countries, where it is widely used to treat parasitic diseases. Resistance to amoxicillin ranges from 0% in The Netherlands to 18% in Mexico to 72% in Shanghai, China. Resistance to clarithromycin ranges from 1.7% in The Netherlands to 10% to 12% in the United States to 24% in Mexico.
Tetracycline has been added more recently to antibiotic regimens, and resistance rates are 0% in The Netherlands, 5.3% in Korea, and 58.8% in Shanghai, China, with the disturbing possibility that tetracycline-resistant H. pylori may exhibit cross-resistance with metronidazole. Metronidazole resistance in H. pylori may decrease the effectiveness of therapy by 37.7%, and clarithromycin resistance may decrease the effectiveness of therapy by 55%.25 The widespread use of systemic metronidazole and tetracycline is difficult to justify in the management of a relatively trivial, mechanically responsive disease such as periodontitis when such a practice may promote resistance in a microbial pathogen responsible for very serious diseases such as peptic ulcer and gastric cancer.
The current therapy for HIV infection entails highly active antiretroviral therapy (HAART) (see Chapter 40) with a combination of drugs that interfere with several steps in viral replication, including reverse transcriptase inhibitors, protease inhibitors, and the new integrase inhibitors that prevent HIV from integrating into the genome of the host cell. Difficulties have arisen with this therapeutic approach because the virus provides for reservoirs of replication-competent HIV in resting CD4 T lymphocytes throughout many years of intensive HAART. It is estimated that more than 60 years of HAART may be necessary to eradicate the virus from these reservoirs.69 More than 50% of HIV-infected individuals in the United States receiving HAART are resistant to one or more of the drugs, and 78% of individuals with measurable viral loads are resistant to at least one drug, encompassing about 100,000 people in the United States.99 From 1994-2000, 14% of new HIV cases had one or more HIV mutations associated with antiretroviral drug resistance; in 2000, it was 27%. Approximately 25% of newly infected, therapy-naïve individuals carry at least one key HIV drug-resistant mutant.