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Principles of Antimicrobial Therapy
CLINICAL PHARMACOLOGY AND THERAPEUTICS
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PRINCIPLES OF ANTIMICROBIAL THERAPY Ted Whittem, BVSc, PhD, and Deborah Gaon, DVM, MS
The use of antimicrobial drugs in the absence of an etiological diagnosis is empirical antimicrobial therapy. 11 Empirical antimicrobial therapy is frequent in veterinary practices. Empirical therapy both is necessary and justified when therapy must be initiated immediately (prior to bacteriology), when microbiological samples fail to yield useable information, and when ethical or economic considerations predominate. Rational empirical antimicrobial therapy demands prior consideration of several specific questions: 1. Does the patient have an infection with an organism for which
therapy is possible? (Etiology) 2. What are the probable infectious organisms? (Etiology) 3. Which anatomic sites are possibly afflicted? (Pathophysiology) 4. Which of the available antimicrobial drugs are likely to be effective in inhibiting (or preferably killing) the likely pathogens, and at what concentration? (Spectrum of Activity) 5. What dose regimen is necessary to achieve these probable effective concentrations at the suspected anatomic location of the infectious process? (Efficacy) 6. Will this proposed regimen contribute to development of antimicrobial resistance by the pathogen or by commensal organisms? (Efficacy) 7. What is the risk of adverse drug reactions or toxicity (benefit:risk ratio)? (Toxicity)
From the Department of Veterinary Biosciences, College of Veterinary Medicine, University of Illinois, Urbana, Illinois
VETERINARY CLINICS OF NORTH AMERICA: SMALL ANIMAL PRACTICE VOLUME 28 • NUMBER 2 • MARCH 1998
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8. Can the client afford the proposed therapy for their animal? (Cost, opportune cost) Rational antimicrobial therapy is achieved when all or most of these questions have been adequately answered. Of paramount importance is that a drug with a suitable spectrum of activity must reach the site of infection in adequate concentrations to kill or inhibit the growth of the pathogen and make it susceptible to phagocytosis (Table 1). An important need in choosing an effective antimicrobial agent, therefore, is knowledge of which bacterial pathogens are involved in the infection. Ideally, identification is made by direct smear and culture of the organism(s). In practice, the choice of antibiotic often is based on knowledge of the probable pathogen most commonly associated with a certain site and clinical signs (Table 2). The lowest concentration of an antimicrobial drug that will inhibit the growth of a particular pathogen is known as that drug-pathogen combination's minimum inhibitory concentration (MIC). 29 Dose regimens that achieve adequate antimicrobial-drug concentrations invariably result in plasma-drug concentration in excess of the known or suspected MIC for the drug-pathogen pair. To ensure that an adequate antimicrobial-drug concentration is reached at the site of infection, the clinician must consider the dose to be used. The dose may be varied according to the known or suspected sensitivity of the pathogen to the chosen drug and with consideration of the ability of the chosen drug to reach the site of the infection. The dose regimen will vary from drug to drug, to ensure the most suitable drug-concentration profile is achieved for optimal efficacy according to known or assumed pharmacokinetic-efficacy correlates. 34 Age, organ system affected, concurrent diseases, and the environment at the site of infection will also influence antibiotic choice, dose, and regimen. For example, adjustment of antibiotic dose or interval may have to be made in patients with renal insufficiency because many antibiotics including 13-lactams, aminoglycosides, fluoroquinolones, and sulfonamides are eliminated by renal excretion (Table 3). If an aminoglycoside were indicated as the drug of last resort, the general recommendation is that the dose remain the same, but the time between doses is increased. 28 In contrast, under the same circumstances, for penicillins it may be more appropriate to decrease the total dose but maintain the same dose interval-2° Appropriate choice of antimicrobial drug and dose regimen often is not sufficient to ensure the success of therapy. Adjunctive treatments are often necessary. For instance, antimicrobial therapy of anaerobic infections such as pyometra, peritonitis, or bite wound abscess is more successful when combined with surgery. 9 Antibiotics can be administered parenterally, orally, rectally, vaginally, and topically. The correct route of administration is important in determining whether the antimicrobial is absorbed and whether it will reach adequate concentrations at the target site.
Table 1. SPECTRUM OF ACTIVITY OF SOME ANTIMICROBIAL DRUGS Antimicrobial Drug 13-Lactams Amoxicillin Amoxicillin + Clavulanate Cephalexin Penicillin G Aminoglycosides Amikacin Gentamicin Neomycin Tetracyclines Doxycycline Oxytetracycline Fluoroquinolones Enrofloxacin Orbifloxacin Macrolides and Lincosamides Clindamycin Erythromycin Lincomycin Tylosin Potentiated Sulfonamides Sulphadiazine + Trimethoprim Miscellaneous Metronidazole Key: 1-'
'-0 '-0
Gram-Positive Aerobes
Gram-Positive Anaerobes
Gram-Negative Aerobes
Gram-Negative Anaerobes
++ ++
++ ++
+ +
+ +
Very Some
+ ++
+ ++
+ +
+ +
Some Very
++ ++ ++
-
No No No
+ +
+ +
+ + + + +
. + +
+ +
++ ++
Others
(3-Lactamase Sensitive
+ +
No No
+ +
No No
+ + +
No No No No
++ ++ ++ ++
++ ++ ++ ++
-
+ + +
+
+
+
+
+
No
++
+
No
++
+ + = reliably useful; + = effective against some isolates; - = not clinically useful; (missing) = unknown.
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Table 2. BACTERIA THAT COMMONLY CAUSE INFECTIONS IN SPECIFIC ORGAN SYSTEMS IN DOGS AND CATS Organ System Canine Urinary tract
Upper respiratory tract
Lower respiratory tract External auditory canal
Common Pathogens Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae, Staphylococcus intermedius, Enterococcus faecalis, Pseudomonas aeruginosa, Leptospira spp. Staphylococcus intermedius, Eschericia coli, Pasteurella multocida, Klebsiella pneumoniae, Pseudomonas aeruginosa Bordetella bronchiseptica, Proteus mirabi/is, Straphylococcus intermedius Staphylococcus intermedius, Proteus mirabilis, Pseudomonas aeruginosa
Oral cavity
Obi igate anaerobes
Gastrointestinal tract
Enterobacteriaceae, Campylobacter spp., Clostridium perfringens, Bacteroides tragi/is, other anaerobes Staphylococcus intermedius, Eschericia coli, Proteus mirabilis, Pseudomonas aeruginosa Staphylococcus intermedius, Eschericia coli Eschericia coli, Staphylococcus intermedius, obligate anaerobes, other enterobacteria
Skin
Bone Septicemia
Feline Urinary tract
Upper respiratory tract Lower respiratory tract
Conjunctiva Oral cavity Skin
Eschericia coli, Proteus mirabi/is, Klebsiella pneumoniae, Pasteurella multocida, Pseudomonas aeruginosa Pasteurella multocida, Eschericia coli, Proteus mirabilis, Pseudomonas aeruginosa Pasteurella multocida, Eschericia coli, Klebsiella pneumoniae Chlamydia psittaci, 13-hemolytic· Streptococci, Pasteurella multocida Obligate anaerobes Pasteurella multocida, Eschericia coli, Proteus mirabilis
Rational Empirical Choices Amoxicillin + Clavulanate Potentiated sulfonamide Cephalexin Aminoglycoside Amoxicillin + Clavulanate Doxycycline Cephalexin Need culture and sensitivity Polymixin B Gentamicin Amoxicillin + Clavulanate Enrofloxacin Amoxicillin Clindamycin Metronidazole Need culture and sensitivity
Amoxicillin + Clavulanate Cephalexin Enrofloxacin Amoxicillin + Clavulanate Cephalexin Ampicillin or Cephalexin/ cefazolin + amikacin or gentamicin or enrofloxacin + metronidazole Amoxicillin + Clavulanate Cephalexin Enrofloxacin Amoxicillin + Clavulanate Cephalexin Gentamicin Amoxicillin + Clavulanate Cephalexin Enrofloxacin Gentamicin Doxycycline Chloramphenicol Amoxicillin Clindamycin Metronidazole Amoxicillin + Clavulanate Enrofloxacin Cephalexin
Data from Aucoin D: Target: The Antimicrobial Reference Guide to Effective Treatment. Port Huron, Ml, North American Compendiums Inc., 1993 and Ferguson DC: Rational empirical antimicrobial therapy in small animals. An International Symposium on Antimicrobial Selection, The North American Veterinary Conference, Orlando, FL, Veterinary Learning Systems Co., Inc., 1994, pp 4-11.
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Table 3. ORGAN PRIMARILY RESPONSIBLE FOR ELIMINATION Antimicrobial Drug
Renal
13-Lactams Aminoglycosides Tetracyclines Doxycycline Other tetracyclines Fluoroquinolones Macrolides and lincosamides Sulfonamides
++ ++
Hepatic Metabolism
++ ++ + ++
Biliary Excretion
Other
±
+ + ++
++ + +
+
MICROBIAL CHARACTERISTICS
Knowledge of the classifying characteristics of the infecting organism can be helpful in predicting how effective certain microbial agents will be in combating an infection. The competent clinician will know for each suspected pathogen its Gram-staining characteristic and whether it is an aerobe, facultative, or obligate anaerobe (Table 4). Gram Positive
Gram-positive bacteria have an outer wall that is comprised of peptidoglycans 50 to 100 molecules thick. 22 Between the peptidoglycan layers are strands of lipoteichoic acid that are anchored to the glycolipid molecules of the cell membrane (Fig. 1). 19 Drugs that disrupt the formation of peptidoglycans can easily reach Table 4. SOME COMMON BACTERIAL PATHOGENS AND THEIR CLASSIFICATION Gram-Positive Aerobes Streptococcus Enterococcus Staphylococcus*t
Gram-Positive Gram-Negative Gram-Negative Anaerobes Aerobes Anaerobes Clostridia
·························································
Borde tel/a Leptospira
Bacteroides* Fusobacterium
Pseudomonas*t Proteust Escherichia coltt
·"Eiiiei-ahaCieif ································ ·kieiisieiiaf ················ ····················· ·"Pasieiireiiat ··································· ·saimoiieilat ··································· ··cam"P"Yioiiac"ier ······························ Nocardia
Nocardia
*Constitutive 13-lactamase producers. tlnducible 13-lactamase production possible.
Others Chlamydia Mycobacterium Mycoplasma
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lipotecheic acid
A
bacterial inner cell membrane
porin channel lipoprotein bacterial outer membrane lipoprotein peptidoglycan
8
bacterial inner cell membrane
Figure 1. Cell wall structures of Gram-positive (A) and Gram-negative (B) bacteria.
their target in Gram-positive organisms, because the peptidoglycan cell wall is at the surface of the organism. 4 The antibiotics that are the most successful at targeting Gram-positive organisms act through this mechanism. Examples of these drugs are the 13-lactam s (penicillin, aminopenicillins, and cephalosporins), vancomycin, and bacitracin. Trimethoprim-sulfa, erythromycin, lincomycin, clindamycin, and chloramphenicol are also effective against many Gram-positive organisms, but these drugs act through mechanisms that do not affect cell wall synthesis. Gram Negative
In contrast to Gram-positive bacteria, those that are Gram-negative have an outer membrane that protects the thinner peptidoglycan layer: the peptidoglycan is merely one to two molecules thick and is linked to the outer membrane by an unusual lipoprotein.19• 22 Embedded in the outer membrane are proteins called porins, which are arranged in triplets to form diffusion pores. These porins allow small hydrophilic molecules,
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such as antibiotics, to easily pass through the outer membrane (see Fig. 1). 29 An exception is vancomycin, which is excluded by the porins and therefore is not effective against Gram-negative organisms. The pores are smaller in some Gram-negative bacteria such as Pseudomonas aeruginosa than in most other Gram-negative genera, thereby excluding many antimicrobial drugs. Therefore, Pseudomonas aeruginosa is more resistant to antimicrobial therapy. 16, 36 Because many antibiotics have difficulty in penetrating the lipid cell membranes, infections caused by Gram-negative bacteria are more challenging to treat. Antibiotics such as penicillin, the macrolides, and bacitracin are generally less potent against Gram-negative bacteria. Aminoglycosides are organic bases that are charged at physiologic pH and therefore would be predicted to have difficulty transiting across lipid membranes. They are able, however, to diffuse through the porin channels of the outer lipid membrane of Gram-negative bacteria and then are actively imported by oxygen-dependent transport proteins within the inner membrane. This allows their accumulation and access to the ribosomal site-of-action?' 25 The tetracyclines, chloramphenicol, polymyxins, fluoroquinolones, and some (3-lactams obtain access to and are effective against Gramnegative bacteria. 4 Anaerobes
Generally, facultative anaerobes are considered pathogenic, whereas strict anaerobes are not. Facultative anaerobes require reduced oxygen tension for growth and do not require molecular oxygen for metabolic activity. The pathogenic anaerobes are classified as Gram-positive and Gram-negative, but in addition to these characteristics, the anaerobes have other unique characteristics that vary their susceptibility to certain antimicrobials. Anaerobes are able to elaborate a variety of toxins and enzymes that can cause extensive tissue necrosis. Bacteroides fragilis, for example, can produce heparinase, deoxyribonuclease, neuraminidase, endotoxins, and 13-lactamases that can inactivate most 13-lactam antibiotics.9 Potentiated sulfonamides are inactivated by exudate and debris and therefore are not considered useful in treating anaerobic infections. The transport mechanism for import of aminoglycosides into cells is oxygendependent, so members of this family of antimicrobials are not active against anaerobes. 4 Chloramphenicol, clindamycin, and metronidazole provide the most reliable activity against pathogenic anaerobes. 9 CHOOSING A DOSE REGIMEN
The mechanism by which antibiotics kill or prevent the growth of bacteria is an important consideration when choosing a dose regimen.
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The mechanism of action is also a convenient way to classify antimicrobials. Knowing the mechanistic class of antimicrobial for any particular drug helps to remember the correct approach to dosing with that drug. Sites of action for several antimicrobial drugs are shown in Figure 2. Inhibition of Bacterial Cell Wall Synthesis Penicillins, Cepha/osporins, Vancomycin, and Bacitracin
13-lactams (penicillins and cephalosporins) bind to penicillin binding proteins (PBP) near the cell surface disrupting peptidoglycan synthesis in cell w all formation that results in cell lysis and deathY The bacterial cell must be growing and replicating for this class of drug to be effective. The binding to PBP is reversible and saturable.38 The bacterial action of the 13-lactams is time-dependent. For the penicillins and cephalosporins to be effective, their concentration in blood must be maintained above the minimum inhibitory concentrations (MIC) for long periods of time.20• 34 Most of these drugs need to be administered more than once daily, as they tend to have short elimination half-lives.15• 26 Excessive serum concentrations will saturate the PBPs and result in no additional benefit. If the 13-lactam concentration falls below the MIC, the bacteria will remain stable, but may show some structural abnormalities. With the exception of Staphylococci spp., 13-lactams do not cause a significant postantibiotic effect.35 A
B
Figure 2. Sites of action of various antimicrobial drugs: A. Cell wall-13-lactams, vancomycin, bacitracin; 8 , Cell membrane, tetracyclines; C, Cell membrane associated enzymes- aminoglycosides; 0 , DNA- metronidazole, sulfonamides, trimethoprim; E, DNA topoisomerase-fluoroquinolones; F, 30S Ribosomal subunit-aminoglycosides, tetracyclines; G, 50S Ribosomal subunit- macrolides, lincosamides, tetracyclines; H, DNA-dependent RNA polymerase-rifampin; J, RNA.
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These factors together suggest that for !3-lactam antibiotics, a dose regimen should be designed such that the drug concentration at the site of infection remains above the MIC for the pathogen throughout the dose interval. In addition, it can be deduced that exceeding the pathogen MIC by a great amount is likely to be of no additional benefit. These deductions have been supported by experimental evidence with in vivo models of bacterial infection. 10' 21 Similar information about dose regimen-efficacy correlations is unavailable for vancomycin and bacitracin, the latter of which is used only topically because of its toxicity. Disruption of Cell Membranes
Polymyxin B, Colistin (Polymixin E), Nystatin, and Amphotericin 8
These drugs insert into cell membranes either as detergents (Polymyxin B, colistin) or by binding to ergosterol (amphotericin B) and thereby disrupt the integrity of the phospholipid membrane. By destroying cell membranes, they allow cytoplasm leakage, leading to cell death. These drugs can act against quiescent cells; they are not dependent on active cell growth. Therefore they can be combined with bacteriostatic antibiotics. Because they require access to the cell membrane, the polymyxins are most effective against Gram-negative organisms. Because ergosterol is a component of fungal cell membranes, nystatin and amphotericin are antifungal drugs. Mammalian cell membranes possess similar components to those of both bacteria and fungi. Therefore, these drugs are not very selectively toxic and also damage host cell membranes. Although the efficacy of these drugs is likely to be concentration dependent, so too is the toxicity. Therefore the clinician has little room to vary from recommended dose regimens. 2, 30 Inhibitors of Protein Synthesis
Aminoglycosides, Tetracyclines, Macro/ides, Lincosamides, and the Chloramphenicol Family
After import into the bacterial cell, the aminoglycosides (e.g., streptomycin, gentamicin, amikacin, tobramycin, kanamicin) bind irreversibly to the 30s ribosomes altering protein synthesis. 32 These drugs also affect cell membrane structural integrity. 6 Together these actions result in bacterial cell death. Because these drugs are actively accumulated by aerobic bacteria and the rib9somal binding is irreversible/1 aminoglycosides continue to inhibit bacterial protein synthesis after the drug is no longer detectable in the bacterial milieu. 35 This effect is known as the postantibiotic effect (PAE). Possibly partly because of this effect, once daily dosing is efficacious despite rapid clearance from the host by renal filtration. 8
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Once daily aminoglycoside therapy is effective so long as the peak drug concentration in the plasma is at least four times the MIC for the pathogen (inhibitory quotient :::::: 4) even though the drug concentration falls below the MIC for long periods. Although it has been postulated that the PAE is partly dependent on the patient's immune system, once daily therapy is efficacious in immunodeficient children. 14 The PAE may also prevent proper cell wall and septum formation or cause the bacteria to be transformed to a less virulent strain. It may be necessary to dose patients with aminoglycosides more frequently than once daily when their infection continues to be seeded from a body cavity or foreign body to which the antibiotic fails to gain access. 37 In contrast to aminoglycosides and their bactericidal action, chloramphenicol, tetracyclines, macrolides, lincosamides, and clindamycin bind reversibly to ribosomes and, under most circumstances in vivo, appear to achieve only bacteriostatic action. 30 There are no good clinical trial data available in which dose regimen-efficacy relationships are described for these drugs. Conventional wisdom suggests that the efficacy of these drugs is likely to relate to the length of time at which the pathogen is exposed to supra-MIC drug concentration. Dose regimens should be designed to ensure rapid attainment of appropriate concentration of drug at the site of the pathogen, perhaps initially using loading doses or parenteral formulations. Initiation of therapy should then be followed by a dose regimen that ensures continued supra-MIC drug concentration at the site of action. Since the pharmacokinetics of these drugs vary greatly from drug to drug and between species, clinicians should carefully follow dose regimens recommended in formularies or on the package insert. Interruption of Nuclei Acid Metabolism Fluoroquinolones, Sulfonamides, Diaminopyrimidines, Metronidazole
The fluoroquinolones (e.g., enrofloxacin, orbifloxacin) inhibit DNA topoisomerase, thereby inhibiting bacterial replication causing cell death. 5 Metronidazole may have a similar action in some bacteria.24 The intensity of exposure to a drug has been quantified as the ratio of the area under the plasma-concentration versus time curve to the MIC (AUC/MIC). For the fluoroquinolones, the intensity of exposure is more closely linked with efficacy than the time above the MIC. Higher once daily dosing has been shown to be more effective than the same daily dose divided into four daily injections, because higher doses given less frequently result in a greater AUC. Higher doses also cause higher peak plasma concentrations. The peak concentration achieved may also be closely related to efficacy but certainly has been related to decreasing the development of resistant strains. 5 For these reasons, the labeled dose regimen for enrofloxacin (in the United States) has recently been changed
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from a dose interval of 12 hours to one of 24 hours. Furthermore, the upper limit of the labeled total daily dose has also been increased, since this is likely to increase efficacy, decrease development of resistance, and can be done without inducing toxicity. Orbifloxacin, another recently introduced fluoroquinolone, should also be administered once daily. Folate is required for DNA synthesis. In bacteria, folate may be either synthesized from para-amino benzoic acid (PABA) or imported directly into the cell as a nutrient (e.g., from pus). Sulfonamides contain a structural analog of PABA that competes with PABA and thereby interferes with bacterial DNA synthesis if insufficient folate is available for uptake, having a bacteriostatic effect. Folate is sequentially reduced to dihydrofolate and tetrahydrofolate in the pathway for thymidine synthesis. These reactions are dependent on enzymes. Diaminopyrimidines such as trimethoprim compete for and inhibit the enzyme dihydrofolate reductase, thereby also inhibiting pyrimidine synthesis. This effect is also bacteriostatic. However, the combination of sulfonamide and trimethoprim is synergistic and bactericidal.23 When sulfonamides are used alone, their bacteriostatic effect is dependent on the maintenance of supra MIC plasma drug concentrations. More frequent dose intervals are better at achieving bacterial elimination. Both the fluoroquinolones and trimethoprim exhibit a PAE. Their effects persist after their plasma concentrations fall below the MICa few hours following dosing, probably because tissue concentrations remain higher at the site of infection. The PAE may be important to these drug's efficacy when administered only once or twice daily. 5' 35 RESISTANCE
Under normal conditions the frequency of mutations in a bacterial population is 1 organism in 1,000,000. The frequency of mutation is influenced by antimicrobial drugs and is thought to be directly correlated to the ratio of the MIC to the antibiotic concentration at the site of infection. This emphasizes the importance of adequate dosing. In treating an infection, the time above the MIC is also an important factor in minimizing development of resistance. Generally, the more chronic the infection and less accessible the site of infection is to the drug, then higher doses and longer treatment times are required to prevent resistance.16' 36 For example, in dogs with experimentally induced peritonitis, standard parenteral doses of cephalothin and aminoglycosides (gentamicin, tobramycin, amikacin, and kanamycin) did not reach the MIC in the ascitic fluid. It was the conclusion of the authors that higher than usual parenteral doses may have to be used in treating peritonitisY Underdosing antibiotics selects for the resistant bacteria by causing the eradication of susceptible population of organisms and overgrowth of resistant subpopulations that are present in small numbers. Resistant subpopulations may also be selected when antimicrobial agents are used
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at placement of intravenous or urinary catheters. This practice can result in a high number of nosocomial infections. Using aseptic techniques when placing a catheter is more effective than prophylactic use of antibiotics. In surgery, prophylactic use of antibiotics is indicated for clear contaminated wounds encountered in gastrointestinal, respiratory, and urogenital therapy.U Animals previously exposed to antimicrobials are more likely to have antimicrobial resistant Staphylococcus aureus. These bacteria were demonstrated to be resistant most frequently to penicillin, then streptomycin followed by tetracycline. 3 Knowing the antimicrobial's family may help to understand the development and ramifications of resistance by bacteria to that drug. Resistance of Staphylococcus spp. to penicillin is most often plasmidmediated. Plasmids are extrachromosomal genetic elements that exist and function independently from chromosomes. Plasmids that confer antimicrobial resistance are known as R-plasmids. These DNA fragments can induce resistance to antimicrobial drugs in a number of ways, including coding for 13-lactamase. Expression of R-plasmids of Grampositive organisms can be induced by exposure to 13-lactam antibiotics. These bacteria then produce 13-lactamase and secrete it extracellularly in large amounts, inactivating some types of 13-lactam antibiotics. 16 Plasmid mediated 13-lactamases are also responsible for the majority of Gram-negative resistance to 13-lactam antibiotics. In contrast to Grampositive bacterial 13-lactamase, Gram-negative bacterial 13-lactamases are strategically confined within the periplasmic space of Gram-negative bacteria (i.e., at the site of action of 13-lactam antibiotics). Gram-negative bacteria also produce a greater variety of lactamases than do Grampositive bacteria. The plasmid mediated 13-lactamase of the Gram-negative bacteria Eschericia coli, Enterobacter spp., Proteus spp., Shigella spp., Pseudomonas aeruginosa, Hemophilus spp. and Klebsiella spp. are effective in destroying both penicillins and cephalosporins. Chromosomes can also confer 13-lactamase resistance to penicillin for Proteus mirabilis and morganii or to cephalosporins for P. aeruginosa, Bacteroides fragilis, Proteus, Escherichia coli, and Enterobacter spp. Chromosomally mediated 13-lactamases of Klebsiella spp. are effective against both penicillins and cephalosporins.4, 16 Therefore, when a clinician suspects an infection is caused by a Gram-positive 13-lactamase producer, such as may be the case when the bacteria is reported resistant to amoxicillin but sensitive to amoxicillinclavulanate, then a cephalosporin may still be a good choice. However, if the 13-lactamase producer is Gram-negative, then it would be better to choose a drug that is a member of an alternate mechanistic class. Both plasmid and chromosomal DNA can code other changes that cause resistance against antimicrobial drugs. Conformational changes to or lack of the ribosomal binding sites may induce resistance to chloramphenicol or streptomycin. In the case· of chloramphenicol, this bacterial resistance is chromosomally conferred as a lack of the 30s subunit binding site. Staphylococcus aureus biochemically modifies eryth-
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romycin, which reduces the affinity of erythromycin for its binding site on the 50s ribosomal subunit. Resistance to erythromycin via this mechanism also confers resistance to other macrolides and lincosamides.36 Resistance to trimethoprim is also plasmid mediated. This plasmid encodes for the production of dihydrofolate reductase, which has little or no affinity for trimethoprim. 12 In contrast to the 13-lactams, macrolides, lincosamides, and trimethoprim, fluoroquinolones do not appear to be as readily affected by transferable plasmid mediated resistance. R-plasmids, which confer resistance to other antibiotics, may be eliminated from the harboring bacteria as a response to exposure to fluoroquinolones. Resistance to fluoroquinolones occurs by genomic mutation, which is an infrequent occurrence. These mutations primarily alter bacterial cell walls decreasing drug penetration. Mutational change also occurs to the structure of the target DNA gyrase. Some of these mutations confer resistance also to cephalosporins, tetracyclines, and chloramphenicol. Other mutations that cause fluoroquinolone resistance can cause hypersusceptibility to 13-lactams, aminoglycosides, and novobiocin. 5• 33 COMBINATION THERAPY Synergism
Synergism between two antimicrobial drugs is an in vitro phenomenon. Two drugs are synergistic for a particular bacterial isolate if the sum of the individual in vitro fractional inhibitory concentrations (FIC) is less than one. The two drugs may also be synergistic if the rate of in vitro bactericidal activity is increased 100-fold for a particular bacterial isolate when the bacterium is exposed to the combination. Finally, synergy is also declared if an increase in the rate of clinical cure from defined infections is demonstrated after combination of two antimicrobial drugs. Antimicrobial drug combinations may also be indifferent or antagonistic.17, 29 Synergy is a real phenomenon that occassionally aids in clinical resolution of disease. However, without laboratory documentation of potential synergy, combination therapy is never indicated simply in the hope of induced synergy. Antimicrobial drugs have been divided into two groups, based on their mechanism of action, to aid prediction of the in vitro effect of combinations/ 8 and this scheme has been more recently revised. 27 Group 1 drugs have bactericidal action at clinically achieved concentrations (e.g., penicillins, cephalosporins, aminoglycosides, vancomycin, and perhaps the fluoroquinolones). Group 2 drugs are merely bacteriostatic at clinically achieved concentrations (e.g., tetracyclines, chloramphenicol, macrolides, and lincosamides). Unfortunately, it has become a generally accepted dogma that group 1 drugs when combined may provide "synergistic" activity and group 2 drugs when combined may cause "indifference," but combination of a group 1 drug with a
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group 2 drug will be likely to cause "antagonism." This dogma is incorrect. Clinicians should not use this scheme to predict synergy, indifference, or antagonism in vivo. The scheme is useful only as a guide to the in vitro investigation of drug combinations. 18 There is no rational potential for the direct clinical application of this schemeY
Increasing Antimicrobial Spectrum
Combination therapy may be rational when used to increase the breadth of antibacterial spectrum. This is especially appropriate for cases with acute, life-threatening bacterial infections of unknown or as-yet unidentified etiology. Occasionally, combination therapy also may be used rationally to decrease the rate or probability of resistance developing to the effective antimicrobial drug. Generally, bacteria can be divided into four major groups: Grampositive aerobes, Gram-positive anaerobes, Gram-negative aerobes, and Gram-negative anaerobes. If these four groups are displayed in a classical 2 X 2 chart, each group is represented by one quadrant. Accordingly, desirable antimicrobial drug combinations are frequently described as achieving "four-quadrant therapy." Typical combinations for four-quadrant therapy include (1) a 13-lactam and an aminoglycoside or a fluoroquinolone and (2) clindamycin and an aminopenicillin or a fluoroquinolone. With the first of these two options, it may be necessary to include metronidazole if Bacteroides fragilis is likely to be one of the pathogens.
TOXICITY
Antibiotic therapy can be of benefit, but it is also important to consider the negative side effects. The consequences of the potential toxicity may preclude the use of a certain drug, especially if it may worsen a pre-existing condition. Antimicrobial toxicities include kidney, nervous system, gastrointestinal, bone marrow, cartilage/joint, eye, and liver. Knowing the antimicrobial's family also helps us to remember and understand the likely manifestations of toxicity (Table 5).
CONCLUSION
It has been 2500 years since the Chinese began applying moldy soybean curd to cure skin infections. Technology today has refined somewhat the benefits of antibiotic forming molds and bacteria and has greatly increased the number of antimicrobial drugs available to combat infection. Understanding the principles fundamental to rational therapy with these drugs will ensure the best of possible outcomes.
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Table 5. MANIFESTATIONS OF ADVERSE REACTION AND TOXICITY TO SOME ANTIMICROBIAL DRUG FAMILIES Antimicrobial Drug
Aminoglycosides
13-Lactams Tetracyclines
Fluoroquinolones Erythromycin Metronidazole
Lincomycin Sulfonamides
Adverse Reactions (Not Dose Related)
Toxicity (Dose Related)
Nephropathy CN VIII damage Neuromuscular blockade Acute hypersensitivity Ulcerative colitis Acute hypersensitivity Gastroenteritis Toxic epidermal necrolysis Hepatopathy Photosensitization Nephropathy Cartilage damage Altered CNS signaling Hepatopathy Abdominal pain CNS intoxication
Enterocolitis Acute hypersensitivity Nephropathy Toxic epidermal necrolysis Hepatopathy Keratoconjunctivitis sicca
Clinco-Pathological Sign
Increased serum creatinine, urinary casts, ataxia, loss of righting reflexes, deafness, flaccid paralysis Diarrhea, vomiting, febrile Diarrhea Hepatic enzyme elevation Dermatitis, erythema Increased serum creatinine Lameness Convulsions Diarrhea, vomiting Anorexia, vomiting Depression Nystagmus, ataxia Tremors, seizures Diarrhea, vomiting Febrile Diarrhea, vomiting, depression, oliguria, increased serum creatinine, urinary casts, crystalluria, blepharospasm, corneal pigmentation, depressed Schirmer tear test
References 1. Aucoin D: Target. The Antimicrobial Reference Guide to Effective Treatment. Port Huron, MI, North American Compendiums Inc., 1993 2. Bennet JE: Antimicrobial agents [continued]: Antifungal agents. In Gilman AG, Rail TW, Nies AS, et a! (eds): Goodman and Gilman's The Pharmacological Basis of Therapeutics. New York, McGraw-Hill, 1992, pp 1165-1181 3. Biberstein IL, Franti CE, Spencer SJ, et a!: Anitmicrobial sensitivity patterns in Staphylococcus aureus from animals. JAm Vet Med Assoc 164:1183-1186, 1974 4. Brown SA: Treatment of Gram-negative infections. Vet Clin North Am: Small Anim Pract 18:1141-1165, 1988 5. Brown SA: Fluoroquinolones in animal health. J Vet Pharmacal Ther 19:1-14, 1996 6. Bryan LE: Cytoplasmic membrane transport and antimicrobial resistance. In Bryan LE (ed): Microbial Resistance to Drugs. Berlin, Springer-Verlag, 1989, pp 35-57 7. Bryan LE, Kwan S: Mechanisms of aminoglycoside resistance of anaerobic bacteria and facultative bacteria grown anaerobically. J Antimicrob Chemother 8 (suppl D):1-8, 1981 8. Campbell BG, Bartholow S, Rosin E: Bacterial killing by use of once daily gentamicin dosage in guinea pigs with Escherichia coli infection. Am J Vet Res 57(11):1627-1630, 1996 9. Dow SW: Management of anaerobic infections: Clinical pharmacology. Vet Clin North Am: Small Anim Pract 18:1167-1182, 1988 10. Drusano GL: Role of pharmacokinetics in the outcome of infections. Antimicrob Agents Chemother 32(3):289-297, 1988 11. Ferguson DC: Rational empirical antimicrobial therapy in small animals. An International Symposium on Antimicrobial Selection, The North American Veterinary Conference. Orlando, Florida, Veterinary Learning Systems Co., Inc., 1994, pp 4-11
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12. Foltzer MA, Reese RE: Trimethoprim-sulfamethoxazole and other sulfonamides. Med Clin North Am 71(6):1177-1194, 1987 13. Gerding DN, Kromhout JP, Sullivan JJ, et a!.: Antibiotic penetrance of ascitic fluid in dogs. Antimicrob Agents Chemother 10(5):850-855, 1976 14. Giacoia GP, Schentag JJ: Pharmacokinetics and nephrotoxicity of continuous intravenous infusion of gentamicin in low birth weight infants. J Pediatr 109:715-719, 1986 15. Greco DS, Brown SA, Gauze JJ, et a!.: Dexamethasone pharmacokinetics in clinically normal dogs during low- and high-dose dexamethasone suppression testing. Am J Vet Res 54(4):580-586, 1993 16. Jacoby GA, Archer GL: New mechanisms of bacterial resistance to antimicrobial agents. N Eng! J Med 324:601-{;12, 1991 17. Jawetz E: The use of combinations of antimicrobial drugs. Ann Rev Pharmacol 8:151170, 1968 18. Jawetz E, Gunnison JB: Studies on antibiotic synergism and antagonism: The scheme of combined antimicrobial activity. Antibiot Chemother 2:243-248, 1952 19. Jawetz E, Melnick JL, Adelberg EA: Review of Medical Microbiology. Los Altos, CA, Lange Medical Publications, 1980 20. Koritz GD, Bevill RF: Role of pharmacokinetics in the outcome of treatment of infections. Acta Vet Scand 87(suppl):18-27, 1991 21. Leggett JE, Fantin B, Ebert S, et al: Comparative antibiotic dose-effect relations at several dosing intervals in murine pneumonitis and thigh-infection models. J Infect Dis 159:281-292, 1989 22. Mandell GL, Sande MA: Penicillins, cephalosporins, and other beta-lactam antibiotics. In Gilman AG, Rail TW, Nies AS, et al (eds): Goodman and Gilman's The Pharmacological Basis of Therapeutics. New York, McGraw-Hill, 1992, pp 1065-1097 23. Mandell GL, Sande MA: Sulfonamides, trimethoprim-sulfamethoxazole, quinolones, and agents for urinary tract infections. In Gilman AG, Rail TW, Nies AS, et a! (eds): Goodman and Gilman's The Pharmacological Basis of Therapeutics. New York, McGraw-Hill, 1992, pp 1047-1064 24. Muller M: Mode of action of metronidazole on anaerobic bacteria and protozoa. Surgery 93:165-171, 1983 25. Nakae R, Nakae T: Diffusion of aminoglycoside antibiotics across the outer membrane of Escherichia coli. Antimicrob Agents Chemother 22:554-559, 1982 26. Petersen SW, Rosin E: Cephalothin and cefazolin in vitro antibacterial activity and pharmacokinetics in dogs. Vet Surg 24:347-351, 1995 27. Rahal JJ: Antibiotic combinations: The clinical relevance of synergy and antagonism. Medicine (Baltimore) 57:179-195, 1978 28. Riviere JE, Coppoc GL: Dosage of antimicrobial drugs in patients with renal insufficiency. J Am Vet Med Assoc 178:70-72, 1981 29. Sande MA, Kapusnik-Uner JE, Mandell GL: Chemotherapy of microbial diseases: Antimicrobial agents: General considerations. In Gilman AG, Rail TW, Nies AS, et a! (eds): Goodman and Gilman's The Pharmacological Basis of Therapeutics. New York, McGraw-Hill, 1992, pp 1018-1046 30. Sande MA, Mandell GL: Antimicrobial agents [continued]: Tetracyclines, chloramphenicol, erythromycin, and other miscellaneous antibacterial agents. In Gilman AG, Rail TW, Nies AS, et a! (eds): Goodman and Gilman's The Pharmacological Basis of Therapeutics. New York, McGraw-Hill, 1992, pp 1117-1145 31. Sande MA, Mandell GL: Antimicrobial agents [continued]: The aminoglycosides. In Gilman AG, Rail TW, Nies AS, eta! (eds): Goodman and Gilman's The Pharmacological Basis of Therapeutics. New York, McGraw-Hill, 1992, pp 1098-1116 32. Shannon K, Phillips 1: Mechanisms of resistance to aminoglycosides in clinical isolates. J Antimicrob Chemother 9:91-102, 1982 33. Smith JT: Mutational resistance to 4-quinolone antibacterial agents. Eur J Clin Microb 3:347-350, 1984 34. Vogelman B, Gudmundsson S, Leggett J, et a!: Correlation of antimicrobial pharmacokinetic parameters with therapeutic efficacy in an animal model. J Infect Dis 158:831847, 1988
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35. Vogelman BS, Craig WA: Post antibiotic effects. J Antimicrob Chemother 15(suppl A):37-46, 1985 36. Weisblum B: Inducible resistance to macrolides, lincosamides and streptogramin type B antibiotics: The resistance phenotype, its biological diversity, and structural elements that regulate expression: A review. J Antimicrob Chemother 16 (suppl A):63-90, 1985 37. Whittem T: Gentamicin, once, twice or thrice a day? In Stiff M, Sellon DC (eds): 15th Annual Veterinary Medical Forum. Lake Buena Vista, FL, American College of Veterinary Internal Medicine, 1997, pp 371-373 38. Zorzi W, Ziou XY, Dardenne 0, et al: Structure of the low-affinity penicillin-binding protein 5 PBPSFM in wild-type and highly penicillin-resistant strains of Enterococcus faecium. J Bacterial 178:4948-4957, 1996
Address reprint requests to Ted Whittem, BVSc, PhD Assistant Professor, Veterinary Clinical Pharmacology Department of Veterinary Biosciences College of Veterinary Medicine University of Illinois 1008 W. Hazelwood Drive Urbana, IL 61802
0195-5616/98 $8.00
+ .00
PRINCIPLES OF ANTIMICROBIAL THERAPY Ted Whittem, BVSc, PhD, and Deborah Gaon, DVM, MS
The use of antimicrobial drugs in the absence of an etiological diagnosis is empirical antimicrobial therapy. 11 Empirical antimicrobial therapy is frequent in veterinary practices. Empirical therapy both is necessary and justified when therapy must be initiated immediately (prior to bacteriology), when microbiological samples fail to yield useable information, and when ethical or economic considerations predominate. Rational empirical antimicrobial therapy demands prior consideration of several specific questions: 1. Does the patient have an infection with an organism for which
therapy is possible? (Etiology) 2. What are the probable infectious organisms? (Etiology) 3. Which anatomic sites are possibly afflicted? (Pathophysiology) 4. Which of the available antimicrobial drugs are likely to be effective in inhibiting (or preferably killing) the likely pathogens, and at what concentration? (Spectrum of Activity) 5. What dose regimen is necessary to achieve these probable effective concentrations at the suspected anatomic location of the infectious process? (Efficacy) 6. Will this proposed regimen contribute to development of antimicrobial resistance by the pathogen or by commensal organisms? (Efficacy) 7. What is the risk of adverse drug reactions or toxicity (benefit:risk ratio)? (Toxicity)
From the Department of Veterinary Biosciences, College of Veterinary Medicine, University of Illinois, Urbana, Illinois
VETERINARY CLINICS OF NORTH AMERICA: SMALL ANIMAL PRACTICE VOLUME 28 • NUMBER 2 • MARCH 1998
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8. Can the client afford the proposed therapy for their animal? (Cost, opportune cost) Rational antimicrobial therapy is achieved when all or most of these questions have been adequately answered. Of paramount importance is that a drug with a suitable spectrum of activity must reach the site of infection in adequate concentrations to kill or inhibit the growth of the pathogen and make it susceptible to phagocytosis (Table 1). An important need in choosing an effective antimicrobial agent, therefore, is knowledge of which bacterial pathogens are involved in the infection. Ideally, identification is made by direct smear and culture of the organism(s). In practice, the choice of antibiotic often is based on knowledge of the probable pathogen most commonly associated with a certain site and clinical signs (Table 2). The lowest concentration of an antimicrobial drug that will inhibit the growth of a particular pathogen is known as that drug-pathogen combination's minimum inhibitory concentration (MIC). 29 Dose regimens that achieve adequate antimicrobial-drug concentrations invariably result in plasma-drug concentration in excess of the known or suspected MIC for the drug-pathogen pair. To ensure that an adequate antimicrobial-drug concentration is reached at the site of infection, the clinician must consider the dose to be used. The dose may be varied according to the known or suspected sensitivity of the pathogen to the chosen drug and with consideration of the ability of the chosen drug to reach the site of the infection. The dose regimen will vary from drug to drug, to ensure the most suitable drug-concentration profile is achieved for optimal efficacy according to known or assumed pharmacokinetic-efficacy correlates. 34 Age, organ system affected, concurrent diseases, and the environment at the site of infection will also influence antibiotic choice, dose, and regimen. For example, adjustment of antibiotic dose or interval may have to be made in patients with renal insufficiency because many antibiotics including 13-lactams, aminoglycosides, fluoroquinolones, and sulfonamides are eliminated by renal excretion (Table 3). If an aminoglycoside were indicated as the drug of last resort, the general recommendation is that the dose remain the same, but the time between doses is increased. 28 In contrast, under the same circumstances, for penicillins it may be more appropriate to decrease the total dose but maintain the same dose interval-2° Appropriate choice of antimicrobial drug and dose regimen often is not sufficient to ensure the success of therapy. Adjunctive treatments are often necessary. For instance, antimicrobial therapy of anaerobic infections such as pyometra, peritonitis, or bite wound abscess is more successful when combined with surgery. 9 Antibiotics can be administered parenterally, orally, rectally, vaginally, and topically. The correct route of administration is important in determining whether the antimicrobial is absorbed and whether it will reach adequate concentrations at the target site.
Table 1. SPECTRUM OF ACTIVITY OF SOME ANTIMICROBIAL DRUGS Antimicrobial Drug 13-Lactams Amoxicillin Amoxicillin + Clavulanate Cephalexin Penicillin G Aminoglycosides Amikacin Gentamicin Neomycin Tetracyclines Doxycycline Oxytetracycline Fluoroquinolones Enrofloxacin Orbifloxacin Macrolides and Lincosamides Clindamycin Erythromycin Lincomycin Tylosin Potentiated Sulfonamides Sulphadiazine + Trimethoprim Miscellaneous Metronidazole Key: 1-'
'-0 '-0
Gram-Positive Aerobes
Gram-Positive Anaerobes
Gram-Negative Aerobes
Gram-Negative Anaerobes
++ ++
++ ++
+ +
+ +
Very Some
+ ++
+ ++
+ +
+ +
Some Very
++ ++ ++
-
No No No
+ +
+ +
+ + + + +
. + +
+ +
++ ++
Others
(3-Lactamase Sensitive
+ +
No No
+ +
No No
+ + +
No No No No
++ ++ ++ ++
++ ++ ++ ++
-
+ + +
+
+
+
+
+
No
++
+
No
++
+ + = reliably useful; + = effective against some isolates; - = not clinically useful; (missing) = unknown.
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Table 2. BACTERIA THAT COMMONLY CAUSE INFECTIONS IN SPECIFIC ORGAN SYSTEMS IN DOGS AND CATS Organ System Canine Urinary tract
Upper respiratory tract
Lower respiratory tract External auditory canal
Common Pathogens Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae, Staphylococcus intermedius, Enterococcus faecalis, Pseudomonas aeruginosa, Leptospira spp. Staphylococcus intermedius, Eschericia coli, Pasteurella multocida, Klebsiella pneumoniae, Pseudomonas aeruginosa Bordetella bronchiseptica, Proteus mirabi/is, Straphylococcus intermedius Staphylococcus intermedius, Proteus mirabilis, Pseudomonas aeruginosa
Oral cavity
Obi igate anaerobes
Gastrointestinal tract
Enterobacteriaceae, Campylobacter spp., Clostridium perfringens, Bacteroides tragi/is, other anaerobes Staphylococcus intermedius, Eschericia coli, Proteus mirabilis, Pseudomonas aeruginosa Staphylococcus intermedius, Eschericia coli Eschericia coli, Staphylococcus intermedius, obligate anaerobes, other enterobacteria
Skin
Bone Septicemia
Feline Urinary tract
Upper respiratory tract Lower respiratory tract
Conjunctiva Oral cavity Skin
Eschericia coli, Proteus mirabi/is, Klebsiella pneumoniae, Pasteurella multocida, Pseudomonas aeruginosa Pasteurella multocida, Eschericia coli, Proteus mirabilis, Pseudomonas aeruginosa Pasteurella multocida, Eschericia coli, Klebsiella pneumoniae Chlamydia psittaci, 13-hemolytic· Streptococci, Pasteurella multocida Obligate anaerobes Pasteurella multocida, Eschericia coli, Proteus mirabilis
Rational Empirical Choices Amoxicillin + Clavulanate Potentiated sulfonamide Cephalexin Aminoglycoside Amoxicillin + Clavulanate Doxycycline Cephalexin Need culture and sensitivity Polymixin B Gentamicin Amoxicillin + Clavulanate Enrofloxacin Amoxicillin Clindamycin Metronidazole Need culture and sensitivity
Amoxicillin + Clavulanate Cephalexin Enrofloxacin Amoxicillin + Clavulanate Cephalexin Ampicillin or Cephalexin/ cefazolin + amikacin or gentamicin or enrofloxacin + metronidazole Amoxicillin + Clavulanate Cephalexin Enrofloxacin Amoxicillin + Clavulanate Cephalexin Gentamicin Amoxicillin + Clavulanate Cephalexin Enrofloxacin Gentamicin Doxycycline Chloramphenicol Amoxicillin Clindamycin Metronidazole Amoxicillin + Clavulanate Enrofloxacin Cephalexin
Data from Aucoin D: Target: The Antimicrobial Reference Guide to Effective Treatment. Port Huron, Ml, North American Compendiums Inc., 1993 and Ferguson DC: Rational empirical antimicrobial therapy in small animals. An International Symposium on Antimicrobial Selection, The North American Veterinary Conference, Orlando, FL, Veterinary Learning Systems Co., Inc., 1994, pp 4-11.
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Table 3. ORGAN PRIMARILY RESPONSIBLE FOR ELIMINATION Antimicrobial Drug
Renal
13-Lactams Aminoglycosides Tetracyclines Doxycycline Other tetracyclines Fluoroquinolones Macrolides and lincosamides Sulfonamides
++ ++
Hepatic Metabolism
++ ++ + ++
Biliary Excretion
Other
±
+ + ++
++ + +
+
MICROBIAL CHARACTERISTICS
Knowledge of the classifying characteristics of the infecting organism can be helpful in predicting how effective certain microbial agents will be in combating an infection. The competent clinician will know for each suspected pathogen its Gram-staining characteristic and whether it is an aerobe, facultative, or obligate anaerobe (Table 4). Gram Positive
Gram-positive bacteria have an outer wall that is comprised of peptidoglycans 50 to 100 molecules thick. 22 Between the peptidoglycan layers are strands of lipoteichoic acid that are anchored to the glycolipid molecules of the cell membrane (Fig. 1). 19 Drugs that disrupt the formation of peptidoglycans can easily reach Table 4. SOME COMMON BACTERIAL PATHOGENS AND THEIR CLASSIFICATION Gram-Positive Aerobes Streptococcus Enterococcus Staphylococcus*t
Gram-Positive Gram-Negative Gram-Negative Anaerobes Aerobes Anaerobes Clostridia
·························································
Borde tel/a Leptospira
Bacteroides* Fusobacterium
Pseudomonas*t Proteust Escherichia coltt
·"Eiiiei-ahaCieif ································ ·kieiisieiiaf ················ ····················· ·"Pasieiireiiat ··································· ·saimoiieilat ··································· ··cam"P"Yioiiac"ier ······························ Nocardia
Nocardia
*Constitutive 13-lactamase producers. tlnducible 13-lactamase production possible.
Others Chlamydia Mycobacterium Mycoplasma
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lipotecheic acid
A
bacterial inner cell membrane
porin channel lipoprotein bacterial outer membrane lipoprotein peptidoglycan
8
bacterial inner cell membrane
Figure 1. Cell wall structures of Gram-positive (A) and Gram-negative (B) bacteria.
their target in Gram-positive organisms, because the peptidoglycan cell wall is at the surface of the organism. 4 The antibiotics that are the most successful at targeting Gram-positive organisms act through this mechanism. Examples of these drugs are the 13-lactam s (penicillin, aminopenicillins, and cephalosporins), vancomycin, and bacitracin. Trimethoprim-sulfa, erythromycin, lincomycin, clindamycin, and chloramphenicol are also effective against many Gram-positive organisms, but these drugs act through mechanisms that do not affect cell wall synthesis. Gram Negative
In contrast to Gram-positive bacteria, those that are Gram-negative have an outer membrane that protects the thinner peptidoglycan layer: the peptidoglycan is merely one to two molecules thick and is linked to the outer membrane by an unusual lipoprotein.19• 22 Embedded in the outer membrane are proteins called porins, which are arranged in triplets to form diffusion pores. These porins allow small hydrophilic molecules,
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such as antibiotics, to easily pass through the outer membrane (see Fig. 1). 29 An exception is vancomycin, which is excluded by the porins and therefore is not effective against Gram-negative organisms. The pores are smaller in some Gram-negative bacteria such as Pseudomonas aeruginosa than in most other Gram-negative genera, thereby excluding many antimicrobial drugs. Therefore, Pseudomonas aeruginosa is more resistant to antimicrobial therapy. 16, 36 Because many antibiotics have difficulty in penetrating the lipid cell membranes, infections caused by Gram-negative bacteria are more challenging to treat. Antibiotics such as penicillin, the macrolides, and bacitracin are generally less potent against Gram-negative bacteria. Aminoglycosides are organic bases that are charged at physiologic pH and therefore would be predicted to have difficulty transiting across lipid membranes. They are able, however, to diffuse through the porin channels of the outer lipid membrane of Gram-negative bacteria and then are actively imported by oxygen-dependent transport proteins within the inner membrane. This allows their accumulation and access to the ribosomal site-of-action?' 25 The tetracyclines, chloramphenicol, polymyxins, fluoroquinolones, and some (3-lactams obtain access to and are effective against Gramnegative bacteria. 4 Anaerobes
Generally, facultative anaerobes are considered pathogenic, whereas strict anaerobes are not. Facultative anaerobes require reduced oxygen tension for growth and do not require molecular oxygen for metabolic activity. The pathogenic anaerobes are classified as Gram-positive and Gram-negative, but in addition to these characteristics, the anaerobes have other unique characteristics that vary their susceptibility to certain antimicrobials. Anaerobes are able to elaborate a variety of toxins and enzymes that can cause extensive tissue necrosis. Bacteroides fragilis, for example, can produce heparinase, deoxyribonuclease, neuraminidase, endotoxins, and 13-lactamases that can inactivate most 13-lactam antibiotics.9 Potentiated sulfonamides are inactivated by exudate and debris and therefore are not considered useful in treating anaerobic infections. The transport mechanism for import of aminoglycosides into cells is oxygendependent, so members of this family of antimicrobials are not active against anaerobes. 4 Chloramphenicol, clindamycin, and metronidazole provide the most reliable activity against pathogenic anaerobes. 9 CHOOSING A DOSE REGIMEN
The mechanism by which antibiotics kill or prevent the growth of bacteria is an important consideration when choosing a dose regimen.
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The mechanism of action is also a convenient way to classify antimicrobials. Knowing the mechanistic class of antimicrobial for any particular drug helps to remember the correct approach to dosing with that drug. Sites of action for several antimicrobial drugs are shown in Figure 2. Inhibition of Bacterial Cell Wall Synthesis Penicillins, Cepha/osporins, Vancomycin, and Bacitracin
13-lactams (penicillins and cephalosporins) bind to penicillin binding proteins (PBP) near the cell surface disrupting peptidoglycan synthesis in cell w all formation that results in cell lysis and deathY The bacterial cell must be growing and replicating for this class of drug to be effective. The binding to PBP is reversible and saturable.38 The bacterial action of the 13-lactams is time-dependent. For the penicillins and cephalosporins to be effective, their concentration in blood must be maintained above the minimum inhibitory concentrations (MIC) for long periods of time.20• 34 Most of these drugs need to be administered more than once daily, as they tend to have short elimination half-lives.15• 26 Excessive serum concentrations will saturate the PBPs and result in no additional benefit. If the 13-lactam concentration falls below the MIC, the bacteria will remain stable, but may show some structural abnormalities. With the exception of Staphylococci spp., 13-lactams do not cause a significant postantibiotic effect.35 A
B
Figure 2. Sites of action of various antimicrobial drugs: A. Cell wall-13-lactams, vancomycin, bacitracin; 8 , Cell membrane, tetracyclines; C, Cell membrane associated enzymes- aminoglycosides; 0 , DNA- metronidazole, sulfonamides, trimethoprim; E, DNA topoisomerase-fluoroquinolones; F, 30S Ribosomal subunit-aminoglycosides, tetracyclines; G, 50S Ribosomal subunit- macrolides, lincosamides, tetracyclines; H, DNA-dependent RNA polymerase-rifampin; J, RNA.
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These factors together suggest that for !3-lactam antibiotics, a dose regimen should be designed such that the drug concentration at the site of infection remains above the MIC for the pathogen throughout the dose interval. In addition, it can be deduced that exceeding the pathogen MIC by a great amount is likely to be of no additional benefit. These deductions have been supported by experimental evidence with in vivo models of bacterial infection. 10' 21 Similar information about dose regimen-efficacy correlations is unavailable for vancomycin and bacitracin, the latter of which is used only topically because of its toxicity. Disruption of Cell Membranes
Polymyxin B, Colistin (Polymixin E), Nystatin, and Amphotericin 8
These drugs insert into cell membranes either as detergents (Polymyxin B, colistin) or by binding to ergosterol (amphotericin B) and thereby disrupt the integrity of the phospholipid membrane. By destroying cell membranes, they allow cytoplasm leakage, leading to cell death. These drugs can act against quiescent cells; they are not dependent on active cell growth. Therefore they can be combined with bacteriostatic antibiotics. Because they require access to the cell membrane, the polymyxins are most effective against Gram-negative organisms. Because ergosterol is a component of fungal cell membranes, nystatin and amphotericin are antifungal drugs. Mammalian cell membranes possess similar components to those of both bacteria and fungi. Therefore, these drugs are not very selectively toxic and also damage host cell membranes. Although the efficacy of these drugs is likely to be concentration dependent, so too is the toxicity. Therefore the clinician has little room to vary from recommended dose regimens. 2, 30 Inhibitors of Protein Synthesis
Aminoglycosides, Tetracyclines, Macro/ides, Lincosamides, and the Chloramphenicol Family
After import into the bacterial cell, the aminoglycosides (e.g., streptomycin, gentamicin, amikacin, tobramycin, kanamicin) bind irreversibly to the 30s ribosomes altering protein synthesis. 32 These drugs also affect cell membrane structural integrity. 6 Together these actions result in bacterial cell death. Because these drugs are actively accumulated by aerobic bacteria and the rib9somal binding is irreversible/1 aminoglycosides continue to inhibit bacterial protein synthesis after the drug is no longer detectable in the bacterial milieu. 35 This effect is known as the postantibiotic effect (PAE). Possibly partly because of this effect, once daily dosing is efficacious despite rapid clearance from the host by renal filtration. 8
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Once daily aminoglycoside therapy is effective so long as the peak drug concentration in the plasma is at least four times the MIC for the pathogen (inhibitory quotient :::::: 4) even though the drug concentration falls below the MIC for long periods. Although it has been postulated that the PAE is partly dependent on the patient's immune system, once daily therapy is efficacious in immunodeficient children. 14 The PAE may also prevent proper cell wall and septum formation or cause the bacteria to be transformed to a less virulent strain. It may be necessary to dose patients with aminoglycosides more frequently than once daily when their infection continues to be seeded from a body cavity or foreign body to which the antibiotic fails to gain access. 37 In contrast to aminoglycosides and their bactericidal action, chloramphenicol, tetracyclines, macrolides, lincosamides, and clindamycin bind reversibly to ribosomes and, under most circumstances in vivo, appear to achieve only bacteriostatic action. 30 There are no good clinical trial data available in which dose regimen-efficacy relationships are described for these drugs. Conventional wisdom suggests that the efficacy of these drugs is likely to relate to the length of time at which the pathogen is exposed to supra-MIC drug concentration. Dose regimens should be designed to ensure rapid attainment of appropriate concentration of drug at the site of the pathogen, perhaps initially using loading doses or parenteral formulations. Initiation of therapy should then be followed by a dose regimen that ensures continued supra-MIC drug concentration at the site of action. Since the pharmacokinetics of these drugs vary greatly from drug to drug and between species, clinicians should carefully follow dose regimens recommended in formularies or on the package insert. Interruption of Nuclei Acid Metabolism Fluoroquinolones, Sulfonamides, Diaminopyrimidines, Metronidazole
The fluoroquinolones (e.g., enrofloxacin, orbifloxacin) inhibit DNA topoisomerase, thereby inhibiting bacterial replication causing cell death. 5 Metronidazole may have a similar action in some bacteria.24 The intensity of exposure to a drug has been quantified as the ratio of the area under the plasma-concentration versus time curve to the MIC (AUC/MIC). For the fluoroquinolones, the intensity of exposure is more closely linked with efficacy than the time above the MIC. Higher once daily dosing has been shown to be more effective than the same daily dose divided into four daily injections, because higher doses given less frequently result in a greater AUC. Higher doses also cause higher peak plasma concentrations. The peak concentration achieved may also be closely related to efficacy but certainly has been related to decreasing the development of resistant strains. 5 For these reasons, the labeled dose regimen for enrofloxacin (in the United States) has recently been changed
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from a dose interval of 12 hours to one of 24 hours. Furthermore, the upper limit of the labeled total daily dose has also been increased, since this is likely to increase efficacy, decrease development of resistance, and can be done without inducing toxicity. Orbifloxacin, another recently introduced fluoroquinolone, should also be administered once daily. Folate is required for DNA synthesis. In bacteria, folate may be either synthesized from para-amino benzoic acid (PABA) or imported directly into the cell as a nutrient (e.g., from pus). Sulfonamides contain a structural analog of PABA that competes with PABA and thereby interferes with bacterial DNA synthesis if insufficient folate is available for uptake, having a bacteriostatic effect. Folate is sequentially reduced to dihydrofolate and tetrahydrofolate in the pathway for thymidine synthesis. These reactions are dependent on enzymes. Diaminopyrimidines such as trimethoprim compete for and inhibit the enzyme dihydrofolate reductase, thereby also inhibiting pyrimidine synthesis. This effect is also bacteriostatic. However, the combination of sulfonamide and trimethoprim is synergistic and bactericidal.23 When sulfonamides are used alone, their bacteriostatic effect is dependent on the maintenance of supra MIC plasma drug concentrations. More frequent dose intervals are better at achieving bacterial elimination. Both the fluoroquinolones and trimethoprim exhibit a PAE. Their effects persist after their plasma concentrations fall below the MICa few hours following dosing, probably because tissue concentrations remain higher at the site of infection. The PAE may be important to these drug's efficacy when administered only once or twice daily. 5' 35 RESISTANCE
Under normal conditions the frequency of mutations in a bacterial population is 1 organism in 1,000,000. The frequency of mutation is influenced by antimicrobial drugs and is thought to be directly correlated to the ratio of the MIC to the antibiotic concentration at the site of infection. This emphasizes the importance of adequate dosing. In treating an infection, the time above the MIC is also an important factor in minimizing development of resistance. Generally, the more chronic the infection and less accessible the site of infection is to the drug, then higher doses and longer treatment times are required to prevent resistance.16' 36 For example, in dogs with experimentally induced peritonitis, standard parenteral doses of cephalothin and aminoglycosides (gentamicin, tobramycin, amikacin, and kanamycin) did not reach the MIC in the ascitic fluid. It was the conclusion of the authors that higher than usual parenteral doses may have to be used in treating peritonitisY Underdosing antibiotics selects for the resistant bacteria by causing the eradication of susceptible population of organisms and overgrowth of resistant subpopulations that are present in small numbers. Resistant subpopulations may also be selected when antimicrobial agents are used
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at placement of intravenous or urinary catheters. This practice can result in a high number of nosocomial infections. Using aseptic techniques when placing a catheter is more effective than prophylactic use of antibiotics. In surgery, prophylactic use of antibiotics is indicated for clear contaminated wounds encountered in gastrointestinal, respiratory, and urogenital therapy.U Animals previously exposed to antimicrobials are more likely to have antimicrobial resistant Staphylococcus aureus. These bacteria were demonstrated to be resistant most frequently to penicillin, then streptomycin followed by tetracycline. 3 Knowing the antimicrobial's family may help to understand the development and ramifications of resistance by bacteria to that drug. Resistance of Staphylococcus spp. to penicillin is most often plasmidmediated. Plasmids are extrachromosomal genetic elements that exist and function independently from chromosomes. Plasmids that confer antimicrobial resistance are known as R-plasmids. These DNA fragments can induce resistance to antimicrobial drugs in a number of ways, including coding for 13-lactamase. Expression of R-plasmids of Grampositive organisms can be induced by exposure to 13-lactam antibiotics. These bacteria then produce 13-lactamase and secrete it extracellularly in large amounts, inactivating some types of 13-lactam antibiotics. 16 Plasmid mediated 13-lactamases are also responsible for the majority of Gram-negative resistance to 13-lactam antibiotics. In contrast to Grampositive bacterial 13-lactamase, Gram-negative bacterial 13-lactamases are strategically confined within the periplasmic space of Gram-negative bacteria (i.e., at the site of action of 13-lactam antibiotics). Gram-negative bacteria also produce a greater variety of lactamases than do Grampositive bacteria. The plasmid mediated 13-lactamase of the Gram-negative bacteria Eschericia coli, Enterobacter spp., Proteus spp., Shigella spp., Pseudomonas aeruginosa, Hemophilus spp. and Klebsiella spp. are effective in destroying both penicillins and cephalosporins. Chromosomes can also confer 13-lactamase resistance to penicillin for Proteus mirabilis and morganii or to cephalosporins for P. aeruginosa, Bacteroides fragilis, Proteus, Escherichia coli, and Enterobacter spp. Chromosomally mediated 13-lactamases of Klebsiella spp. are effective against both penicillins and cephalosporins.4, 16 Therefore, when a clinician suspects an infection is caused by a Gram-positive 13-lactamase producer, such as may be the case when the bacteria is reported resistant to amoxicillin but sensitive to amoxicillinclavulanate, then a cephalosporin may still be a good choice. However, if the 13-lactamase producer is Gram-negative, then it would be better to choose a drug that is a member of an alternate mechanistic class. Both plasmid and chromosomal DNA can code other changes that cause resistance against antimicrobial drugs. Conformational changes to or lack of the ribosomal binding sites may induce resistance to chloramphenicol or streptomycin. In the case· of chloramphenicol, this bacterial resistance is chromosomally conferred as a lack of the 30s subunit binding site. Staphylococcus aureus biochemically modifies eryth-
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romycin, which reduces the affinity of erythromycin for its binding site on the 50s ribosomal subunit. Resistance to erythromycin via this mechanism also confers resistance to other macrolides and lincosamides.36 Resistance to trimethoprim is also plasmid mediated. This plasmid encodes for the production of dihydrofolate reductase, which has little or no affinity for trimethoprim. 12 In contrast to the 13-lactams, macrolides, lincosamides, and trimethoprim, fluoroquinolones do not appear to be as readily affected by transferable plasmid mediated resistance. R-plasmids, which confer resistance to other antibiotics, may be eliminated from the harboring bacteria as a response to exposure to fluoroquinolones. Resistance to fluoroquinolones occurs by genomic mutation, which is an infrequent occurrence. These mutations primarily alter bacterial cell walls decreasing drug penetration. Mutational change also occurs to the structure of the target DNA gyrase. Some of these mutations confer resistance also to cephalosporins, tetracyclines, and chloramphenicol. Other mutations that cause fluoroquinolone resistance can cause hypersusceptibility to 13-lactams, aminoglycosides, and novobiocin. 5• 33 COMBINATION THERAPY Synergism
Synergism between two antimicrobial drugs is an in vitro phenomenon. Two drugs are synergistic for a particular bacterial isolate if the sum of the individual in vitro fractional inhibitory concentrations (FIC) is less than one. The two drugs may also be synergistic if the rate of in vitro bactericidal activity is increased 100-fold for a particular bacterial isolate when the bacterium is exposed to the combination. Finally, synergy is also declared if an increase in the rate of clinical cure from defined infections is demonstrated after combination of two antimicrobial drugs. Antimicrobial drug combinations may also be indifferent or antagonistic.17, 29 Synergy is a real phenomenon that occassionally aids in clinical resolution of disease. However, without laboratory documentation of potential synergy, combination therapy is never indicated simply in the hope of induced synergy. Antimicrobial drugs have been divided into two groups, based on their mechanism of action, to aid prediction of the in vitro effect of combinations/ 8 and this scheme has been more recently revised. 27 Group 1 drugs have bactericidal action at clinically achieved concentrations (e.g., penicillins, cephalosporins, aminoglycosides, vancomycin, and perhaps the fluoroquinolones). Group 2 drugs are merely bacteriostatic at clinically achieved concentrations (e.g., tetracyclines, chloramphenicol, macrolides, and lincosamides). Unfortunately, it has become a generally accepted dogma that group 1 drugs when combined may provide "synergistic" activity and group 2 drugs when combined may cause "indifference," but combination of a group 1 drug with a
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group 2 drug will be likely to cause "antagonism." This dogma is incorrect. Clinicians should not use this scheme to predict synergy, indifference, or antagonism in vivo. The scheme is useful only as a guide to the in vitro investigation of drug combinations. 18 There is no rational potential for the direct clinical application of this schemeY
Increasing Antimicrobial Spectrum
Combination therapy may be rational when used to increase the breadth of antibacterial spectrum. This is especially appropriate for cases with acute, life-threatening bacterial infections of unknown or as-yet unidentified etiology. Occasionally, combination therapy also may be used rationally to decrease the rate or probability of resistance developing to the effective antimicrobial drug. Generally, bacteria can be divided into four major groups: Grampositive aerobes, Gram-positive anaerobes, Gram-negative aerobes, and Gram-negative anaerobes. If these four groups are displayed in a classical 2 X 2 chart, each group is represented by one quadrant. Accordingly, desirable antimicrobial drug combinations are frequently described as achieving "four-quadrant therapy." Typical combinations for four-quadrant therapy include (1) a 13-lactam and an aminoglycoside or a fluoroquinolone and (2) clindamycin and an aminopenicillin or a fluoroquinolone. With the first of these two options, it may be necessary to include metronidazole if Bacteroides fragilis is likely to be one of the pathogens.
TOXICITY
Antibiotic therapy can be of benefit, but it is also important to consider the negative side effects. The consequences of the potential toxicity may preclude the use of a certain drug, especially if it may worsen a pre-existing condition. Antimicrobial toxicities include kidney, nervous system, gastrointestinal, bone marrow, cartilage/joint, eye, and liver. Knowing the antimicrobial's family also helps us to remember and understand the likely manifestations of toxicity (Table 5).
CONCLUSION
It has been 2500 years since the Chinese began applying moldy soybean curd to cure skin infections. Technology today has refined somewhat the benefits of antibiotic forming molds and bacteria and has greatly increased the number of antimicrobial drugs available to combat infection. Understanding the principles fundamental to rational therapy with these drugs will ensure the best of possible outcomes.
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Table 5. MANIFESTATIONS OF ADVERSE REACTION AND TOXICITY TO SOME ANTIMICROBIAL DRUG FAMILIES Antimicrobial Drug
Aminoglycosides
13-Lactams Tetracyclines
Fluoroquinolones Erythromycin Metronidazole
Lincomycin Sulfonamides
Adverse Reactions (Not Dose Related)
Toxicity (Dose Related)
Nephropathy CN VIII damage Neuromuscular blockade Acute hypersensitivity Ulcerative colitis Acute hypersensitivity Gastroenteritis Toxic epidermal necrolysis Hepatopathy Photosensitization Nephropathy Cartilage damage Altered CNS signaling Hepatopathy Abdominal pain CNS intoxication
Enterocolitis Acute hypersensitivity Nephropathy Toxic epidermal necrolysis Hepatopathy Keratoconjunctivitis sicca
Clinco-Pathological Sign
Increased serum creatinine, urinary casts, ataxia, loss of righting reflexes, deafness, flaccid paralysis Diarrhea, vomiting, febrile Diarrhea Hepatic enzyme elevation Dermatitis, erythema Increased serum creatinine Lameness Convulsions Diarrhea, vomiting Anorexia, vomiting Depression Nystagmus, ataxia Tremors, seizures Diarrhea, vomiting Febrile Diarrhea, vomiting, depression, oliguria, increased serum creatinine, urinary casts, crystalluria, blepharospasm, corneal pigmentation, depressed Schirmer tear test
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Address reprint requests to Ted Whittem, BVSc, PhD Assistant Professor, Veterinary Clinical Pharmacology Department of Veterinary Biosciences College of Veterinary Medicine University of Illinois 1008 W. Hazelwood Drive Urbana, IL 61802