Inoculum Size

The larger the bacterial inoculum at the target site, the greater the concentration (number of molecules) of antimicrobial necessary to kill the organism. Further, more CFUs are more likely to produce greater amounts of enzymes or other materials that can destroy the drug. The “inoculum effect” of ESBL resistance describes the increasing MIC of the organisms toward cephalosporins at a larger (10⁷) compared with smaller (10⁵) inoculum.⁹² In addition, the larger the inoculum, the greater the risk that spontaneous mutation will contribute to resistance or virulence. Note that resistance and virulence do not necessarily co-exist. In general, emerging resistance appears to be associated with decreased rather than increased virulence, although increasingly studies are identifying exceptions. For example, community-acquired infections may be associated with increased virulence, but less resistance. For example, although hospital-acquired infections tend to be caused by nonvirulent organisms, community-acquired infections reflect virulent organisms that can infect even the overtly healthy patient. Concern regarding MRSA reflects, in part, its apparent acquisition of virulence factors that have facilitated its transition from a hospital to community-acquired infection.

Virulence Factors

The degree of pathogenicity of bacteria (virulence) will affect antimicrobial efficacy indirectly by facilitating infection. The ability of microbes to cause disease reflects the size of the inocululm, the effectiveness of host defense mechanisms, and the intrinsic pathogenicity of the microbes resulting from the presence of virulence factors. Like biochemical mechanisms of resistance, virulence factors generally involve proteins encoded by DNA of chromosomal or shared (e.g., plasmids, transduction) origin. Contributing to the negative impact of virulence factors is host response to their effects. Virulence factors facilitate adhesion to host cell surfaces, colonization (e.g., urease of Helicobacter pylori, which protects it from gastric acidity), invasion (facilitated by disruption of host cell membranes or stimulation of endocytosis), immunosuppression (e.g., antibody-binding proteins), or bacterial toxins that cause local, distant, or both (e.g., endotoxin) host damage. Pathogen attachment to host cells is a crucial early step in mucosal infections and is facilitated in epithelial tissues by bacterial adherence. Adherence is a specific two-phase process involving bacterial virulence factors called adhesins and complementary receptors of the host epithelial cells.^93,94 Adhesins are generally found on the surface of microbes, (e.g., bacterial fimbriae) and along with other virulence factors facilitating infection, may be targets for alternative (to antimicrobial)therapy. Species differences exist among the types of receptors in the host epithelial cells. The predominant receptor type in humans is glycolipid in nature, and its presence varies with blood cell types, implying individual variation in susceptibility to bacterial adherence in several body systems. Bacterial adherence is discussed with regard to specific body systems in Chapter 8.

Another virulence factors that facilitate infection are invasins. Invasins are enzymes that damage physical barriers presented by tissue matrices or cell membranes, facilitating rapid bacterial spread. Examples include clostridial hyaluronidase, which is able to destroy connective tissue, and lecithinases and phospholipases of clostridial and gram-positive organisms. Bacteria have developed siderophores, which are specialized virulence factors that mediate the release or scavenging of iron critical for microbial virulence. Bacteria also have developed specialized transport systems that secrete toxic materials into the extracellular matrix. It is not clear whether the efflux proteins that transport toxins are related to those that transport drugs (see the discussion of resistance). Bacteria also facilitate invasion through materials (e.g., proteins, “slime”) that prevent phagocytosis or, if the microbe is phagocytized, preclude intracellular killing. Examples include lytic enzymes of gram-positive cocci or exotoxin A produced by P. aeruginosa. Toxins include both endotoxins (discussed in depth later) and exotoxins. Bacterial exotoxins are among the most potent toxins known, acting on either the cell surface (e.g., E. coli hemolysins, “superantigens” of S. aureus or Streptococcus pyogenes); membrane; or, once the membrane is penetrated, intracellular targets (e.g., A/B toxins).

Biofilm

Among the most effective and probably least appreciated protective microbial factors is biofilm. Bacteria exist in either a planktonic (free floating) or sessile (attached) state; while it is the former state that characterizes C&S testing, but it is the latter state that enables persistence of the resident population, as well as the formation of biofilm.^95-97 Biofilm is defined as a biopolymer, matrix-enclosed bacterial population in which bacteria adhere either to one another or to a surface.⁹⁵ The outer layer of the biofilm may lose water such that it is hardened, thus providing better protection from the environment, including exposure to antimicrobials. The inner sactum of the biofilm is largely aqueous, composed of glycocalyx or slime (e.g., Staphylococcus spp.). In addition to passive diffusion, aqueous pores permeate the structure, allowing movement of nutrients and metabolic debris. Biofilm populations containing normal microflora in the skin or mucous membranes (e.g., urinary bladder) are lost with shedding of the skin (or bladder) surface or by the excretion of mucus; new cells and mucus are rapidly colonized by biofilm-forming bacteria. Microbes released from the surface may colonize new surfaces and subsequently produce new biofilms and new (e.g., persistent or recurring) infections. Bacterial communication during biofilm formation is sophisticated, involving quorum-sensing systems that ultimately may be targets of microbial therapy.⁹⁶ Biofilm may facilitate and protect growth of normal or pathogenic flora on foreign surfaces and can facilitate subsequent translocation of microbes to otherwise sterile tissues. Persistent, chronic bacterial infections may reflect biofilm-producing bacteria; persistent inflammation associated with immune complexes contributes to clinical signs. Dental plaque is a prototypic example of the impact that biofilm might have on preventing antimicrobial penetration. Cystic fibrosis associated with Pseudomonas is a disease in which biofilm contributes to mortality. Pathogens associated with biofilm in veterinary medicine include, but are by no means limited to, Acinetobacter, Actinobacillus, Klebsiella, P. aeruginosa, and Staphylococcus (aureus and pseudintermedius).⁹⁵ Glycocalyx may contribute to protective mechanisms of other organisms as well (e.g., sulfur granules and Nocardia; Figure 6-14). Not all pathogens associated with biofilm cause infection (e.g., urinary catheters). However, because they ultimately may be the source of infection, clinical resolution may not be possible until the biofilm is destroyed. Yet, its nature is difficult to predict based on the planktonic growth of individuals in cultures compared to the consortium that occurs in vivo.⁹⁷ Catheters (urinary or intravascular), orthopedic fixation devices, and materials used in wound management are examples of surfaces on which biofilm might develop.

Figure 6-14 An example of combined host and microbial factors that negatively impacts therapy, Nocardia causes a marked inflammatory response by the host. Additionally, the organism causes secretion of calcium that combines with its biofilm, resulting in the formation of “sulfur” granules that protect the organisms from drug penetrations.

Antimicrobial Resistance

The role of resistance in therapeutic failure of antimicrobials is well established.^23,98 The use of antimicrobials increasingly is associated with emergence of resistance. For each class of antimicrobial drugs approved for use in human medicine, resistance generally has emerged within 1 to 2 decades of use. Clinically relevant resistance toward sulfonamides, the first class of antimicrobials approved in the United States (1930s) was documented by the 1940s. Penicillins, tetracyclines, streptomycin (aminoglycoside), and erythromycin (macrolides) were all approved within a 10-year span, with resistance documented within 5 years for methicillin versus approximately 10 years for streptomycin. Resistance to nalidixic acid, the progenitor of fluoroquinolones (approved in 1950), took 3 decades to emerge, perhaps convincing manufacturers that resistance to fluoroquinolones would emerge very slowly. However, resistance to norfloxacin, the first fluoroquinolone approved in the United States, took less than 3 years to emerge, despite the fact that the lack of plasmid-mediated resistance was among the attributes of this class. Resistance to extended-spectrum cephalosporins emerged within 4 years of approval and to amoxicillin–clavulanic acid, within 5 years. Resistance to vancomycin, specifically developed to treat MRSA, emerged in its second decade of use.

Factors Contributing to the Emergence of Resistance

Development of antimicrobial resistance is facilitated by several factors¹¹¹; among the most important is exposure to antimicrobials. In the individual patient, single-dose ciprofloxacin prophylaxis increased the prevalence of ciprofloxacin-resistant fecal E. coli from 3% to 12% in humans.¹¹² Ciprofloxacin treatment for prostatitis resulted in posttreatment fecal colonization with quinolone-resistant E. coli that was genetically distinct from the infection-causing strains after treatment in 50% of the patients.¹¹³ Our laboratory has demonstrated that standard doses of either amoxicillin or enrofloxacin given orally will cause close to 100% of fecal E. coli to become resistant to the treatment drug within 3 to 9 days of therapy; for enrofloxacin the isolates generally are multidrug resistant. As with MRSA or MRSI (S. intermedius), the advent of resistance by E. coli and other gram-negative organisms has been associated with increased cephalosporin use.¹

The gastrointestinal flora offers a natural environment that exemplifies the impact of antimicrobials on selection pressure. The normal flora of the gastrointestinal tract is extremely diverse, with anaerobes predominating. Among the aerobes, E. coli are the major gram-negative and Enterococcus the major gram-positive organisms.¹⁰¹ Environmental microbes maintain an ecologic niche through suppression of the competition by either consumption of nutrients or secretion of antibiotics. Therefore commensal organisms are constantly being exposed to antibiotics, and are “primed” to develop resistance.¹⁰¹ However, the microbes producing the antibiotic, as well as surrounding normal flora, are resistant to the antibiotic. Thus genes for resistance develop along with genes directing antibiotic production.

Rapid microbial turnover in the gastrointestinal tract supports the development of resistance by ensuring active DNA replication and thus mutation potential (see previous discussion). Chromosomal (DNA) mutations (10^-14 to 10^-10 per cell division) are DNA mistakes that have been missed by bacterial repair mechanisms. These mistakes occur spontaneously and randomly, regardless of whether the antibiotic is present. If the mutation that confers resistance to an antimicrobial occurs in the presence of the antimicrobial when it is administered to the patient, the surviving mutant, reflecting its single-step mutation, confers a low level of resistance (see the discussion of mutant prevention concentration). The MIC of the organism is likely to increase. Further microbial turnover and continued therapy can lead to multistep mutations and rapid emergence of high-level resistance characterized by increasingly higher MIC. Stepwise mutations can lead to specific resistance such as that demonstrated toward fluorinated quinolones (stepwise mutation in the DNA gyrase gene). Nonspecific mechanisms of resistance, including that shared among organisms, are more likely to result in MDR. Microflora of the gastrointestinal tract can serve as a reservoir of resistance genes; a single drug, via integrons, plasmids, and transposons, facilitates the rapid transfer of MDR among organisms. The gastrointestinal environment exemplifies a pattern whereby resistance can emerge as a result of a combination of selection pressure and mutation. Clinically, similar mechanisms of emerging resistance are likely to occur at sites of infection.

Mutant Prevention Concentration

Drlica and coworkers¹¹⁴ have hypothesized the mutant selection window, (see Figure 6-15) comprised of a lower threshold represented by the culture MIC of the infecting organism and an upper threshold or boundary, the MPC. Should a dose be designed such that drug concentrations fall within this window (i.e., between the MIC and MPC) at the site of infection, the mutant isolate is likely to emerge as a resistant colony. The practical application of the hypothesis explains the observed behavior of mycobacterium organisms toward fluoroquinolones (FQs). Increasing concentrations of the FQs inhibits the nonresistant (wild-type) organisms and colony numbers rapidly decrease. But this period of decline is followed by a plateau period of minimal or no growth. During this plateau phase, remaining resistant isolates recover and start to multiply again. The resistance of this emerging, second population presumably reflect a single-step (chromosomal or plasmid-mediated) mutation that resulted in an increase in the MIC to low-level resistance (e.g., MIC is close to the breakpoint). However, when these first-step mutants are exposed to even higher drug concentrations, a second rapid decline in numbers occurs, this time reflecting inhibition of the mutated, resistant organisms. Again, once sufficient bacteria recover, a second plateau occurs as the first-step mutants mutate. This stepwise or multistep mutation confers high level resistance (MIC exceeds the breakpoint several fold) that can be overcome only by very high concentrations of the FQ. The mutant selection window, which is to be avoided with initial therapy, describes drug concentrations on either side of the initial plateau for the single-step mutants. The lower boundary is defined by those drug concentrations sufficiently high to remove the majority of the wild-type competitors (MIC), whereas the higher boundary (the MPC) is defined by the concentrations necessary to inhibit the least susceptible (most resistant) isolates (the single-step mutants).¹¹⁵ Above this concentration, a second mutation step (which is very rare) would be required for a population of resistant organisms to develop; the risk of this happening is reduced by preventing microbial turnover (i.e., killing all isolates).

Figure 6-15 Stepwise mutation can emerge as a result of selection pressure induced by antimicrobial therapy that targets the minimum inhibitory concentration (MIC) of the infecting microbe. The mutant prevention concentration (MPC) is the concentration of drug that is necessary to inhibit first-step mutants, or the MIC of the least susceptible isolate in a resident population of pathogens. As the resident population or inoculum of wild (nonresistant) pathogen isolates reaches 10^8-10 colony-forming units (CFUs), some isolates will spontaneously mutate such that resistance emerges to the drug of interest. However, when cultured, the MIC reported for the population is likely to represent the mode (the most commonly reported MIC), which in a normally distributed population, is also the MIC₅₀ for the population. In contrast, the MIC of the first-step mutant will be the high end of the population MIC range. This is the concentration that should be targeted to inhibit the entire population—that is, the MPC. If the dosing regimen is designed to target the the mutant selection window, that is, the MIC of the wild population rather than the MPC (the MIC of the first-step mutant)—treatment with the drug will inhibit all isolates at or below the MIC. The void in isolates will allow the remaining, more resistant first-step mutants to recover, particularly in patients not sufficiently healthy to suppress recovering microbes. As this new population expands, a second distribution curve emerges. If recultured, the MIC of the second first-step mutant population will be higher than the wild population. If the population reaches a sufficient size (e.g., 10⁸ CFUs), a second, spontaneous mutation is likely to occur, resulting in a new, higher MPC. Targeting the MPC is particularly important when using drugs for which resistance emerges in response to mutations.

On the basis of this observation, Drlica and coworkers contend that MIC-based strategies used to design dosing regimens readily select for resistant mutants.¹¹⁵ Their contention is based on the observation that only one resistant mutation is needed for bacteria to grow in the presence of an antimicrobial and that infections generally contain an adequate number of CFUs for several first-step resistant mutants to be present prior to treatment. They coined the term MPC as an in vitro measure of preferred antimicrobial concentration target. If the MPC (rather than the MIC) is achieved at the site of infection, the risk of resistance is minimized because isolates that exceed the MPC concentration must have undergone a second concurrent resistance mutation step prior to therapy. As such, the MPC, not the MIC, would be the concentration targeted at the site of infection in the patient. Indeed, simply achieving the reported MIC of the infecting microbe at the site of infection is probably the approach that is most likely to yield clinically resistant organisms. Accordingly, consideration should be given to assuring that “dead bugs don’t mutate.” If the least susceptible of the isolates is inhibited with the dosing regimen, then the recovering population should not be resistant.

Drlica¹¹⁵ has demonstrated that MPCs do not correlate to MICs. In vitro, the MPC would be defined in vitro as the (lowest) drug concentration (in the media) that yields no recovered organisms when over 10¹⁰ CFUs (mimicking bacterial load in the patient) are plated. Currently, determining the MPC is costly, requiring multiple testing steps and large numbers of cells; for example, standard culture procedures are based on 10⁶ CFUs, whereas determination of the MPC requires at least 10^8-10 CFUs. However, an MPC-based strategy to dosing clinically makes sense and should be an effective means of blocking the growth of first-step resistant mutants. Such a strategy would force wild-type cells to acquire two resistance mutations for growth, an event that is rare. Experimental in vitro data¹¹⁴ have confirmed that MPC levels of an FQ do indeed inhibit strains that harbor first-step gyrA mutations (the mechanism of microbial resistance to FQ).¹¹⁶ Application of the MPC is most appropriate for drugs and organisms that develop resistance by chromosomally mediated point mutations (e.g., the fluorquinolones).¹¹⁷ However, the spirit of targeting the MPC might be assumed even for other drugs, in order to minimize the impact of selection pressure on emergent resistant populations. The mutant selection window can be narrowed if more than two bacterial sites are targeted, such as might occur with combination antimicrobial therapy, or with drugs that simultaneously target more than one site (e.g., the FQs).¹¹⁴

Biochemical Mechanisms of Resistance

Bacteria often respond to the presence of the antimicrobial by altering their physiology such that resistance occurs, often to multiple drugs. Microbes develop antimicrobial resistance by two primary mechanisms: modification of the target site or altered intracellular drug concentration. Methods by which intracellular drug concentration can be decreased include changes in porin sizes for gram-negative organisms (e.g., most drugs; see Figure 6-3). Porins are transmembrane proteins (e.g., OmpF) that form an aqueous channel that allows passive movement of large hydrophilic molecules. Porins are one of the few means by which drugs can gain access to intracellular targets. A change in porin size (i.e., by the addition of side chains that filter out drugs) or number increases antimicrobial resistance, as is demonstrated by the loss of the OprD protein that imparts resistance to imepenem by Pseudomonas spp. Closely associated with the porin proteins are efflux proteins that pump drug out of the organism; the pumps often are associated with porin proteins (e.g., FQs and tetracyclines). Most of these pumps are fueled by energy associated with proton exchange, the most notable in gram negative organisms being of the RND (resistance nodulation division) family. The best characterized in this family is the Acr-AB/TolC system, which is a complex bacterial stress response system that allows bacteria to pump out toxic molecules.¹⁰¹ These and other pump systems are often characterized by a wide range of substrate specificities and, along with porins, are a common mechanism whereby an isolate can express multidrug resistance. In contrast, a number of microbes generate enzymes that destroy antimicrobials (e.g., aminoglycoside acetylases, beta-lactamases that destroy penicillins or cephalosporins, transferases that destroy chloramphenicol); in such instances resistance conferred by these mechanisms is generally limited to a single drug or drug class. Enzymatic inactivation is more likely for natural drugs to which microbes have previously been exposed (and thus presented with a greater opportunity to develop enzymes). In contrast, enzymes are less likely to destroy synthetic drugs.¹¹⁸ However, plasmid-mediated enzymatic destruction of FQs has recently been described, once again highlighting the resourcefulness of bacteria.¹¹⁹ Changes in target structure are another major mechanism of resistance. Example targets that have been modified include, but are not limited to, cell wall proteins (e.g., penicillin-binding proteins [PBs], particularly for MRSA [PB2] or Enterococcus [PB5]), or binding sites (i.e., on ribosomes, as for aminoglycosides, or DNA gyrase for FQs).^120,121 Organisms often are characterized by more than one mechanism of resistance. Multiple mechanisms are well documented for some organisms against selected beta-lactams and have been described against FQs (e.g., altered DNA gyrase and increased efflux pumps) and others. Resistance can be induced, as is exemplified by beta-lactamase formation in Staphylococcus spp. which greatly increases in the presence of a beta-lactam antibiotic, or for fluoroquinolone, for which efflux pump activity is markedly upregulated. Discussion of specific mechanisms of resistance will be addressed with the appropriate drugs (see Chapter 7).

Avoiding Antimicrobial Resistance

Among the approaches to reducing resistance are pharmacologic manipulations and changes in antimicrobial use practices. Pharmaceutical manufacturers have been able to manipulate antimicrobial drugs in a variety of ways such that resistance is minimized, and these options can be selected in an attempt to minimize resistance. For example, bacterial resistance has been decreased by synthesizing smaller molecules that can penetrate smaller porins (e.g., the extended-spectrum penicillins ticarcillin and piperacillin); synthesizing larger molecules that force the microbe to develop more than one point mutation (e.g., later-generation FQs), “protecting” the antimicrobial from enzymatic destruction (e.g., with clavulanic acid, which diverts the beta-lactamase from the penicillin); modifying the compound so that it is more difficult to destroy (e.g., amikacin, which is a larger and more difficult to reach molecule than gentamicin and carbapenems, later generation cephalosporins); and developing lipid-soluble compounds that are more able to achieve effective concentrations at the site of infection (e.g., doxycycline compared with other tetracyclines). Increasingly, drug design–based tactics will be implemented to minimize emergent resistance. Increasingly the role of the practitioner is equally important. A three “D”s approach might reduce the risk of emergent resistance: De-escalate antimicrobial use, design a treatment regimen that minimizes resistance (dead bugs don’t mutate), and decontaminate the environment through proper hygiene. These approaches are exemplified by strategies implemented by intensive care units to reduce antimicrobial resistance that often involve a multitiered approach (Box 6-3). Actions include the following:

Risk factors for emerging resistance in the hospital or community setting include but are not limited to increased antimicrobial use, host factors such as severity of illness and length of stay, and lack of adherence to infection control practices.¹⁰⁷ Consequently, among the de-escalation efforts implemented in human hospital and community environments is restricted antimicrobial use. In humans the increasing presence of drug-resistant bacterial infections among hospitalized patients is linked to the greater numbers of patients receiving inappropriate antimicrobial treatment.¹²³ A recent on-line report found that in human medicine, antimicrobials were often prescribed despite infection being an infrequent cause of the illness (i.e., pharyngitis). Further, the chosen antimicrobial often was inappropriate for those bacteria potentially causing infection in the treated body system.¹²⁴ Accordingly, reducing inappropriate antimicrobial use has become a priority in human medicine.

Among the more rational paradigms for antibacterial de-escalation, is an approach to empirical antimicrobial use in the hospital setting for patients with serious bacterial infections.¹²³ Such antimicrobial de-escalation attempts to balance the need to provide appropriate initial antibacterial treatment while limiting the emergence of antimicrobial resistance. The goal of de-escalation in this setting is to prescribe an initial antimicrobial regimen that will cover the most likely bacterial pathogens associated with infection while minimizing the risk of emerging antimicrobial resistance.¹²³ The three-pronged approach includes narrowing the antimicrobial regimen through culture, assessing isolate susceptibility for dose determination, and choosing the shortest course of therapy clinically acceptable. Judicious antimicrobial use combined with restricted use of ceftazidime led to a decreased antimicrobial resistance to beta-lactams, in general, in a human teaching hospital environment.¹²⁶ Note that this strategy does not exclude the use of “big gun” antimicrobials. The approach of withholding use of high-impact drugs (e.g., meropenem or vancomycin) in patients whose need for effective therapy is critical to avoid emerging resistance that might limit drug use in later patients may not be rational or in the best interest of the patient. A more appropriate approach is to use the drug correctly. However, routine use of less powerful drugs is appropriate but only if these alternatives are just as effective. Regardless of the choice, once the decision is made to use an antimicrobial, attention must be paid to dosing regimens that minimize the advent of resistance by ensuring that infecting microbes are eradicated.

Another strategy to decrease the impact of antimicrobial use on resistance is a decreased duration of therapy (see the discussion of enhancing antimicrobial efficacy). One study in human critical care patients found that reducing the duration of antimicrobial therapy from 14 days to 10 days decreased the emergence of resistance.¹²⁷ Increasingly, clinical trials will focus on demonstrating efficacy of shorter (i.e., < 5 to 7 days) treatment regimens.

Host Factors That Facilitate Drug Efficacy

Host factors may also facilitate antimicrobial efficacy. Among the most important host factors are local and systemic defenses ranging from compounds that directly target microbes to healthy tissues that provide mechanical barriers and a competent immune system. The role of host defenses are beyond the scope of this chapter but cannot be underemphasized.

Other host factors that facilitate therapy include the accumulation of the drug in active form at the site of infection, which may facilitate antimicrobial efficacy and decrease the risk of resistance. Obvious examples include drugs that undergo renal or biliary excretion. For such drugs urine or bile concentrations (respectively) may exceed PDC thirtyfold to several hundredfold (see the discussion of treatment of urinary tract infections, Chapter 8). Another site of drug concentration is the phagocytic leukocyte (WBC), both in peripheral circulation and at the site of inflammation. Active concentrations of some antimicrobials (e.g., macrolides, lincosamides, and FQs) may increase concentrations 20 to 100 or more times the PDC.^28,132-137 Phagocytic accumulation may facilitate treatment of intracellular infections (e.g., Brucella spp., cell wall–deficient organisms, intracellular parasites, and facultative intracellular organisms such as Staphylococcus spp.). Thus drugs that achieve only bacteriostatic concentrations in plasma may become bactericidal inside the cell, particularly against organisms that locate and survive inside cells (Figure 6-18). Additionally, accumulated drug released by dying phagocytes at the site of infection may increase concentrations to which the infecting microbe is exposed. Accumulation of drug inside WBC has been assumed as an explanation of the disconnect of azithromycin efficacy in pulmonary infections despite low PDCs.⁹¹ Note, however, that drug accumulation does not necessarily enhance drug efficacy. Often, the accumulated drug is sequestered into subcellular organelles, where it cannot reach the organism. In addition, the drug may become otherwise inactivated once inside the cell. The different mechanisms of action of these drugs may not occur in an anaerobic environment, and concentrations by the WBCs might be impaired in an anaerobic environment. The FQs are an example of a class of drugs whose uptake by WBCs is facilitated in an acidic environment; these drugs are distributed throughout the cytosol, where they remain active. The drug will leave the WBCs and enter a drug-free environment and thus may facilitate drug concentrations at the site of infection. Phagocytic WBCs with accumulated enrofloxacin delivered drug to inflamed tissue cages in dogs, demonstrating that accumulation may increase therapeutic response.¹³⁷ Drugs that do not accumulate in WBCs include the beta-lactams, aminoglycosides, and metronidazole. Drugs that are moderately accumulated in WBCs include chloramphenicol (onefold to fivefold) and selected sulfonamides (threefold to fivefold).¹³⁸

Figure 6-18 The intracellular location of organisms presents a barrier to drug penetration. Some organisms are obligate intracellular organisms, whereas others, such as Staphyloccocus spp., demonstrated cytologically (A) and by special stain of infected skin (B), may survive phagocytosis, serving to reinfect tissue once the phagocytic white blood cell has died.

Another potential facilitating host factor is infection at a site that is topically accessible. In such situations several 1000-fold concentrations of the MIC may be reached with topical administration. The rationale for collecting C&S data for such infections might be controversial, but identification of the organism and some indication of susceptibility is prudent, particularly if initial therapy fails.

Mechanisms of Drug Action

Knowledge of the mechanism of action (see Figure 6-19) of a particular antimicrobial is important for several reasons:

Figure 6-19 Targets of antimicrobial actions for the different classes of antimicrobial drugs. The number in parentheses refers to the major mechanism(s) of acquired resistance (other mechanisms also exist; see Chapter 7): 1 = enzymatic destruction (e.g., beta-lactamases for beta-lactams, acetylases for phenicols); 2 = increased efflux pump activity (may be associated with altered porin influx in gram-negative isolates); 3 = altered targeted site (e.g., mutations in DNA gyrase for fluoroquinolones or penicillin-binding proteins for gram-positive isolates); 4 = interfering protein and 5 = increased production of targeted metabolite. Decreased porin size is a common mechanism of resistance associated with increased efflux pump activity for many gram-negative isolates.

The cell wall is an important target for several antimicrobials, protecting the hypertonic intracellular environment of the organism from the hypotonic extracellular environment.²³ A variety of proteins located in the cell wall (penicillin-bound proteins) are important in the formation of the cell wall during division of growth of the organisms. These proteins are the target of several antimicrobial agents. Destruction of the peptidoglycan layer, which provides support to the cell wall, increases the permeability of the cell wall to the hypotonic environment, resulting in osmotic lysis of the cell. Intracellular structures are also major targets for various antimicrobial agents. Binding of ribosomes, the site of protein synthesis in the cell, can either inhibit protein formation or result in the formation of faulty proteins that eventually prove detrimental to the organism. The nuclear material of microbes is another target: Interference with cellular DNA inhibits cellular division, as well as initial cellular functions. Generally, impaired DNA synthesis results in cell death. Other intracellular targets include selected metabolic pathways such as folic acid synthesis, which, when interfered with, prevents formation of materials vital to the microorganism.

Drug Disposition

Nonantimicrobial Effects of Antimicrobials

A number of antimicrobials influence various aspects of the immune system. The phagocytosis of drugs (e.g., macrolides, lincosamides, and FQs) was previously discussed.^{23,132-134,138} In addition to accumulation in WBCs, antimicrobials can influence WBC function. However, the effect can be variable. The negative effect of antimicrobials on phagocytic function has been well established, although the clinical relevance of this effect is less clear.¹⁴⁹ Functions that are targeted include chemotaxis (increased, decreased, or unchanged by clindamycin, erythromycin, chloramphenicol, and lincomycin and decreased or unchanged by gentamicin), phagocytosis (increased by erythromycin and chloramphenicol and decreased by tobramycin and polymyxin B), oxidative burst (increased by clindamycin, cefotaxime, and quinolones and decreased by cefotaxime, trimethoprim/sulfonamides, chloramphenicol, and erythromycin), bacterial killing (increased by cefotaxime and decreased by sulfonamides and aminoglycosides), and cytokine production or activity (interleukin 1 [IL-1] increased by cefotaxime and cefaclor and IL-10 by erythromycin; IL-1 and tumor necrosis factor decreased by cefoxitin, erythromycin, and ciprofloxacin).¹³⁸ Apoptosis of neutrophils may be accelerated.¹⁵⁰

The clinical relevance of these potentially beneficial effects on phagocyte function is not clear, but relevance is supported by some studies. For example, long-term use of azithromycin appears to improve lung function in children with cystic fibrosis and is increasingly being included in its therapeutic regimen; the disease appears to progress more rapidly if azithromycin is not added to therapy. This effect of macrolides appears to target inflammation, because the effect occurs at concentrations below the MIC of the infecting organisms. Potential mechanisms include a reduction in IL-1β, IL-8, and neutrophils in bronchoalveolar lavage fluid.^151,152 In addition to the antiinflammatory effects, macrolides appear to decrease Pseudomonas virulence by reducing the number of pili, thus altering adherence to tracheal epithelium, altering membrane proteins, and decreasing alginate formation.^153,154

Antimicrobial Effects of Nonantimicrobial Drugs

Antimicrobial effects have been described for a number of nonantimicrobial drugs at plasma concentrations achieved when the drug is used for noninfective indications. For example, a number of phenothiazines, including those with antihistaminergic effects, are antibacterial. Because these effects occur both in vitro and in vivo, the effects cannot be attributed simply to immunomodulation. Chlorpromazine is antimycotic at concentrations much higher than can be achieved safely in plasma, but its accumulation over a hundredfold in macrophages containing phagocytized pathogens facilitates effective therapy at recommended doses.¹⁵⁵ The less psychotically active thioridazine enhances the antimycotic activities of rifampin and streptomycin; between 2 and 3 months of use has been promoted as adjuvant therapy. Trifluoperazine and prochlorperazine inhibit S. aureus at concentrations of 10 to 50 μg/mL and selected other microbes (Shigella, Vibrio) at the same or higher concentrations and have demonstrated inhibitory effects in an animal model.^156,157 Selected cardioactive drugs, including oxyfedrine and dobutamine, exhibit antimicrobial effects, again toward selected microbes.¹⁵⁸ Amlodipine has broad antibacterial efficacy at concentrations as low as 5 to 10 μg/mL, with S. aureus being the most susceptible and gram-negative organisms (E. coli, Klebsiella, and Pseudomonas) requiring higher concentrations.¹⁵⁹ Other drugs with demonstrated antimicrobial effects include the antispasmodic drug dicyclomine¹⁶⁰ and selected nonsteroidal antiinflammatories.¹⁶¹ Among the dietary supplements with recognized antibacterial effects are the flavones. Flavone dietary supplements exhibited antibacterial activity to a variety of microbes in a mouse infection model.^162,163 Chitosans have demonstrated efficacy toward a number of bacterial organisms, particularly gram-negative isolates at concentrations as low as 0.05 μg/mL.¹⁶⁴ Several antifungal drugs have antibacterial properties, which are addressed in Chapter 9.

Adverse Drug Events and Antimicrobials

Actions that minimize host toxicity enhance therapeutic success. However, host cells are eukaryotic, whereas the bacteria are prokaryotic. As such, targets of antibacterial therapy are sufficiently different from mammalian cells that, as a class, antibacterials (but not antifungals) tend to be safe. For example, beta-lactam antibiotics are among the safest antimicrobials because they target cell walls, a structure not present in mammalian cells. Often, even if cellular structures are present in both microbe and host, differences in the structure will result in different antimicrobial binding properties. For example, sulfonamides and FQs tend to be safe because the antimicrobials have a much greater affinity for the bacterial target enzymes than the mammalian enzymes. As with other drugs, the incidence of predictable (type A) drug reactions to most antimicrobial therapy correlates with maximum or peak PDC. However, aminoglycoside-induced nephrotoxicity and ototoxicity are an exception; toxicity tends to be related to duration of exposure and is more likely if minimum or trough PDCs are above a maximum level.^76,165,166 Occasionally, toxicity of antimicrobials does reflect their mechanism of action, if the microbial target occurs in mammalian cells and is structurally similar (see Chapter 7). For example, colistin and polymyxin target both microbial and host cell membranes. Administration of either drug is associated with a high incidence of nephrotoxicity (probably because drug is concentrated in renal tubular cells), and subsequently their use generally is limited to the topical route of administration. Drugs that inhibit protein synthesis by binding to ribosomes (e.g., tetracyclines, chloramphenicol) may cause (limited) antianabolic effects in the host at sufficiently high doses. For most antimicrobial drugs, host toxicity may occur through mechanisms unrelated to its mechanism of action, but as a result of targeting structures in host cells. Aminoglycosides cause nephrotoxicity and ototoxicity, not because of their ribosomal inhibition (their antibacterial mechanism of action) but because they actively accumulate in renal tubular (or otic hair) cells (as they do in bacterial organisms) and in lysosomes causing lysosomal disruption. Topical application is more likely to cause ototoxicity with aminoglycoside and other drugs (see Chapters 4 and 7). FQs cause retinal degeneration in cats, through mechanisms yet to be defined. Tilmicosin causes (potentially lethal) beta-adrenergic stimulation; the caustic nature of doxycycline can cause esophageal erosion in cats. Allergies are a less common adverse reaction caused by antimicrobials. Some drugs cause anaphylactoid reactions as a result of direct mast cell degranulation. True allergic reactions should be differentiated from anaphylactoid reactions (more common with intravenous administration of FQs). The latter may occur with the first dose and may be dose dependent. Anaphylactoid reactions can be minimized by administration of a small first dose before therapy. In contrast, drug-induced allergies generally require previous administration or a duration of therapy sufficient to allow antibody formation to the drug, which acts as a hapten (generally 10 to 14 days). Few drug allergies have been documented in animals. Among the most notorious are reactions to the potentiated sulfonamides.

Among the adverse reactions associated with antimicrobial use are those associated with drug interactions. Those most clinicaly relevant involve drug metabolizing enzymes. Examples of drugs that inhibit the metabolism of other drugs are the macrolides; chloramphenicol; and for selected drugs, the fluoroquinolones. In contrast, rifampin is an inducer. Increasingly, drugs that alter drug metabolizing enzymes are emerging as drugs that compete for or alter drug transport proteins (e.g., P-glycoprotein). Drug interactions involving antimicrobials are discussed with each class (see Chapter 8).

Adverse reactions to antimicrobials may reflect their antimicrobial success. Many orally administered drugs cause disruption of normal gastrointestinal microflora (see previous discussions). For example, the author has detected emergence of Clostridium perfringens in dogs treated with fosfomycin. Streptococcus spp. are generally associated with opportunistic infections. However, infections caused by members of this genus (S. pyogenes in humans and Streptococcus canis in animals) are associated with streptococcal toxic shock syndrome (STSS) and necrotizing fasciitis (NF).¹⁶⁷ These syndromes appear to reflect the presence of lysogenic bacteriophage-encoded superantigen genes encoded in the bacterial organisms.¹⁶⁷ The superantigen genes are powerful inducers of T-cell proliferation; the presence of the superantigens then causes release of host cytokines in quantities that may be sufficient to cause lethal effects. In one study a bacteriophage-encoded streptococcal superantigen gene was identified in the majority of S. canis isolates. Induction of these genes can lead to bacterial lysis and subsequent release of proinflammatory and other destructive cytokines. Indeed, use of the FQs has been associated with STSS and NF in dogs (see Chapter 7).¹⁶⁸

Release of endotoxin is another example of seeming therapeutic success potentially leading to therapeutic failure (Figure 6-20). However, the clinical relevance of endotoxin release may be species dependent. Endotoxin release is a side effect of antimicrobials that occurs with therapeutic success, and it may influence antimicrobial selection for the patient infected with a large number of gram-negative organisms.⁸⁴ Endotoxins cause further release of cytokines and other mediators of septic shock (see Chapter 8). Most of these effects are mediated by the inner lipid A component of the LPS molecule that becomes exposed after antimicrobial therapy. In human patients suffering from endotoxic shock, outcome of antimicrobial therapy has been related to plasma endotoxin levels. A number of antimicrobials cause release of endotoxin from gram-negative organisms. Attempts have been made to correlate the amount of endotoxin released to the class of antimicrobial and specifically to its mechanism of action.

Figure 6-20 Among the adverse reactions of antimicrobial therapy is release of bacterial toxins. The risk of damage to the host is greater with a large inoculum. In this example, rapid death of gram-negative organisms can result in rapid release of endotoxin. Drugs whose mechanism results in osmotic lysis (e.g., penicillins) are more likely to be associated with sufficient endotoxin release to cause harm to the patient.

Continued bacterial growth or rapid cell lysis and death have been suggested as important criteria for endotoxin release after antimicrobial therapy. In contrast, the rate of bacterial killing and antimicrobial efficacy do not appear to be related to the rate and amount of endotoxin release. The amount of endotoxin release varies among the antimicrobial classes and even within the classes. Release can be related to mechanism of action. Among the drugs traditionally used to treat septicemia, aminoglycosides have been associated with the least and beta-lactams with the greatest endotoxin release (with imipenem or meropenem causing the least amount of endotoxin release among the beta-lactams).¹⁶⁹ The different amounts of endotoxin released by beta-lactams may reflect different affinities of the drugs for different penicillin-binding proteins. In vitro studies indicate that those beta-lactam antibiotics that specifically bind penicillin-binding protein (PBP)-3 are associated with endotoxin release, whereas those that bind PBP-2 cause little to no endotoxin release.¹⁷⁰ The difference may reflect the fact that PBP-3 appears to form a complex with PBP-1, 4, and 7;¹⁷¹ binding of PBP-3 might thus affect a larger component of cell wall synthesis compared to binding of another PBP. The release of endotoxin by quinolones varies depending on the study. However, in a study of mouse E. coli peritonitis, imipenem (or meropenem) and ciprofloxacin caused less endotoxin release than did cefotaxime.⁸⁴ Selected third-generation cephalosporins also appear to be associated with less endotoxin release: In a study of septicemic patients with acute pyelonephritis, the amount of endotoxin released did not differ among cefuroxime, ciprofloxacin, or netilmicin and each was deemed safe in the septicemic patient.¹⁷²

The release of endotoxin may also be dose (concentration) dependent. For example, endotoxin release is greater at half the recommended dose of ciprofloxacin (3 mg/kg versus 7 mg/kg ciprofloxacin) according to the previously described model.⁸⁴ Actions that might minimize the sequelae of endotoxin release after antimicrobial therapy have not been established. Presumably, administering a dose more slowly may decrease the rate of endotoxin release. Binding and subsequent inactivation of endotoxin by antimicrobials have been documented, particularly for cationic antimicrobials (e.g., quinolones, aminoglycosides, and polymyxin).^84,173

Selecting the Route

Drugs may be selected on the basis of their route of administration. Not all drugs are available for parenteral or oral administration. Parenteral, and particularly intravenous, administration is indicated for life-threatening infections or whenever tissue concentrations must be maximized. Parenteral drugs are also indicated for the vomiting animal. Oral drugs are indicated for long-term use, outpatient therapy, and treatment of gastrointestinal tract illness. Topical therapy may be selected to enhance drug delivery while minimizing toxicity. Topical therapy with lipid-soluble drugs might, however, best be limited to situations in which systemic therapy of the same drug is implemented, thus preventing development of subtherapeutic drug concentrations in tissues other than the site of topical application, as might occur if topical administration alone is implemented.

Combination Antimicrobial Therapy

Combination therapy can be used to achieve a broad antimicrobial spectrum for empirical therapy, treat a polymicrobial infection involving organisms not susceptible to the same drugs, reduce the likelihood of antimicrobial resistance, and reduce the risk of adverse drug reactions by minimizing doses of potentially toxic antimicrobials.^23,24,26,137 Rational combination antimicrobial therapy may be the single most effective action taken to enhance antimicrobial efficacy for the chronic or serious infection. Primary reasons to avoid combination therapy include increases in risk of suprainfection, risk of toxicity (if both drugs are potentially toxic), high cost, and inconvenience to the patient.²⁴

Synergism and Antagonism

Antimicrobials to be used in combination therapy should be selected rationally and based on target organisms as well as on mechanism of action (Table 6-8). Combinations might result in antagonistic, additive, or synergistic antimicrobial effects (Figure 6-21).¹⁸⁰ Generally, these effects are defined by in in vitro systems; clinical relevance is more difficult to establish. Also, the combined effects of two or more antimicrobials are likely to differ with the organism. Avoidance of antagonism is particularly important for patients with inadequate host defenses.^23,24,26,180 In general, bacteriostatic drugs that inhibit ribosomes and thus microbial growth (e.g., chloramphenicol, tetracyclines, erythromycin) should not be combined with drugs whose mechanism of action depends on protein synthesis such as growth of the organism (e.g., beta-lactams) or formation of a target protein. The bactericidal activity and continued degradation or destruction of the microbial target of beta-lactams and FQs depend on continued synthesis of bacterial proteins. Antagonistic effects have been well documented between beta-lactam antibiotics and inhibitors of ribosomal activity. The degree of antagonism between FQs and growth inhibitors is controversial; antagonism has been reported with the use of ciprofloxacin and chloramphenicol,¹⁸⁰ but impaired efficacy was not detected in other studies.¹⁸¹ Antagonism between chloramphenicol and gentamicin has also been documented.¹⁸⁰ Occasionally, the combination of a bacteriostatic ribosomal inhibitor and a drug whose efficacy depends on rapid growth might enhance efficacy, even though the “-cidal” drug will act only in a “static” fashion. For example, chloramphenicol enhances the efficacy of ampicillin toward Salmonella typhimurium and Staphylcoccus spp., presumably because it inhibits the production of beta-lactamases by the organisms that might otherwise destroy ampicllin.

Table 6-8 Examples of Synergistic Drug Combinations

Drug One	Drug Two	Organisms
Dicloxacillin	Ampicillin, penicillin, cephalothins	Escherichia coli, Klebsiella, Pseudomonas aeruginosa
β-Lactam: cephalothin, ampicillin, piperacillin, cefotaxime, cefamandole	Aminoglycoside: gentamicin, amikacin	Escherichia coli, Pseudomonas, aeruginosa, enterococci, others
Chloramphenicol	Ampicillin	Salmonella typhimurium, Staphylococcus aureus (effect is bacteriostatic in nature)
Penicillin	Gentamicin	Bacteroides melaninogenicus
Imipenem	Vancomycin	Staphylococcus aureus
β-Lactam, vancomycin	Aminoglycoside	Staphylococcus aureus
Trimethoprim/sulfonamide	Imipenem, amikacin	Nocardia asteroides (effect is bacteriostatic)
Imipenem	Trimethoprim/sulfonamide, cefotaxime	Nocardia asteroides (effect is bacteriostatic)
Ethambutol	Rifampin, aminoglycosides, ciprofloxacin (enrofloxacin), clarithromycin	Mycobacterium avium (effect is bacteriostatic)

From Wiedemann B, Atkinson BA: Susceptibility to antibiotics: species incidence and trends. In Lorian V, editor: Antibiotics in laboratory medicine, Baltimore, 1996, Williams & Wilkins, pp. 900-1168.

Figure 6-21 Combining antimicrobials can have different sequelae. Antagonistic antimicrobial combinations most commonly result when a drug that inhibits bacterial growth is combined with a drug whose action depends on rapid cell growth. Drugs that act at the same site may be antagonistic, additive, or synergistic (e.g., beta-lactams, depending which penicillin binding protein is targeted).

KEY POINT 6-31

The appropriate combination of two drugs characterized by resistance may render the microbe susceptible.

Chemical antagonism is also possible between two or more antimicrobials (see Chapter 2).^181,182 Aminoglycosides and quinolones are chemically inactivated by penicillins at sufficient concentrations. Ticarcillin has been used therapeutically to reduce the risk of toxicity in a patient overdosed with an aminoglycoside.¹⁸³ Chemical antagonism is unlikely in most clinical uses of these drugs. The risk of antagonism is increased, however, with simultaneous intravenous use of high doses of both ticarcillin and aminoglycosides, such as might occur if aminoglycosides are administered once daily. Potential chemical interactions between other antimicrobials should be identified before combination therapy. Certainly, antimicrobials should not be mixed in the same syringe or intravenous line unless a lack of antagonism has been confirmed.¹⁸²

Drugs that have the same mechanism of action may act in an additive or synergistic fashion. For example, chloramphenicol and clindamycin bind the same 50S ribosomal subunit and will antagonize each other. Because tetracyclines bind to the 30S ribosomal subunit, combination with antimicrobials that target the 50S subunit might be considered (e.g., the phenicols, macrolides, and lincosamides) if there is scientific support. One study indicates an in vitro synergistic effect of the combined use of doxycycline and azithromycin against P. aeruginosa.^183a

Additive effects probably occur when active metabolites are produced from an active parent compound, such as metabolism of enrofloxacin to ciprofloxacin.¹⁸⁴ Antagonistic effects might occur, however, if the drugs compete for a limited number of target sites (e.g., chloramphenicol and erythromycin). In contrast, synergistic actions might occur if the antimicrobial targets are subtly different. For example, a combination of different beta-lactams generally results in additive antimicrobial activity. If the two antimicrobials target different PBPs, however, their combined effect may actually be synergistic (“double beta-lactam therapy”).^185,186 In contrast, combinations of other beta-lactam antibiotics (including combining selected cephalosporins) are antagonistic.¹⁸⁶ The different sequelae of combined beta-lactam therapy might be caused by the PBPs targeted by each drug.

Synergism between antimicrobials can occur if the two antimicrobials kill bacteria through independent mechanisms or through sequential pathways toward the same target.^180,187 The combination of trimethoprim and a sulfonamide exemplifies synergism resulting from sequential actions in the same metabolic pathway (see discussion of potentiated sulfonamides) (see Chapter 7). Clavulanic acid “draws” the beta-lactamase activity of the microorganism away, allowing the protective beta-lactam to impair cell wall synthesis. Synergism between beta-lactams and aminoglycosides exemplifies synergism resulting from killing by independent pathways. Synergism is expected because their mechanisms of action complement one another, but efficacy is enhanced further because aminoglycoside movement into the bacteria is enhanced by increased cell wall permeability induced by the beta-lactam (Figure 6-22).^180,188 Indeed, aminoglycoside activity against enterococci is adequate only when used synergistically with a cell wall–active antimicrobial, such as beta-lactams and vancomycin. Synergism also has been demonstrated against some strains of Enterobacteriaceae; P. aeruginosa; staphylococci, including MRSA; and other microorganisms. However, these organisms are not always inhibited by the combination of aminoglycoside and cell wall–active compounds. Indeed, antagonism has been described between aminoglycosides and beta-lactams against an MRSA, presumably because of induction of an aminoglycoside-modifying enzyme. Enhanced movement in a bacteria may occur with other drugs (e.g., potentiated sulfonamides, FQs) when combined with beta-lactams (see Figure 6-22). Rifampin is another drug for which combined use enchances antimicrobial efficacy of a number of drugs.

Figure 6-22 The combination of any number of drugs with a beta-lactam may result in synergistic antimicrobial effects. The prototypic example is a beta-lactam combined with an aminoglycoside, a class of water-soluble drugs whose movement through the cell to target ribosomes is limited. Changes in the cell wall permeability associated with the beta-lactam exposes the hypertonic (compared with the host) intracellular cytoplasm to the isotonic host, resulting in the influx of solutes into the organism. Intracellular access is thus facilitated for drugs also in the environment. Together, the two drugs are now more likely to kill the microbe. Such synergism has been documented in vitro between beta-lactams and a number of drugs, particularly those classified as bactericidal.

Combination therapy is a powerful tool for enhancing efficacy (Figure 6-23) as well as preventing resistance. Occasionally, the combination of drugs, which by themselves would not be expected to have efficacy against organisms not included in their spectrum, may exhibit efficacy against the organisms. For example, azithromycin and clarithromycin may exhibit synergistic effects with several other drugs against P. aeruginosa. When studied in patients with cystic fibrosis, the most active combinations demonstrating synergy were azithromycin combined with sulfadiazine/trimethoprim or doxycycline. Azithromycin occasionally demonstrated synergism against P. aeruginosa when combined with timentin, piperacillin/tazobactam, ceftazidime, meropenem, imipenem, ciprofloxacin, travofloxacin, chloramphenicol, and tobramycin.¹⁸⁹ In the treatment of S. aureus, clindamycin inhibits early rapid killing of amikacin but acts synergistically with it at 24 to 48 hours.¹⁹⁰

Figure 6-23 Atypical mycobacterium in a cat is associated with marked inflammation, including deposition of fibrous tissue deposition. This cat was successfully treated with a combination of sulfadiazine/trimethoprim and enrofloxacin after 3 months of therapy.

Polymicrobial Infections

Combination antimicrobial therapy may be selected because of the presence of a polymicrobial infection (Figure 6-24).^23,24,77,191 Aminoglycosides or FQs are often combined with beta-lactams, metronidazole, or clindamycin to target both aerobic gram-positive and gram-negative infections or infections caused by both aerobes and anaerobes. The combined use of selected antimicrobials may result in therapy effective against a given microbe when either drug alone was ineffective.

Figure 6-24 Polymicrobial infections may require combination therapy. The quinolones and aminoglycosides offer excellent aerobic gram-negative coverage; the beta-lactams (especially penicillins), metronidazole, and clindamycin offer excellent gram-positive and anaerobic coverage.

Type of Surgery

Surgical wounds are classified as clean, clean-contaminated, contaminated, or dirty. Clean wounds are made under aseptic conditions, are closed primarily, and are not drained. Prophylactic antimicrobial therapy is not warranted for most clean procedures because bacterial contamination is minor, and the patient’s competence helps prevent wound infection. Possible indications for the use of antimicrobials in clean surgical procedures are when the consequences of infection would be catastrophic (e.g., total joint replacement) or when surgical implants are used.

Clean-contaminated wounds include those made in the gastrointestinal, genitourinary, or respiratory tract without significant intraoperative spillage. Also, clean procedures in which a break in sterile procedure occurred are considered clean-contaminated. Clean-contaminated wounds may benefit from prophylactic antimicrobial therapy, and consideration of the following factors seems appropriate when contemplating the use of perioperative antimicrobial therapy: number of resident bacteria encountered, amount of spillage expected, and impact of disease condition on bacterial colonization. Resident bacterial numbers vary depending on the site of the tract incised and the nature of disease. In the normal gastrointestinal tract, resident bacteria are numerous in the oropharyngeal cavity, distal ileum, and colon. Numbers are normally much lower in the distal esophagus, stomach, and most of the small intestine. The normal genitourinary tract above the distal urethra has low bacterial populations. The normal trachea and bronchi also have relatively sparse flora. Although amount of spillage cannot always be predicted preoperatively, prophylactic antimicrobials are probably indicated if the risk of intraoperative spillage seems high. Diseases, in general, tend to modify both bacterial numbers (usually increased numbers) and populations (usually more virulent forms).

Contaminated wounds include those in which there is acute, nonpurulent inflammation or those in which gross contamination from a hollow viscus occurs. Antimicrobial prophylaxis is generally warranted when surgery is performed on contaminated wounds. Also, the presence of extensive tissue damage or accumulation of blood within wounds may warrant prophylactic drug administration, because bacterial colonization is usually promoted.

Dirty or infected wounds benefit from irrigation with antiseptics. Chlorhexidine (0.05%) is an effective wound disinfectant for infected wounds. Use of antimicrobials (systemically, topically, or both) is generally indicated before surgery to treat an infected or dirty wound. Such use is more appropriately termed therapeutic antimicrobial therapy.

Potential Pathogens Encountered

The most frequently encountered pathogenic bacterial contaminants of surgical wounds are Staphylococcus spp. and E. coli. The most common skin bacteria are Staphylococcus spp., although many other organisms may be present as transient, topical flora. The oropharynx has a mixed population of gram-positive organisms (especially Staphylococcus spp., Streptococcus spp., and Actinomyces pyogenes), gram-negative organisms (Proteus, Pasteurella, Pseudomonas, and E. coli), and anaerobic organisms. The stomach and small intestine have very few organisms normally present, whereas the distal ileum and large intestine have large numbers of gram-negative (especially E. coli and Klebsiella, Pseudomonas, and Salmonella spp.) and anaerobic organisms. Potential pathogens encountered in the genitourinary tract include both gram-positive and gram-negative organisms (especially Staphylococcus and Streptococcus spp., E. coli, and Proteus and Pseudomonas spp.). Pathogens of the respiratory tract (especially lower respiratory tract) include both gram-positive organisms (Staphylococcus spp., Streptococcus spp., and A. pyogenes) and gram-negative organisms (Pseudomonas spp., E. coli, and Klebsiella, Pasteurella, and Enterobacter spp.).

Host Competence

Host resistance may be compromised systemically or locally. Patients with systemic immunodeficiency often have chronic, recurrent, or partially responsive infections. Prophylactic antimicrobial therapy is probably indicated for such patients regardless of the surgical procedure to be performed. Secondary immunodeficiencies have been associated with a variety of diseases, including hepatic or renal failure, hyperadrenocorticism, diabetes mellitus, and neoplasia. Other factors that may affect systemic host competence include advanced age, severe malnutrition, obesity, immunosuppressive drugs, and splenectomy.

Local factors of importance in the maintenance of host competence include tissue perfusion and tissue trauma. The competence of local defense mechanisms may be affected adversely by obstruction, neoplasia, ulceration, and hemorrhage. For example, the bacterial flora of a stagnant loop of jejunum caused by intestinal obstruction resembles that of the normal distal ileum (i.e., large numbers of resident bacteria). For the purposes of selecting perioperative antimicrobials, the clinician should accurately assess host competence before the surgical procedure.

Pharmacologic and Antibacterial Properties

The primary goal to be achieved by administration of prophylactic antimicrobial agents is to produce adequate concentrations of antimicrobial at the surgical incision site at the time of wound contamination. Also important is the concept that the major risk of contamination is at the time of surgery until a fibrin seal develops between wound edges (approximately 3 to 5 hours postoperatively). Factors of importance in the use of perioperative antimicrobials are absorption (timing and route of administration), distribution, and elimination characteristics. Absorption issues are of least concern with intravenously administered antimicrobials. For most antimicrobials distribution is relatively rapid and complete within 30 to 60 minutes after intravenous administration. The concentration of drug achieved in the tissue correlates with the concentration of free drug in the serum. Highly protein-bound drugs (i.e., little free drug in the serum) achieve lower tissue concentrations than do weakly bound agents (e.g., cefazolin, gentamicin, and ampicillin). Other factors such as lipid solubility, pH, and local environment may also influence tissue penetration of the drug. Elimination of most antimicrobials is principally by way of the kidneys. The rate of elimination determines the dosing interval that is selected. More rapidly eliminated drugs require more frequent administration. Cefazolin, for example, should be administered at 2-hour intervals during the surgical procedure to maintain adequate tissue and serum levels.

The following prophylactic antimicrobial regimen seems appropriate: an intravenous dose of drug given 30 to 60 minutes before incision (i.e., at anesthetic induction) and another dose given at the completion of the procedure. If the surgical procedure lasts longer than 3 hours, an additional intraoperative dose of antimicrobial should be given approximately 2 to 3 hours after the initial dose. There is no rationale for continuing antibiotic administration longer than 24 hours after surgery in the absence of documented infection. If infection is documented, therapeutic antimicrobial therapy is initiated.

The selected drug should be bactericidal for the pathogens that are most likely to contaminate the surgical site. First-generation cephalosporins (e.g., cefazolin) are generally as effective as and less expensive than second- and third-generation cephalosporins. Surgery of the lower gastrointestinal tract may require a more elaborate schedule of prophylactic drug administration, partly because of the presence of anaerobic organisms. A second-generation cephalosporin (e.g., cefoxitin) or an aminoglycoside/anaerobic combination (e.g., amikacin and clindamycin or gentamicin and amoxicillin) should be administered systemically. The use of oral antimicrobials for prophylaxis may not be prudent, in part, because peak concentrations are likely to be less than with intravenous administration, even if bioavailability is close to 100% (and many are not).

Inappropriate perioperative antimicrobial use has been shown to increase the incidence of complications. Examples of inappropriate perioperative antimicrobial use include use of antimicrobials for clean surgical procedures, initiation of prophylactic antimicrobials postoperatively, and continuation of antimicrobial administration for longer than 24 hours. Each of these actions risks the occurrence of one or more of the following complications: reduced efficacy, suprainfection, selection of resistant bacterial pathogens, greater client cost, and a potential for higher incidence of drug-associated complications.

Although surgical prophylaxis has been integrated into the perioperative surgical plans for veterinary patients, surprising little information supports its use. In one controlled study of dogs (n = 329) and cats (n = 544) undergoing clean and clean-contaminated surgical procedures, the postoperative infection rate did not differ in placebo (9.4%) compared with the cephalexin-pretreated group (8.9%).¹⁹² In another study investigating the impact of flushing in dogs undergoing total ear canal ablation, organisms were characterized by a higher incidence of antimicrobial resistance to cefazolin,²⁹ suggesting that cefazolin may not be a rational choice in all presurgical candidates.