Goodman and Gilman Manual of Pharmacology and Therapeutics

Section VII
Chemotherapy of Microbial Diseases

chapter 48
General Principles of Antimicrobial Therapy

ANTIMICROBIAL CHEMOTHERAPY

Microorganisms of medical importance fall into 4 categories: bacteria, viruses, fungi, and parasites. Likewise, antibiotics are broadly classified as (1) antibacterial, (2) antiviral, (3) antifungal, and (4) antiparasitic agents. Antimicrobial molecules should be viewed as ligands whose receptors are microbial proteins. The microbial proteins targeted by the antibiotic are essential components of biochemical reactions in the microbes, and interference with these physiological pathways kills the microorganisms. The biochemical processes commonly inhibited include cell wall synthesis in bacteria and fungi, cell membrane synthesis, synthesis of 30s and 50s ribosomal subunits, nucleic acid metabolism, function of topoisomerases, viral proteases, viral integrases, viral envelope fusion proteins, folate synthesis in parasites, and parasitic chemical detoxification processes.

Classification of an antibiotic is based on:

• The class and spectrum of microorganisms it kills

• The biochemical pathway it interferes with

• The chemical structure of its pharmacophore

The relationship between antimicrobial concentration and effect on a population of organisms is modeled using the standard Hill-type curve for receptor and agonist (see Chapters 2 and 3), characterized by 3 parameters: the inhibitory concentration 50, or IC50 (also termed EC50), a measure of the antimicrobial agent’s potency; the maximal effect, Emax; and H, the slope of the curve, or Hill factor. In antimicrobial therapy, the relationship is often expressed as an inhibitory sigmoid Emax model, to take into account the control bacterial population without treatment (Econ) as a fourth parameter (Equation 48–1 and Figure 48–1), where E is the effect as measured by microbial burden.

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Figure 48–1 Inhibitory sigmoid Emax curve. CFU, colony-forming unit.

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THE PHARMACOKINETIC BASIS OF ANTIMICROBIAL THERAPY

PENETRATION OF ANTIMICROBIAL AGENTS INTO ANATOMIC COMPARTMENTS. In many infections, the pathogen causes disease not in the whole body, but in specific organs. Antibiotics are often administered far away from these sites of infection. Therefore, in choosing an antimicrobial agent for therapy, a crucial consideration is whether the drug can penetrate to the site of infection.

For example, the antibiotic levofloxacin achieves skin tissue/plasma peak concentration ratio of 1.4, epithelial lining fluid to plasma ratio of 2.8, and urine-to-plasma ratios of 67. In clinical trials with levofloxacin, the failure rate of therapy was 0% in patients with urinary tract infections, 3% in patients with pulmonary infections, and 16% in patients with skin and soft tissue infections. Clearly, the poorer the penetration into the anatomical compartment, the higher the likelihood of failure. Penetration of a drug into an anatomical compartment depends on the physical barriers that the molecule must traverse, the chemical properties of the drug, and the presence of multidrug transporters. The physical barriers are usually due to layers of epithelial and endothelial cells, and the type of junctions formed between these cells. Penetration across this physical barrier generally correlates with the octanol-water partition coefficient of the antimicrobial agent, a measure of its hydrophobicity. Hydrophobic molecules get concentrated in the cell membrane bilayer, whereas hydrophilic molecules tend to concentrate in the blood, the cytosol, and other aqueous compartments (see Figure 2–3).

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Figure 48–3 Changes in sigmoid Emax model with increases in drug resistance. Increase in resistance may show changes in IC50 (panel A: the IC50 increases from 70 [orange line] to 100 [green line], to 140 [blue line]) or decrease in Emax (panel B: efficacy decreases from full response [orange line] to 70% [green line]).

Membrane transporters comprise another barrier; they actively export drugs from the cellular or tissue compartment back into the blood (see Chapter 5). A well-known example is the P-glycoprotein. Although the octanol-water partition coefficient would favor lipophilic molecules to transverse across cell barriers, P-glycoprotein exports structurally unrelated amphiphilic and lipophilic molecules of 3-4 kDa, reducing their effective penetration. Antimicrobial agents that are P-glycoprotein substrates include HIV protease inhibitors, the antiparasitic agent ivermectin, the antibacterial agent telithromycin, and the antifungal agent itraconazole.

The CNS is guarded by the blood-brain barrier. The movement of antibiotics across the blood-brain barrier is restricted by tight junctions that connect endothelial cells of cerebral micro-vessels to one another in the brain parenchyma, as well as by protein transporters. Antimicrobial agents that are polar at physiological pH generally penetrate poorly; some, such as penicillin G, are actively transported out of the cerebrospinal fluid (CSF) and achieve CSF concentrations of only 0.5-5% of that achieved in plasma. However, the integrity of the blood-brain barrier is diminished during active bacterial infection; tight junctions in cerebral capillaries open, leading to a marked increase in the penetration of even polar drugs.

The eye, the epithelial lining fluid of the lung, and biofilms and vegetations on artificial heart valves and indwelling catheters pose special problems for drug penetration and effective therapy.

PHARMACOKINETIC COMPARTMENTS. Once an antibiotic has penetrated to the site of infection, it may be subjected to processes of elimination and distribution that differ from those in the blood. These sites where the concentration-time profiles differ from each other are considered separate pharmacokinetic compartments, thus, the human body is viewed as multicompartmental. The concentration of antibiotic within each compartment is assumed to be homogeneous. The model is also defined as open or not open; an open model is one in which the drug is eliminated out of the body from the compartment (e.g., kidneys). The order of the process must also be specified (see Chapter 2): a first-order process is directly correlated to concentration of drug D, or [D]1, as opposed to zero order, which is independent of [D] and reflects a process that is saturated at ambient levels of D.

Suppose a patient has pneumonia with the pathogen in the lung epithelial lining fluid (ELF). The patient ingests an antibiotic that is absorbed via the GI tract (g) into blood or central compartment (compartment 1), as a first-order input. In this process, the transfer constant from the GI tract to central compartment is termed the absorption constant and is designated ka. The antibiotic in the central compartment is then delivered to the lungs where it penetrates into the ELF (compartment 2). However, it also penetrates into other tissues of the body peripheral to the site of infection, termed theperipheral compartment (compartment 3). Thus, we have 4 compartments (including g, a specific compartment, the GI tract, from the set of initial absorption compartments labeled “p” in Figure 48–2), each with its own concentration-time profile, as shown in Figure 48–2. The penetration of drug from compartment 1 to 2 is based on the penetration factors discussed earlier and is defined by the transfer constant k12. However, the drug also redistributes from compartment 2 back to 1, defined by transfer constant k21. A similar process between the blood and peripheral tissues leads to transfer constants k13and k31. The drug may also be lost from the body (i.e., open system) via the lungs and other peripheral tissues (e.g., kidneys or liver) at a rate proportional to the concentration.

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Figure 48–2 Diagrammatic depiction of a multicompartment model.

Antibiotic concentrations within each compartment change with time (the changes are described using standard differential equations). If X is the amount of antibiotic in a compartment, SCL the drug clearance, and Vc the volume of central compartment, then equations for absorption compartment (Equation 48–2), central compartment (Equation 48–3), site of infection or compartment 2 (Equation 48–4), and peripheral compartment (Equation 48–5) are as follows:

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Such models have been used in conjunction with population pharmacokinetics to describe and model a plethora of antimicrobials used to treat bacteria, fungi, viruses, and parasites. Recently, the models have been refined to include sub-populations of the pathogen (killed, inhibited, or resistant to the antibiotic) and other refinements described in Chapter 48 of the 12th edition of the parent text.

POPULATION PHARMACOKINETICS AND VARIABILITY IN DRUG RESPONSE. When multiple patients are treated with the same dose of a drug, each patient will achieve pharmacokinetic parameters that differ from others. This is termed between-patient variability. Even when the same dose is administered to the same patient on 2 separate occasions, the patient may achieve a different concentration-time profile of the drug between the 2 occasions. This is termed inter-occasion or within-patient variability. The variability is reflected at the level of the compartmental pharmacokinetic parameters such as ka, k12, k21, SCL, Vc, and so on. Even when a recommended dose is administered, the drug may fail to reach a therapeutic concentration in some patients. In other patients, the drug may reach high and toxic concentrations. Such variability could be due to genetic variability. In addition, weight, height, and age also lead to variability. Furthermore, patients may have comorbid conditions such as renal and liver dysfunction, which may lead to variability. Drug interactions are an important source of variability with potentially dangerous consequences (see Chapters 5 and 6). Even when such factors are accounted for, there remains residual variability due to computational noise, assay variability, and unexplainable factors. The common practice of using an “average” value of data or “naive pooling” may prevent recognition of subgroups of patients at risk for therapeutic failure or increased toxicity of antibiotic. Knowledge of covariates associated with pharmacokinetic variability leads to better dose adjustments, or switching therapy from one antibiotic to another, or changing concomitant medications.

IMPACT OF SUSCEPTIBILITY TESTING ON SUCCESS OF ANTIMICROBIAL AGENTS

Once the microbial species causing the disease has been identified, a rational choice of the class of antibiotics likely to work in the patient can be made. The microbiology laboratory then plays a second role, which is to perform susceptibility testing to narrow down the list of possible antimicrobials that could be used.

Millions of individuals across the globe get infected by many different isolates of the same species of pathogen. Evolutionary processes cause each isolate to be slightly different from the next, so that each will have a unique susceptibility to antimicrobial agents. As the microorganisms divide within the patient, they may undergo further evolution. Therefore, we expect that there will be a wide distribution of concentrations of antimicrobial agents that can kill pathogens. Often, this distribution is Gaussian, with a skew that depends on where the patient lives. These factors will affect the shape of the inhibitory sigmoid Emax model curve described by Equation 48–1.

With changes in susceptibility, the sigmoid Emax curve shifts in 1 of 2 basic ways. The first is a shift to the right, an increase in IC50, as shown in Figure 48–3A. This means that much higher concentrations than before are now needed to show specific effect. Susceptibility tests for bacteria, fungi, parasites, and viruses have been developed to determine whether these shifts have occurred at a sufficient magnitude to warrant higher doses of drug to achieve particular effect. The change in IC50 may become so large that it is not possible to overcome the concentration deficit by increasing the antimicrobial dose without causing toxicity to the patient. At that stage, the organism is now “resistant” to the particular antibiotic. A second possible change in the curve is a decrease in Emax (Figure 48–3B), such that increasing the dose of the antimicrobial agent beyond a certain point will achieve no further effect; i.e., changes in the microbe are such that eradication of the microbe by the particular drug can never be achieved. This occurs because the available target proteins have been reduced or the microbe has developed an alternative pathway to overcome the biochemical inhibition.

Bacteria. Dilution tests for bacterial drug susceptibility employ antibiotics in serially diluted drug concentrations on solid agar or in broth medium that contains a culture of the test microorganism. The lowest concentration of the agent that prevents visible growth after 18-24 h of incubation is known as the minimum inhibitory concentration (MIC).

Fungi. For fungi that are yeasts (i.e., Candida), susceptibility testing methods are similar to those used for bacteria. However, the definitions of MIC differ based on drug and the type of yeast, so there are cutoff points of 50% decreases in turbidity compared to controls at 24 h, or 80% at 48 h, or total clearance of the turbidity. Susceptibility tests and MICs for triazoles have been extensively shown to correlate with clinical outcomes.

Viruses. In HIV phenotypic assays, the patient’s HIV-RNA is extracted from plasma, and genes for targets of antiretroviral drugs such as reverse transcriptase and protease are amplified. The genes are then inserted into a standard HIV vector that lacks analogous gene sequences to produce a recombinant virus, which is co-incubated with drug of interest in a mammalian cell viability assay. Growth is compared to a standardized wild-type control virus. For HIV reverse transcriptase, e.g., <4-fold increase in IC50 is defined as “sensitive,” 4- to 10-fold increase in I IC50 is “intermediate,” and >10-fold increase is “resistant.” Further use has been made of the viral IC50 to establish the inhibitory quotient (IQ). The IQ is the ratio of plasma concentration of antiviral drug to the IC50. The phenotypic IQ is the ratio of plasma trough concentration to the IC50.

Parasites. Susceptibility testing for parasites, especially malaria, has been performed in the laboratory. The tests are similar to the broth tests for bacteria, fungi, and viruses. Plasmodium species in the patient’s blood are cultured ex vivo in the presence of different dilutions of antimalarial drug. A sigmoid Emax curve for effect versus drug concentration is used to identify IC50 and Emax. These tests are primarily used in the research setting and not for individualization of therapy.

BASIS FOR SELECTION OF DOSE AND DOSING SCHEDULE

Although susceptibility testing is central to decision making, it does not completely predict patient response. Microorganisms in patients are exposed to dynamic concentrations of drug, and antibiotics are prescribed at a certain schedule (e.g., 3 times a day) so that there is a periodicity in the fluctuations of drug at the site of infection. Thus, the microbe is exposed to a particular shape of the concentration-time curve, an important determinant of the efficacy of the antibiotic for which we can write 3 corollaries:

1. In determining therapeutic outcomes, it is important to apply knowledge of susceptibility (MIC or EC90) of the organism to the antimicrobial agent and index drug exposure to MIC.

2. The optimal dose of the antibiotic for a patient is the dose that achieves IC80 to IC90 exposures at the site of infection.

3. Optimal microbial kill by the antibiotic may be best achieved by maximizing certain shapes of the concentration-time curve, using the fact that certain dosing schedules maximize the antimicrobial effect (see below).

As an example, consider an antibiotic with a serum t1/2 of 3 h that is being used to treat a bloodstream infection by a pathogen with an MIC of 0.5 mg/L, administered with a dosing interval of 24 h (that is, a once-daily schedule). Figure 48–4A depicts the concentration-time curve of the antibiotic, with definitions of peak concentration (CPmax), area under the curve (AUC), and the fraction of the dosing interval for which the drug concentration remains above the MIC (T > MIC), as shown. The AUC is a measure of the total concentration of drug and is calculated by taking an integral between 2 time points, 0-24 h (AUC0-24) in this case. With a change in the dosing schedule of the same antibiotic amount by splitting it into 3 equal doses administered at 0, 8, and 16 h, the shape of the concentration-time curves changes to that shown in Figure 48–4B. Because the same cumulative dose has been given for the dosing interval of 24 h, the AUC0-24will be similar whether it was given once a day or 3 times a day. However, the CPmax will decrease by one-third when the total dose is split into thirds and administered more frequently (see Figure 48–4B). Thus, when a dose is fractionated and administered more frequently, theCPmax/MIC ratio decreases. In contrast, the time that the drug concentration persists above MIC (T > MIC) increases with the more frequent dosing schedule.

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Figure 48–4 Effect of different dose schedules on shape of concentration-time curve. The total AUC for the fractionated dose in curve B is determined by adding AUC0-8h, AUC8-16h, and AUC16-24h, which adds up to the same AUC0-24h in curve A. The time that the drug concentration exceeds MIC in curve B is also determined by adding up T1 MIC, T2 MIC, and T3 MIC, which results in a fraction greater than that for curve A.

Some classes of antimicrobial agents kill best when concentration persists above MIC for longer durations of the dosing interval. Indeed, increasing the drug concentration beyond 4 and 6 times the MIC does not increase microbial kill. Two good examples are β-lactam antibacterials (e.g., penicillin) and the antifungal agent 5-fluorocytosine (5-FC). In fact, there are usually good biochemical explanations for this pattern for the drugs. The clinical implication, however, is that a drug optimized by T > MIC should be dosed more frequently, or have its t1/2 prolonged by other drugs, so that drug concentrations persist above MIC (or EC95) as long as possible. Thus, the effectiveness of penicillin is enhanced when it is given as a continuous infusion. HIV protease inhibitors are often “boosted” with ritonavir; this “boosting” inhibits the metabolism of the protease inhibitors by CYPs 3A4 and 2D6, thereby prolonging time above EC95.

Conversely, the peak concentration is paramount for some antimicrobial agents. Persistence of concentration above MIC has less relevance for these drugs, meaning that these drugs can be dosed more intermittently. Aminoglycosides are a prime example of this class: they used to be given 3 times a day but are highly effective when given once a day. These CPmax/MIC-linked drugs are administered less frequently due to their long duration of post-antibiotic effect (PAE). In other words, effect continues long after antibiotic concentrations decline below the MIC. Consider rifampin. The entry of rifampin into Mycobacterium tuberculosis increases with increased concentration in the bacillus microenvironment. Once inside the bacteria, the drug’s macrocyclic ring binds the β subunit of DNA-dependent RNA polymerase (rpoB) to form a very stable drug-enzyme complex within 10 min, a process not enhanced by longer incubation of drug and enzyme. The PAE of the rifampin is long and concentration dependent.

There is a third group of drugs for which the dosing schedule has no effect on efficacy but the cumulative dose matters. Thus, it is the ratio of total concentration over time (AUC)-to-MIC that matters and not the time that concentration persists above a certain threshold. Antibacterial agents such as daptomycin fall into this class. These agents also have a good PAE. The AUC/IC50 explains why the nucleoside analogue reverse transcriptase inhibitors tenofovir and emtricitabine have been combined into 1 pill, administered once a day for the treatment of AIDS.

The shape of concentration-time curve that optimizes resistance suppression is often different from that which optimizes microbial kill. In many instances, the drug exposure associated with resistance suppression is much higher than for optimal kill. The optimal dose should be designed to achieve a high probability of exceeding the EC80 microbial PK/PD (pharmacokinetic/pharmacodynamic) index, or index associated with suppression of resistance, given the population pharmacokinetic variability and the MIC distribution of clinical microbe isolates. The population pharmacokinetic variability enables integration of pharmacogenetics, anthropometric measures, and residual variability into the decision to choose optimal dose. Once that has been achieved, the dose schedule is chosen according to whether efficacy is driven by AUC/MIC (or AUC/EC95), CPmax/MIC, or T > MIC. Duration of therapy is then chosen, based on best available evidence.

TYPES AND GOALS OF ANTIMICROBIAL THERAPY

A useful way to organize the types and goals of antimicrobial therapy is to consider where along the disease progression timetable therapy is initiated (Figure 48–5); therapy can beprophylactic, preemptive, empirical, definitive, or suppressive.

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Figure 48–5 Antimicrobial therapy-disease progression timeline.

PROPHYLACTIC THERAPY. Prophylaxis involves treating patients who are not yet infected or have not yet developed disease. The goal of prophylaxis is to prevent infection in some patients or to prevent development of a potentially dangerous disease in those who already have evidence of infection. The main principle behind prophylaxis is targeted therapy.

Prophylaxis is used in immunosuppressed patients such as those with HIV-AIDS or are post-transplantation and on anti-rejection medications. In these groups of patients, specific antiparasitic, antibacterial, antiviral, and antifungal therapy is administered based on the well-defined pattern of pathogens that are major causes of morbidity during immunosuppression. A risk-to-benefit analysis is used to determine choice and duration of prophylaxis. In AIDS patients, prophylaxis is discontinued when the CD4 count climbs above 200 cells/mm3. Infections for which prophylaxis is given include Pneumocystis jiroveci, Mycobacterium avium-intracellulare, Toxoplasma gondii, Candida species, Aspergillus species, Cytomegalovirus, and other Herpesviridae.

Chemoprophylaxis is used to prevent wound infections after various surgical procedures. Several factors are important for the use of antibiotics for surgical prophylaxis. First, antimicrobial activity must be present at the wound site at the time of its closure. Thus, infusion of the first antimicrobial dose should begin within 60 min before surgical incision and should be discontinued within 24 h of the end of surgery. Second, the antibiotic must be active against the most likely contaminating microorganisms for that type of surgery. Chemoprophylaxis can be justified in dirty or contaminated surgical procedures (e.g., resection of the colon), where the incidence of wound infections is high. In clean surgical procedures, which account for ~75% of the total, the expected incidence of wound infection is <5%, and antibiotics should not be used routinely. When the surgery involves insertion of a prosthetic implant (e.g., prosthetic valve, vascular graft, prosthetic joint), cardiac surgery, or neurosurgical procedures, the complications of infection are so drastic that most authorities currently agree to chemoprophylaxis for these indications.

Prophylaxis may be used to protect healthy persons from acquisition of or invasion by specific microorganisms to which they are exposed. This is termed postexposure prophylaxis. Examples include rifampin administration to prevent meningococcal meningitis in people who are in close contact with a case, prevention of gonorrhea or syphilis after contact with an infected person, and macrolides after contact with confirmed cases of pertussis. Post-exposure prophylaxis is recommended in those patients inadvertently exposed to HIV infection.

Mother-to-child transmission of HIV and syphilis are important public health problems. Anti-retroviral therapy is administered for HIV prophylaxis during the pregnancy and peripartum periods. Prophylactic therapy for syphilis during pregnancy is effective in reducing neonatal death and infant neurological, auditory, and bone malformations.

PRE-EMPTIVE THERAPY. Pre-emptive therapy is early, targeted therapy in high-risk patients who already have a laboratory or other test indicating that an asymptomatic patient has become infected. The principle is that delivery of therapy prior to development of symptoms (presymptomatic) aborts impending disease, and the therapy is for a short and defined duration.

This has been applied in the therapy for cytomegalovirus after both hematopoietic stem cell transplants and after solid organ transplantation.

EMPIRICAL THERAPY IN THE SYMPTOMATIC PATIENT. Once a patient is symptomatic, should the patient be treated immediately? The first consideration in selecting an antimicrobial is to determine if the drug is indicated. The reflex action to associate fever with treatable infections and prescribe antimicrobial therapy without further evaluation is irrational and potentially dangerous.

The diagnosis may be masked if therapy is started and appropriate cultures are not obtained. Antimicrobial agents are potentially toxic and may promote selection of resistant microorganisms. For some diseases, the cost of waiting a few days for microbiological evidence of infection is low. If the risks of waiting are high, based either on the patient’s immune status or other known risk factors for poor outcome, initiation of optimal empirical antimicrobial therapy should rely on the clinical presentation and clinical experience. The most valuable and time-tested method for immediate identification of bacteria is examination of the infected secretion or body fluid with Gram stain. In malaria-endemic areas, or in travelers returning from such an area, a simple thick and thin blood smear may mean the difference between a patient’s receiving appropriate lifesaving therapy or dying while on wrong therapy for presumed bacterial infection. Similarly, neutropenic patients with fever have high risks of mortality, and, when febrile, they are presumed to have either a bacterial or fungal infection; thus, a broad-spectrum combination of antibacterial and antifungal agents covering common infections encountered in granulocytopenic patients is recommended. Performance of cultures is still mandatory with a view to modify antimicrobial therapy with culture results.

DEFINITIVE THERAPY WITH KNOWN PATHOGEN. Once a pathogen has been isolated and susceptibilities results are available, therapy should be streamlined to a narrow targeted antibiotic. Monotherapy is preferred to decrease the risk of antimicrobial toxicity and selection of antimicrobial-resistant pathogens. Proper antimicrobial doses and dose schedules are crucial to maximizing efficacy and minimizing toxicity. In addition, the duration of therapy should be as short as is necessary.

Combination therapy is an exception, rather than a rule. Once a pathogen has been isolated, there should be no reason to use multiple antibiotics, except when evidence overwhelmingly suggests otherwise. Using 2 antimicrobial agents where one is required leads to increased toxicity and unnecessary damage to the patient’s otherwise protective fungal and bacterial flora. Special circumstances where evidence is in favor of combination therapy include:

• Preventing resistance to monotherapy

• Accelerating the rapidity of microbial kill

• Enhancing therapeutic efficacy by use of synergistic interactions

• Reducing toxicity (i.e., when sufficient efficacy of 1 antibacterial agent can be achieved at doses that are toxic to the patient and a second drug is coadministered to permit lowering dose of first drug)

Clinical situations for which combination therapy is advised include antiretroviral therapy for AIDS, antiviral therapy for hepatitis B and C, the treatment of tuberculosis, Mycobacterium avium-intracellulare and leprosy, fixed-dose combinations of antimalarial drugs, the treatment of Cryptococcus neoformans with flucytosine and amphotericin B, during empirical therapy for patients with febrile neutropenia, and advanced AIDS with fever. The combination of a sulfonamide and an inhibitor of dihydrofolate reductase, such as trimethoprim, is synergistic owing to the blocking of sequential steps in microbial folate synthesis.

POST-TREATMENT SUPPRESSIVE THERAPY. In some patients, after the initial disease is controlled by the antimicrobial agent, therapy is continued at a lower dose if the infection is not completely eradicated and the immunological or anatomical defect that led to the original infection is still present.

This is common in AIDS patients and post-transplant patients, for example. The goal is more as secondary prophylaxis. Nevertheless, risks of toxicity from long durations of the therapy are still real. In this group of patients, the suppressive therapy is eventually discontinued if the patient’s immune system improves.

MECHANISMS OF RESISTANCE TO ANTIMICROBIAL AGENTS

Today, significant resistance is emerging to every major class of antibiotic. Two major factors are associated with emergence of antibiotic resistance: evolution and clinical/environmental practices. Pathogens will evolve to develop resistance to the chemical warfare to which we subject them. This evolution is mostly aided by poor therapeutic practices by healthcare workers, as well as indiscriminant use of antibiotics for agricultural and animal husbandry purposes.

Antimicrobial resistance development may develop due to:

• Reduced entry of antibiotic into pathogen

• Enhanced export of antibiotic by efflux pumps

• Release of microbial enzymes that destroy the antibiotic

• Alteration of microbial proteins that transform prodrugs to the effective moieties

• Alteration of target proteins

• Development of alternative pathways to those inhibited by the antibiotic

Mechanisms by which such resistance develops can include acquisition of genetic elements that code for the resistant mechanism, mutations that develop under antibiotic pressure, or constitutive induction.

RESISTANCE DUE TO REDUCED ENTRY OF DRUG INTO PATHOGEN. The outer membrane of gram-negative bacteria is a permeable barrier that excludes large polar molecules from entering the cell. Small polar molecules, including many antibiotics, enter the cell through protein channels called porins. Absence of, mutation in, or loss of a favored porin channel can slow the rate of drug entry into a cell or prevent entry altogether, effectively reducing drug concentration at the target site. If the target is intracellular and the drug requires active transport across the cell membrane, a mutation or phenotypic change that slows or abolishes this transport mechanism can confer resistance.

For example, Trypanosoma brucei is treated with suramin and pentamidine during early stages, but with melarsoprol and eflornithine when CNS disease (sleeping sickness) is present. Melarsoprol is actively taken up by trypanosome P2 protein transporter. When the parasite either lacks the P2 transporter, or has a mutant form, resistance to melarsoprol and cross-resistance to pentamidine occur due to reduced uptake.

RESISTANCE DUE TO DRUG EFFLUX. Microorganisms can overexpress efflux pumps and then expel antibiotics to which the microbes would otherwise be susceptible. There are 5 major systems of efflux pumps that are relevant to antimicrobial agents:

• The multidrug and toxic compound extruder (MATE)

• The major facilitator superfamily (MFS) transporters

• The small multidrug resistance (SMR) system

• The resistance nodulation division (RND) exporters

• The ATP binding cassette (ABC) transporters

Efflux pumps are a prominent mechanism of resistance for parasites, bacteria, and fungi. One of the tragic consequences of resistance emergence has been the development of drug resistance byPlasmodium falciparum. Drug resistance to most antimalarial drugs is mediated by an ABC transporter encoded by Plasmodium falciparum multidrug resistance gene 1 (Pfmdr1). Point mutations in the Pfmdr1 gene lead to drug resistance and failure of chemotherapy.

RESISTANCE DUE TO DESTRUCTION OF ANTIBIOTIC. Drug inactivation is a common mechanism of drug resistance. Bacterial resistance to aminoglycosides and to β-lactam antibiotics usually is due to production of an aminoglycoside-modifying enzyme or β-lactamase, respectively.

RESISTANCE DUE TO REDUCED AFFINITY OF DRUG TO ALTERED TARGET STRUCTURE. Common consequences of single point and multiple point mutations are alterations in amino acid composition and conformation of the target protein. This change leads to a reduced affinity of drug for its target, or of a prodrug for the enzyme that converts the prodrug to active drug.

Such alterations may be due to mutation of the natural target (e.g., fluoroquinolone resistance), target modification (e.g., ribosomal protection type of resistance to macrolides and tetracyclines), or acquisition of a resistant form of the native, susceptible target (e.g., staphylococcal methicillin resistance caused by production of a low-affinity penicillin-binding protein). In HIV, resistance mutations associated with reduced affinity are encountered in protease inhibitors, integrase inhibitors, fusion inhibitors, and nonnucleoside reverse transcriptase inhibitors. Similarly, point mutations in the β-tubulin gene of worms and protozoa lead to modification of the tubulin and resistance to benzimidazoles.

DRUG DEPENDENCE IN BACTERIA. An uncommon situation occurs when an organism not only becomes resistant to an antimicrobial agent but subsequently starts requiring it for growth.

Enterococcus, which easily develops vancomycin resistance, can, after prolonged exposure to the antibiotic, develop vancomycin-requiring strains.

RESISTANCE DUE TO ENHANCED EXCISION OF INCORPORATED DRUG. Nucleoside reverse transcriptase inhibitors such as zidovudine are 2′-deoxyribonucleoside analogs that are converted to their 5′-triphosphate form and compete with natural nucleotides. These drugs are incorporated into the viral DNA chain and cause chain termination. When resistance emerges via mutations at a variety of points in the reverse transcriptase gene, phosphorolytic excision of the incorporated chain-terminating nucleoside analog is enhanced.

HETERO-RESISTANCE AND VIRAL QUASI-SPECIES. Hetero-resistance is said to be present when a subset of the total microbial population is resistant, despite the total population being considered susceptible on testing. A subclone that has alterations in genes associated with drug resistance is expected to reflect the normal mutation rates and occur at between 10–6 and 10–5 colonies.

In bacteria, hetero-resistance has been described especially for vancomycin in Staphylococcus aureus, vancomycin in Enterococcus faecium, colistin in Acinetobacter baumannii-calcoaceticus, rifampin, isoniazid, and streptomycin in M. tuberculosis, and penicillin in Streptococcus pneumoniae. Increased therapeutic failures and mortality may occur in patients with hetero-resistant staphylococci and M. tuberculosis. In fungi, hetero-resistance leading to clinical failure has been described for fluconazole in Cryptococcus neoformans and Candida albicans.

In viruses, replication is more error prone than replication in bacteria and fungi. Viral evolution under drug and immune pressure occurs relatively easily, commonly resulting in variants or quasi-species that may contain drug-resistant subpopulations. This is not often termed hetero-resistance, but the principle is the same as described for bacteria and fungi. These minority quasi-species that are resistant to antiretroviral agents are associated with failure of antiretroviral therapy.

EVOLUTIONARY BASIS OF RESISTANCE EMERGENCE

DEVELOPMENT OF RESISTANCE VIA MUTATION SELECTION. Mutation and antibiotic selection of the resistant mutant are the molecular basis for resistance for many bacteria, viruses, and fungi. Mutations may occur in the gene encoding

• The target protein, altering its structure so that it no longer binds the drug

• A protein involved in drug transport

• A protein important for drug activation or inactivation

• In a regulatory gene or promoter affecting expression of the target, a transport protein, or an inactivating enzyme

Mutations are not caused by drug exposure. They are random events that confer a survival advantage when drug is present. Any large population of drug susceptible bacteria is likely to contain rare mutants that are only slightly less susceptible than the parent. However, suboptimal dosing strategies lead to selective kill of the more susceptible population, which leaves the resistant isolates to flourish.

In some instances, a single-step mutation results in a high degree of resistance. In other circumstances, however, sequential acquisition of more than 1 mutation leads to clinically significant resistance. As an example, the combination of pyrimethamine and sulfadoxine inhibits Plasmodium falciparum’s folate biosynthetic pathway via inhibition of dihydrofolate reductase (DHFR) by the pyrimethamine and inhibition of dihydropteroate synthetase (DHPS) by sulfadoxine. Clinically meaningful resistance occurs when there is a single point mutation in the DHPS gene accompanied by at least a double mutation in the DHFR gene.

HYPERMUTABLE PHENOTYPES. Protecting genetic information from disintegrating and also maintaining flexibility sufficient for genetic changes that lead to adaptation to the environment are essential to life. This is accomplished principally by the insertion of the correct base pair by DNA polymerase III, proofreading by the polymerase, and postreplicative repair. The development of a defect in any of these repair mechanisms leads to mutations in many genes; such isolates are termed mutator (Mut) phenotypes and may include mutations in genes causing antibiotic resistance.

This second-order selection of hypermutable (mutator) alleles based on alterations in DNA repair genes has been implicated in the emergence of multidrug resistant strains of M. tuberculosis Beijing genotype.

RESISTANCE BY EXTERNAL ACQUISITION OF GENETIC ELEMENTS. Drug resistance may be acquired by mutation and selection, with passage of the trait vertically to daughter cells. For mutation and selection to be successful in generating resistance, the mutation cannot be lethal and should not appreciably alter virulence. For the trait to be passed on, the original mutant or its progeny also must disseminate and replicate.

Drug resistance more commonly is acquired by horizontal transfer of resistance determinants from a donor cell, often of another bacterial species, by transduction, transformation, or conjugation. Resistance acquired by horizontal transfer can disseminate rapidly and widely either by clonal spread of the resistant strain or by subsequent transfers to other susceptible recipient strains. Horizontal transfer of resistance offers several advantages over mutation selection. Lethal mutation of an essential gene is avoided; the level of resistance often is higher; the gene, which still can be transmitted vertically, can be mobilized and rapidly amplified within a population by transfer to susceptible cells; and the resistance gene can be eliminated when it no longer offers a selective advantage.

HORIZONTAL GENE TRANSFER. Horizontal transfer of resistance genes is facilitated by mobile genetic elements that include plasmids and transducing phages. Other mobile elements—transposable elements, integrons, and gene cassettes—also participate in the process. Transposable elements are of 3 general types: insertion sequences, transposons, and transposable phages. Only insertion sequences and transposons are important for resistance.

Insertion sequences are short segments of DNA encoding enzymatic functions (e.g., transposase and resolvase) for site-specific recombination with inverted repeat sequences at either end. They can copy themselves and insert themselves into a chromosome or a plasmid. Insertion sequences do not encode resistance, but they function as sites for integration of other resistance-encoding elements (e.g., plasmids or transposons). Transposons are insertion sequences that also code for other functions, 1 of which can be drug resistance. Because transposons move between chromosome and plasmid, the resistance gene can “hitchhike” with a transferable element out of the host and into a recipient. Transposons are mobile elements that excise and integrate in the bacterial genomic or plasmid DNA.Integrons are not formally mobile and do not copy themselves, but they encode an integrase and provide a specific site into which mobile gene cassettes integrate. Gene cassettes encode resistance determinants, usually lacking a promoter, with a downstream repeat sequence. The integrase recognizes this repeat sequence and directs insertion of the cassette into position behind a strong promoter that is present on the integron. Integrons may be located within transposons or in plasmids, and therefore may be mobilizable, or located on the chromosome.

Transduction is acquisition of bacterial DNA from a phage (a virus that propagates in bacteria) that has incorporated DNA from a previous host bacterium within its outer protein coat. If the DNA includes a gene for drug resistance, the newly infected bacterial cell may acquire resistance. Transduction is particularly important in the transfer of antibiotic resistance among strains of S. aureus. Transformation is the uptake and incorporation into the host genome by homologous recombination of free DNA released into the environment by other bacterial cells. Transformation is the molecular basis of penicillin resistance in pneumococci and Neisseria. Conjugation is gene transfer by direct cell-to-cell contact through a sex pilus or bridge. This mechanism for the spread of antibiotic resistance is important because multiple resistance genes can be transferred in a single event. Conjugation with genetic exchange between nonpathogenic and pathogenic microorganisms probably occurs in the GI tracts of humans and animals. The efficiency of transfer is low; however, antibiotics can exert a powerful selective pressure to allow emergence of the resistant strain. Genetic transfer by conjugation is common among gram-negative bacilli, and resistance is conferred on a susceptible cell as a single event. Enterococci also contain a broad range of host-range conjugative plasmids that are involved in the transfer and spread of resistance genes among gram-positive organisms.