Antimicrobial drug factors include mechanism of action, drug disposition, and resistance. Veterinarians in the US should take particular note of the Veterinary Feed Directive and are encouraged to consult the most current US FDA regulations.
Mechanism of Action
Antimicrobials can be grouped into general categories based on the pharmacokinetics that optimize antimicrobial activity. Whether a drug is categorized as concentration dependent or time dependent is important for designing a dosing regimen.
Concentration-dependent drugs are more effective when peak drug concentrations at the site of infection exceed the MIC of the infecting microbe by 10 times or more. Concentration-dependent drugs also have a long postantimicrobial effect, particularly toward gram-negative organisms. This effect results in continued inhibition of microbial growth after brief exposure to the drug. For such drugs, the dose rather than the dosing interval is important.
For time-dependent drugs, dosing regimens should be designed such that drug concentrations remain above the MIC for most of the dosing interval. For such drugs, the dose may need to be increased to surpass the MIC of the organisms; however, the interval should be designed to maintain the concentrations.
The mechanism of action (MOA) of antimicrobial agents determines whether the antimicrobial action is likely to be bactericidal or bacteriostatic, and whether the relationship between plasma drug concentration and organism minimum inhibitory concentration (MIC) is concentration dependent or time dependent. The MOA often is related to the mechanisms by which resistance emerges. For some antimicrobials, the MOA also relates to the mechanisms of toxicity. If combination treatment is to be considered, drugs with MOAs that complement one another should be chosen.
Inhibition of cell wall synthesis: beta-lactams (penicillins, cephalosporins, and cephamycins), glycopeptides (vancomycin), bacitracin, fosfomycin
Impairment of cell membrane function: polymyxin B, colistin
Inhibition of protein synthesis through binding either to a single ribosomal subunit (tetracyclines, chloramphenicol, macrolides, lincosamides) or to both ribosomal subunits (aminoglycosides)
Inhibition of DNA synthesis and replication: novobiocin, quinolones, metronidazole
Inhibition of DNA-dependent RNA polymerase: rifamycins
Inhibition of folic acid and, consequently, DNA synthesis: sulfonamides, trimethoprim
Drug disposition (absorption, distribution, metabolism, and excretion) of antimicrobials can affect the efficacy of treatment and should influence the design of the dosing regimen.
Many antimicrobials are administered orally. Exceptions include those destroyed by GI acidity (eg, some beta-lactams, particularly penicillins) or microbes (in ruminants) and those insufficiently unstable for oral preparations.
Food intake can affect the absorption of some drugs, most notably tetracyclines (except doxycycline) and the fluoroquinolones. A drug that has a very high oral bioavailability in one species cannot be assumed to have a similarly high oral bioavailability in another. For example, the oral bioavailability of ciprofloxacin is good in humans but fair or negligible in dogs and horses.
Most antimicrobials must potentially reach many body tissues. Antimicrobials that have a distribution limited to extracellular fluid (ie, water-soluble drugs) include the beta-lactams, fosfomycin, aminoglycosides, and some members of the sulfonamides and tetracyclines. In general, drugs that are water-soluble distribute well to the extracellular fluid of most organs, but there are exceptions. For example, only ~30% of amoxicillin reaches tracheobronchial secretions, and distribution into sanctuaries is limited. Doses of such drugs should be increased, particularly for isolates deemed susceptible on antimicrobial susceptibility testing but with MICs approaching the CLSI break point (see Interpretation of Culture and Susceptibility Testing Interpretation of Culture and Susceptibility Testing Treatment should be aimed at a specific pathogen whenever feasible, and the pathogen should be identified before administration of the antimicrobial is initiated. Care must be taken when predicting... read more ). Lipid-soluble drugs distribute to a greater variety of tissues and are able to reach intracellular organisms. Lipid-soluble drugs include the fluoroquinolones, macrolides, clindamycin, many sulfonamides, and doxycycline and minocycline. Some drugs are characterized by volumes of distribution that exceed 1 L/kg, indicating that the drug accumulates or becomes trapped at some sites.
Several antimicrobials undergo hepatic metabolism, and some drugs (eg, ceftiofur and enrofloxacin) are metabolized to an active metabolite that can contribute substantively to antimicrobial activity. Some drugs undergo substantive to exclusive excretion in the bile, such as the macrolides, minocycline (and some of doxycycline), and clindamycin. For such drugs, care must be taken in species with a GI microbiota that is subject to antimicrobial disruption. Many drugs are excreted by the kidneys and subsequently concentrated in the urine, including beta-lactams, aminoglycosides, most fluoroquinolones, and several tetracyclines.
Virulence and Resistance
Antimicrobial resistance is a common reason for failure of treatment. Resistance is a natural response to exposure to toxins, including antimicrobials. Although resistance is not desirable in an infecting pathogen, resistance by itself is not the problem. Rather, the problem is the virulent organism that causes harm to the animal. The acquisition of virulence factors is the reason for illness. A virulent organism possessing multidrug resistance (MDR) is particularly problematic.
Bacterial resistance to drugs can be either inherent or acquired.
Genes that encode inherent resistance are present in all strains of an organism, and gene expression is independent of antimicrobial exposure. For example, bacterial wall–defective variants (eg, L-forms, spheroblasts, and protoplasts) are inherently resistant to cell wall inhibitors. Selected gram-negative bacteria (eg, Pseudomonas aeruginosa) have very small porins that prevent cell wall access to some drugs—another example of inherent resistance.
Acquired resistance occurs in only selected strains of an organism, usually emerging as a result of exposure to an antimicrobial. Genes for resistance can be acquired either by spontaneous mutation or through sharing of genetic material via plasmids or transposons.
Mutations usually occur by chance, and the likelihood that at least one CFU in any given population will be resistant to any chosen drug increases as the population reaches 107 CFUs. Because mutations conferring resistance are transmitted to daughter cells, resistance will remain in the population genome (vertical resistance). Selection pressure results in a residual population that expresses resistance, even after exposure to the drug has been discontinued. However, mutated bacteria, which are often physiologically impaired, will not be selected for and will diminish with time. Mutated cells may also be less virulent.
Generally, mutations confer resistance to only one drug or drug class. There are exceptions: resistance to fluorinated quinolones Quinolones, Including Fluoroquinolones, Use in Animals Quinolone carboxylic acid derivatives are synthetic antimicrobial agents. Nalidixic acid and its congener oxolinic acid have been used for treatment of urinary tract infections for years, whereas... read more is an example where a mutation emerges in the presence of one drug but targets multiple drugs. Emergence of MDR in this case reflects the MOA of fluorinated quinolones, which result in damaged DNA. Subsequent mutations include those that alter regulators of efflux pumps. A marked increase in efflux activity results in MDR.
Acquired resistance also can be shared horizontally by transfer from one organism to another. Such resistance emerges in response to the presence of the drug. Shared resistance occurs rapidly, often during the course of treatment and often resulting in the transmission of multiple genes targeting multiple drugs.
Transfer via plasmids is the most recognized mechanism of shared resistance. Plasmids are composed of extrachromosomal DNA (ie, DNA not vital to cell function). Plasmids are replicons, meaning they are capable of replicating autonomously in the host. A single CFU may contain multiple plasmids, each of which, in turn, may carry multiple genes conferring resistance to multiple drugs. Plasmids are easily shared between gram-negative organisms and, less commonly, between gram-positive organisms. Plasmids can also be transferred between gram-positive and gram-negative organisms. Although plasmid-mediated resistance can occur rapidly, plasmids are generally shed by the bacterium after the drug is no longer present.
Multiple mechanisms exist whereby plasmids can enter a bacterial cell. The most common method is conjugation, in which DNA passes from the donor cell to the recipient via a bridge formed during direct cell-to-cell contact. This is the most sophisticated form of transmission, in that the donor must have the necessary surface appendage (sex pilus) to form the bridge that is coded for by a resistance transfer factor on the plasmid.
Transformation, which occurs in only a limited number of bacteria, is accomplished by the passage of naked DNA from donor to recipient.
Transduction involves transfer via a bacteriophage that inserts itself into recipient bacteria. Phage-mediated transduction occurs in some gram-positive species (especially Staphylococcus aureus) as well as gram-negative species.
One mechanism whereby genetic sequences can be transferred between extrachromosomal (plasmid) and chromosomal DNA is thetransposon, which is a DNA sequence that can change position in a genome. Transposons can carry chromosomal DNA from one bacterial cell to a plasmid and back. These transpositional sequences may be carried on gene cassettes. Integrons are gene-capturing systems found in plasmids, chromosomes, and transposons. Integrons can carry genes imparting antimicrobial resistance; generally, such resistance affects multiple drugs. After being incorporated into chromosomal or plasmid DNA, the genes are subsequently expressed or disseminated even further.
In addition to resistance genes, these cassettes may include virulence factors, as exemplified in certain strains of methicillin-resistant Staphylococcus aureus (MRSA) . Methicillin resistance reflects acquisition of the mec gene, encoding for a mutated penicillin-binding protein-2, which prevents binding by beta-lactam drugs. Because of acquisition of a virulence gene that facilitates infectivity, MRSA has transitioned from hospital-acquired infections, occurring only in the immunocompromised, to community-acquired infections.
Acquired resistance reflects three major cellular mechanisms:
Intracellular drug concentrations can be decreased. Multiple mechanisms can accomplish this change. Drug movement into the microbial cell can be prevented by decreasing porin number or size or, more commonly, by transporting the drug out of the cell through efflux transport pumps located in the cell membrane. These mechanisms result in resistance to multiple drug classes.
Microbes can produce enzymes that destroy certain drugs. This mechanism generally targets only drugs of a single class. Examples include beta-lactamases and enzymes that target fosfomycin, aminoglycosides, or phenicols. These enzymes can be expressed constitutively or expression can be induced on exposure to the drug. In general, the addition of larger R groups on the drug molecule sterically hinders the ability of destructive enzymes to reach the vulnerable site of the drug molecule.
Microbes can acquire mutations that change the target so that it no longer binds to the drug. Examples include mutated penicillin-binding proteins that confer methicillin resistance to staphylococci, mutations in DNA gyrases that confer resistance to fluoroquinolones, and mutations in ribosomal subunits that confer resistance to various ribosomal inhibitors. Such mutations generally cause single-drug resistance.
Other, less common mechanisms include alternative metabolic pathways that circumvent the effect of the drug (eg, sulfonamides) or increased synthesis of a key metabolic intermediate that would thus require higher concentrations of the drug (eg, para-aminobenzoic acid in sulfonamide resistance).
Acquired resistance manifests as an increase in the MIC for the drug in question. Notably, resistance can emerge in an isolate that is deemed susceptible on antimicrobial susceptibility testing based on the CLSI criteria if the increase in MIC has not exceeded the break points determined for that drug (see Interpretation of Culture and Susceptibility Testing Interpretation of Culture and Susceptibility Testing Treatment should be aimed at a specific pathogen whenever feasible, and the pathogen should be identified before administration of the antimicrobial is initiated. Care must be taken when predicting... read more ).
For recurrent infections, identifying the underlying cause is likely to be paramount to avoiding antimicrobial resistance, including MDR.
Among the clinically relevant gram-negative organisms that develop MDR are Escherichia coli and Klebsiella, through the production of extended-spectrum beta-lactamases. Gram-positive organisms in which MDR has developed include MRSA and its canine and feline counterpart, methicillin-resistant Staphylococcus pseudintermedius (MRSP). Enterococcus is another gram-positive organism for which MDR is both natural and emerging. Clostridioides difficile is an example of an obligate anaerobic organism for which MDR is clinically important.
Two major aspects of antimicrobial use are of particular concern to veterinarians: the likelihood of causing a pathogenic organism to become resistant to current antimicrobial treatment, and the likelihood of commensal organisms, regardless of location in the body, becoming resistant to future antimicrobial treatment. Although designing the dosing regimen such that all infecting organisms are killed minimizes the risk of acquired resistance in the patient, any antimicrobial use contributes to global concerns regarding antimicrobial resistance. Historically, veterinary use of antimicrobials has focused on agricultural (eg, food-animal) applications. However, concern is shifting to include use in companion animals, particularly in light of greater awareness of the risk that resistant commensal organisms will be transferred between pets and humans.
The role of antimicrobial treatment in the advent of antimicrobial resistance is well recognized. An approach that may help minimize the risk of emerging resistance is following the components of antimicrobial stewardship known as the three Ds—decontamination, de-escalation, and design of a dosing regimen.
Decontamination includes attentiveness to hygiene not only in the hospital but also in the home environment. This decreases the transmission of potentially resistant microbes.
De-escalation includes avoiding inappropriate or unnecessary systemic antimicrobial use. For example, administration of antimicrobials in low concentrations (as in animal feeds) or with improper dosing regimens may lead to acquired resistance in a given population.
Identifying the need to treat with systemic antimicrobials is important. Clinical signs consistent with infection (eg, fever, inflammation, pain, and neutrophilia) are not diagnostic of infection, and the presence of bacteria is not necessarily an indication for systemic antimicrobial treatment. Bacteria may reflect normal microbiota, which must be distinguished from pathogenic microbes. Nonvirulent organisms may act as commensals, preventing infection with more virulent pathogens.
Results of microbial culture may reflect a poorly collected sample. Growth of multiple (three or more) organisms may reflect contamination, whereas growth of a single organism is more indicative of a pathogen.
The extent of growth may be used to support infection; for example, for urine collected by cystocentesis, bacterial counts > 1,000 CFUs/mL are typical for urinary tract infections. A wiser approach to treating subclinical bacteriuria might be to avoid administering systemic antimicrobials until clinical signs become evident.
Infection alone does not justify systemic treatment with antimicrobials. For example, in horses, the necessity for antimicrobial treatment in every case of salmonellosis Salmonellosis in Animals Salmonellosis is infection with Salmonella spp bacteria. It affects most animal species as well as humans and is a major public health concern. The clinical presentation can range from... read more or infection by Rhodococcus equi is coming under question.
The use of topical treatment should be considered when possible; not only can much higher concentrations be achieved at the site of infection, but the systemic impact of antimicrobials on microbiota can be minimized. For example, despite the approval of antimicrobials to treat feline abscesses, most cases might be better managed by local treatment.
De-escalation might also involve shortening the duration of treatment. Identifying the most appropriate duration can be problematic. Short-term treatment at high doses and short intervals should be sufficient to kill the infecting microbe, negating the need for longer-duration treatment at lower concentrations or shorter intervals that might facilitate resistance. For slower-growing organisms or nonhealing tissues, however, longer durations might be more prudent. The longterm use of antimicrobials for infections associated with an underlying cause is particularly problematic. Resistance is more likely in patients undergoing such treatment, particularly if dosing regimens are (inadvertently) designed for promotion rather than for avoidance of resistance. Shorter durations of appropriately designed dosing regimens can be just as effective as longer-term treatment.
De-escalation also might be accomplished by initiating treatment with a higher-tier drug and switching to a lower-tier drug as soon as possible. Reasons that an antimicrobial might be considered a higher-tier drug, with use supported by culture and susceptibility data, versus a lower-tier drug, which might be used empirically, include the following:
Spectrum. Drugs with a more narrow spectrum are reserved for problematic infection (eg, aminoglycosides for gram-negative infections, vancomycin for MRSA).
Mechanisms of resistance that emerge if treatment fails (eg, drugs causing emergence of extended-spectrum beta-lactamases or MDR).
Importance to human health (vancomycin, linezolids, third- and fourth-generation cephalosporins, fluoroquinolones, etc).
The use of antimicrobials in veterinary medicine that are deemed necessary for human health is coming under increasing scrutiny, particularly for empiric treatment. Therefore, a number of these drugs are prohibited from use, or are prohibited from extralabel use in food animals.
Design of a dosing regimen should be approached such that the entire population of the infecting organism is killed by the chosen antimicrobial. This requires not only an understanding of the relationship between plasma or tissue drug concentrations and the MIC of the infecting microbe but also a willingness to modify routine recommended dosing regimens as needed for individual patients. Tailoring dosing regimens requires delineation of host, microbial, and drug factors that might affect antimicrobial treatment, either negatively or positively. Even if a microbe has historically been considered susceptible, the amount of drug required to effectively inhibit its growth is likely to be greater now than it was when the drug was originally approved. For example, procaine penicillin is labeled for use in food animals at 6,600 IU/kg, but current MICs require doses > 12,000 IU/kg, necessitating extralabel use of this drug.
Successful treatment of infection (ie, resolution of clinical signs) does not prevent resistance from developing. In healthy, immunocompetent animals, adequate decrease of pathogen burden might be sufficient for the host to overcome residual microbial growth; in an animal at risk, however, this residual growth may emerge as a resistant population after an initial response. The more at risk the animal is in being unable to overcome a residual resistant population, the more important it is that dosing regimens be designed to kill the microbes. The fact that eradication of microorganisms curtails development of antimicrobial resistance is encapsulated in the expression "dead bugs don't mutate."
The design of an appropriate dosing regimen can be divided into two main components:
The pathogens should be identified and characterized, and their susceptibility to antimicrobials assessed, so that the drug matches the organism as closely as possible, narrowing the spectrum of the drug used. The role of culture and susceptibility testing in selecting the drug and designing the dosing regimen is becoming increasingly important. With notable exceptions, the ability to predict infecting pathogens is limited. Exceptions include respiratory tract infections in food animals, pyoderma in dogs Pyoderma in Dogs and Cats Superficial pyoderma is a bacterial infection confined to the upper layers of the skin and hair follicle. The infection is usually secondary to local trauma, keratinization disorders, parasitic... read more , and, with limitations, urinary tract infections in dogs and cats. For urinary tract infections, however, E coli is a cause in only 50% of cases.
The more complicated the infection, the less likely it is that the infecting pathogen can be predicted. Likewise, even if the correct pathogen is identified, the ability to predict susceptibility in all but the most uncomplicated infections is likely to be limited. This is true both historically and in individual patients. Even historically susceptible organisms are characterized by higher MICs. For example, only 50% of clinical E coliisolates collected from dogs or cats are susceptible to amoxicillin. For a patient that has previously been treated with a particular antimicrobial, assumptions about patterns of susceptibility may no longer be relevant.
After drugs to which the isolates are susceptible are identified, one that is more likely to penetrate the infected tissue should be chosen. The selection process should take into consideration host and microbial responses to infection. Often, for example, the drug must be lipid soluble and be suitable for use in the particular pH environment. A variety of host and microbial factors contribute to antimicrobial failure by presenting barriers to drug penetration. Debris (eg, inflammatory materials, necrotic tissue, and foreign bodies) and biofilms or decreased blood flow and hypoxia contribute to treatment failure. The ability of some bacteria to become intracellular pathogens, such as by resisting killing by phagocytosis, allow them to evade antimicrobial treatment.
Even urinary tract infections may present several barriers. Simply choosing a drug that is renally excreted may not be adequate. Renal function may not be normal, such that urine (and any drug) is not concentrated. Bacteria are in the urine and also inside uroepithelial cells. Particularly with chronic infections, the microbes may be protected by biofilms and in a state of quiescence such that they are less susceptible to many antimicrobial drugs.
Other factors must also be considered when designing a dosing regimen. Immunocompromise as a result of disease, malnutrition, or concurrent drug treatment, or local immunocompromise resulting from invasive procedures, may contribute to failure. The more at risk the patient is in being unable to overcome a residual resistant population, the more important it is that dosing regimens be designed to kill the microbes. Because dosing regimens should be designed to kill, bactericidal drugs should be chosen whenever possible. Bactericidal drugs include beta-lactams (penicillins and cephalosporins), fluoroquinolones, aminoglycosides, and potentiated (not single) sulfonamides. Tetracyclines, chloramphenicol, macrolides (eg, azithromycin), and lincosamides (eg, clindamycin) are bacteriostatic. In general, it is easier to achieve killing concentrations of a bactericidal drug than a bacteriostatic drug. However, the distinction between bactericidal and bacteriostatic is based on in vitro conditions and may not reflect what occurs in the animal.
Special Considerations for Food Animal Veterinarians
The use of antimicrobials in food animals, including as growth promoters, contributes to the transfer of resistance genes among bacteria and ultimately from food animals to humans. In addition, contamination of food with resistant pathogenic bacteria during the processing of food is a concern. Carcasses may be contaminated at slaughter and processing, and subsequent improper handling or cooking of the product may lead to infection in humans. The development of resistant pathogenic bacteria in poultry treated with fluoroquinolones has been documented.
Infection of the human population is of particular concern because the bacterial resistance created in the animal after veterinary use of a drug or drug class may result in resistance to human drugs of the same class. Whereas the organism developing resistance might be nonpathogenic, transfer of the resistance gene to other bacteria in the human GI tract may result in a pathogenic organism that is resistant and ultimately lead to therapeutic failure in humans.
When selecting drug treatments for food animals, veterinarians must be aware of the potential for resistance. Antimicrobial drugs should be used in the context of a valid veterinarian-client-patient relationship and in accordance with the Animal Medicinal Drug Use Clarification Act (AMDUCA; 21 CFR Part 530). Selection should be based on all information available (clinical findings, experience, laboratory data, physical examination findings, and culture and susceptibility data). Pathogens should be identified, and drugs with the narrowest spectrum of activity that is known to be effective against the pathogen should be used. Client education is important to prevent unnecessary use of antimicrobial agents (eg, use of leftover antimicrobial drugs to treat a new occurrence of disease), to advise on proper withdrawal guidelines of any prescribed drugs, and to ensure that drugs of the proper classes are administered at proper doses and via appropriate routes.
The Veterinary Feed Directive
The Veterinary Feed Directive (VFD) amends the Animal Drug Availability Act of 1996 (21 CFR Part 558), and it delineates the FDA's strategy to promote the judicious use of antimicrobials in food animals. Guidance for Industry #213 (GFI #213), released in 2017, called for tightened regulations regarding the use in food animals of antimicrobials that are medically important to humans and ending the use of these antimicrobials for production purposes (ie, growth promotion or feed efficiency). In 2019, an additional Draft Guidance for Industry (GFI #120) was released, further clarifying VFD regulations for small entities. VFD drugs are drugs “intended for use in or on animal feed,” including medicated feed and water in both food animals and animals not intended for consumption. VFD drugs may also be classified as “combination VFD drugs,” in which a combination of drugs may be used together in a VFD feed. A number of medications that, before 2017, had been available over the counter for producers, including tylosin, tetracyclines, sulfonamides, and neomycin, now require a VFD.
A VFD can be issued only under the supervision of a licensed veterinarian, within the framework of a valid veterinary-client-patient relationship as defined by the state in which the issuing veterinarian is licensed and practicing (or by the federal definition in the absence of a state definition). VFDs must be issued in writing, signed by the veterinarian of record, and sent to the distributor and client before fulfillment. VFDs must be written so that they comply with the labeled conditions for use of a VFD drug or combination VFD drug, and they must be kept on file by the veterinarian, with copies kept by the client and distributor, for at least 2 years from the date of issue. For any VFD drugs, extralabel use is prohibited in all food-producing animal species, including minor species and animals that are classified as food animals but are not destined for human consumption (ie, pet animals).
The exact requirements for information on a VFD are available on the FDA website (see Veterinary Feed Directive Requirements for Veterinarians; also see the table Information Required on a Veterinary Feed Directive ), and many drug sponsors have standard prefilled forms that a veterinarian may use to create a VFD. Of particular note, the expiration date for the VFD is defined as the last date that a VFD feed can be fed. This expiration date is determined by the FDA as part of the label approval process, and it cannot be extended. If an expiration date is not part of the VFD label, the VFD may not exceed 6 months after the date of issue. Feeding a VFD feed to animals after the expiration date is prohibited (see 21 CFR Part 558.6(a)(2)).
Three types of products fall under VFD regulations: type A medicated articles, type B medicated feeds, and type C medicated feeds. In addition, VFD drugs are classified as either category I or category II. Category I VFD drugs are considered a low risk of producing unsafe drug residues, so they are not associated with a withdrawal time. Category II VFD drugs are considered to have a risk of unsafe drug residues, so they require a withdrawal time in at least one species for which the product is approved. This classification of VFD drugs is based on zero tolerance because of the risk with human consumption (eg, carcinogenicity) or because of the presence of residues when fed at the lowest recommended label dose in at least one species.
Mixing of category II medicated feeds from type A medicated articles requires a medicated feed mill license. Type A medicated articles are not medicated feeds in themselves, but rather concentrated drug sources that are used by manufacturers to formulate type B and type C medicated feeds. Even though both type B and type C feeds are medicated feeds, only type C may actually be fed to animals. Type B medicated feed products are an intermediate step between type A medicated articles and type C medicated feeds, considered a premix that is then diluted to create type C medicated feeds. Type C feeds may be fed directly to animals or may be used to make other type C feeds. Any type B or type C medicated feeds that contain a VFD drug or VFD drug combination are considered to be VFD feeds. After a VFD drug is ordered by veterinary prescription, a VFD distributor can supply the feed to a producer for administration to animals.