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Examining current measures of antibiotic potency
 Francis S. Mah |
Ocular infection must be treated quickly and aggressively to reduce the risk for developing bacterial DNA mutations that may confer antibiotic resistance. Ophthalmologists consider many factors when selecting an antibiotic agent for the treatment of ocular infections including the in vitro susceptibility, results reported from in vivo or animal testing, randomized clinical trials, and personal experience from patient response to treatment. An optimal antibiotic for the treatment of ocular infection has many characteristics, including being sufficiently bioavailable in the eye and achieving high concentrations in the tissue.
Analyzing in vitro testing
In vitro test results identify more accurately an efficacious systemic antibiotic rather than an ocular antibiotic because high concentrations can be attained in the eye but not throughout the rest of the body. Among antibiotics used in ocular dosing, in vitro susceptibility test results have some predictive value. Studies on fluoroquinolones show that despite higher ocular dosing frequency and high drug concentrations, high-level bacterial resistance found in vitro might accurately predict inefficacy and high-level resistance in vivo through animal experiments.1 However, moderately high-level resistance and intermediate-level resistance shown in vitro can be overcome by higher dosing frequency or concentration, compared with systemic dosing and concentrations.1 In vitro testing can provide excellent information regarding the characteristics of an anti-infective. It shows the spectrum of activity, such as gram-positive and gram-negative coverage. In vitro testing also provides information regarding relative potency as well as differentiates between bactericidal and bacteriostatic and time-dependent vs concentration-dependent agents. However, key information that cannot be obtained through in vitro testing includes pharmacokinetics, or how well the drug enters the tissues, and toxicity to tissues.
The susceptibility of bacteria to drugs is determined by comparing the concentration of antibiotic delivered to the target tissues with the concentration needed to inhibit growth of bacteria. If the concentration of the antibiotic is higher than the concentration needed to inhibit growth, then the bacteria is defined as susceptible. On the other hand, if the concentration needed to inhibit the growth is not exceeded, the bacteria are considered resistant. The data used to determine breakpoints, or the point at which a bacterial isolate is deemed susceptible or resistant, are derived from systemic dosing and the concentration of drugs in tissues after parenteral administration — there are no specific ocular breakpoints. Therefore, because we can dose from once a day up to every 5 minutes, and because the commercial formulations of topical medications are hundreds of times higher than what is achieved in the blood after even an intravenous dose, the breakpoint for ocular bacterial susceptibility and resistance is by definition higher. The information about bacterial susceptibility that is obtained after culturing an eye infection is helpful as a guide, but it is only applicable to systemic infections. Current susceptibility breakpoints used in vitro are based on serum concentrations and systemic infections (Table 1). My colleagues and I used a rabbit model to study the effect of AzaSite (1% azithromycin in DuraSite, Inspire Pharmaceuticals, Inc.) on Pseudomonas aeruginosa, a common gram-negative pathogen, and found that increased frequency of dosing eradicates the bacteria.2 After conducting duplicate studies, trial 1 and trial 2 found azithromycin in DuraSite to be effective against Pseudomonas aeruginosa. This study showed that infections that cannot be treated systemically can potentially be treated in the eye with drug concentrations achieved with topical dosing (Figure).
Table 1. Problems with MIC Breakpoints Based on Systemic Dosing Antibiotic compounds are tested in vitro against recent clinical strains of bacteria to establish susceptibility testing interpretive criteria for the reference broth microdilution or standardized disk diffusion methods of the National Committee for Clinical Laboratory Standards (NCCLS). In the traditional approach, breakpoints are based on achievable serum levels with projected dosing regimens. In this example of azithromycin, MIC breakpoints of < 2 µg/mL for susceptibility and > 8 µg/mL for resistance were selected based on serum levels of 0.4 µg/mL. However, in the case of 1% azithromycin in DuraSite eye drops, breakpoints for ocular pathogens are not always accurate because serum levels are not representative of ocular tissue levels. Source: Zithromax (azithromycin tablets and oral suspension. Full prescribing Information. Pfizer, April 2006.) |
Frequent dosing with topical 1% azithromycin in DuraSite is effective in reducing Pseudomonas aeruginosa colony counts in a NZW rabbit model Figure. Frequent dosing with topical 1% azithromycin in DuraSite was effective in reducing the number of azithromycin-resistant Pseudomonas aeruginosa colony counts by 3 log units in a rabbit model of keratitis. Adapted from data published in: Yates KA, Romanowski EG, Kowalski RP, Mah FS, Gordon YJ. Frequent dosing with topical 1% azithromycin is effective in reducing azithromycin-resistant Psuedomonas aeruginosa colony counts in NZW rabbit model. E-abstract 2677. Presented at: The Association for Research in Vision and Ophthalmology Annual Meeting. May 6-10, 2007; Fort Lauderdale, Fla. |
Several in vitro measures can be used to determine the clinical efficacy of antimicrobials, including minimum bactericidal concentration (MBC), mutation prevention concentration (MPC), and minimum inhibitory concentration (MIC). Minimum bactericidal concentration values measure the antibiotic concentration at which 99.9% eradication of bacterial isolates occurs, whereas MPC values represent the antibiotic concentration at which 100% eradication of isolates occurs. Although these measures seem to be more accurate indicators of an antimicrobial’s ability to eradicate bacterial isolates, they are used less frequently than MIC measures because they are expensive and labor intensive. Mutation prevention concentration testing gives a theoretic profile of antimicrobial characteristics but has not been proven reliable in animal studies or clinical trials. Minimum inhibitory concentration and MBC values are similar for bactericidal medications, ie, those that kill bacteria, such as fluoroquinolones. Minimum bactericidal concentration values provide less information when applied to bacteriostatic agents, ie, those that inhibit bacterial growth, such as macrolides. High concentrations may eradicate certain isolates, but not all are susceptible (Table 2).
Table 2. Interpretation of Laboratory Data [Drug]= drug concentration, CFU=colony-forming units. MIC values are designated by the percent of bacterial isolates that are inhibited. The MBC and MPC are in vitro measures of bactericidal activity. The MPC has not proven reliable in animal studies or clinical trials. |
The science of measuring MICs
Minimum inhibitory concentration testing can determine the susceptibility or resistance of bacteria. The MIC value represents the antimicrobial concentration that will inhibit bacterial growth. Measuring MICs is a relatively easy and inexpensive way to examine therapeutic agents to compare antimicrobial efficacy and the spectrum of coverage, although it should not be the sole indicator for antibiotic use. In vitro culture results can suggest which antibiotic will eradicate a specific infection. Determining MIC value is especially useful when patients do not respond to treatment with an empiric antimicrobial therapy.
Two tests commonly used to measure MIC values are the Etest (epsilometer test, AB BIODISK), which employs gradient diffusion strips, and the broth microdilution test. In the Etest, filter paper is printed with a continuous gradient of antimicrobial and incubated on a bed of bacteria. As the drug diffuses out, a zone of inhibition is produced in the culture, giving a reading for the MIC of anti-infective needed to inhibit the growth of the bacteria. Etests are more efficient than broth microdilution tests because five or six antibiotics can be tested simultaneously using one plate of cultured bacteria.
In the broth microdilution test, equal amounts of bacteria are added to test tubes containing serially diluted concentrations of antibiotic. After incubation, the lowest concentration of antibiotic preventing the appearance of turbidity in the test tube is considered the MIC of the bacteria.
Minimum inhibitory concentration values indicate antibiotic potency and can only be compared within the respective classes of antibiotic. For example, MIC values from fluoroquinolones cannot be compared with values from macrolides and vice versa. When comparing antibiotics that have the same molecular class and mode of eradication, the antibiotic with the lowest MIC is more potent because less of the antibiotic is needed to eradicate the bacteria. Theoretically, a lower MIC suggests that less of an antibiotic agent is needed to inhibit bacterial growth and therefore decreases the chance for selection of bacteria that are resistant to the antibiotic.
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Minimum inhibitory concentration values are designated by the number of bacterial isolates that are inhibited; hence MIC50 indicates the concentration at which the antibiotic inhibits 50% of bacterial isolates that are tested and MIC90 indicates the concentration at which the antibiotic inhibits 90% of bacterial isolates that are tested, and so on. For example, in a sample of 100 different Staphylococcus aureus isolates taken from 100 different patients, the MIC90 is the concentration at which the antibiotic inhibits growth of 90% of the 100 isolates from these 100 different patients. MIC50 values are often slightly lower than MIC90 values because a lower concentration of antibiotic is needed to inhibit 50% of the bacterial isolates compared with 90% of the bacterial isolates tested. MIC50 values indicate the general potency of an antibiotic against a specific type of bacteria. For an infection that has not been treated previously with an empiric antibiotic, MIC50 values can be useful in helping to select a responsive agent. MIC90 values, on the other hand, are more useful in determining how likely a specific type of bacteria will be resistant to the antibiotic. An antibiotic with similar MIC50 and MIC90 values will show minimal resistance. However, if MIC50 and MIC90 values differ widely, the antibiotic will likely encounter bacterial resistance in the clinical setting. The MIC90 can guide clinicians.
With ocular infections, if a pathogen is susceptible to an antibiotic with systemic breakpoint criteria, it will most likely be sensitive to topical dosing in the eye as well. Antibiotic concentrations on the ocular surface tend to be higher than in circulation. However, when high-level bacterial resistance is likely, patient response to treatment must be monitored. If the patient is nonresponsive, the antibiotic should be changed to an agent shown to be active against the specific bacteria isolates. If the patient responds to treatment despite in vitro findings that the MIC is higher than what would be predictive of successful therapy, then it is likely that the higher topical concentration was effective.
Systemic vs topical antibiotic concentrations
Concentrations may differ significantly between systemic and topical antibiotics. For example, the azalide antibiotic azithromycin is shown to achieve high concentrations in the tissue but not in the blood.3 At the clinical development stage plasma or serum levels of azithromycin in some key tissues was near zero after a 500 mg oral dose.4 For some companies, further development of a drug with this profile may have been discontinued. However, azithromycin blood concentrations were low because the antibiotic was concentrated in the tissues. The development of azithromycin was continued because its tissue distribution and elimination profiles resulted in high concentrations that exceeded the MICs of relevant pathogens.5,6 In vitro resistance breakpoints are based on antibiotic concentrations in the blood. However, because ocular antibiotics can achieve higher concentrations, this breakpoint does not accurately represent bacterial resistance in the eye. Ocular antibiotics will likely have a higher resistance breakpoint value than systemic antibiotics. For example, a 1% drop with a concentration of 10 µg/mL equals a concentration of 10,000 µg/mL in the eye. In the blood, serum or tissues, the level may be 10 µg/mL when the same agent is administered orally. Therefore, bacteria identified as resistant in the lung may respond in the eye.
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My colleagues and I conducted rabbit studies1 using 0.3% gatifloxacin, a fourth-generation fluoroquinolone, on methicillin-resistant Staphylococcus aureus (MRSA) with an MIC of 64 µg/mL and a systemic susceptibility breakpoint of 4 µg/mL. These values indicate that the bacteria should be resistant and nonresponsive to antibiotic agents with an MIC breakpoint of 4 µg/mL. In our study, a highly resistant strain of MRSA was eradicated using 0.3% gatifloxacin, which, by systemic measures, should have been ineffective. However, aggressive treatment that involved frequent dosing of gatifloxacin overcame resistance to MRSA and successfully treated the infection in rabbit corneas. The study concluded that aggressive topical therapy can treat ocular infections that are considered resistant to an antibiotic by systemic criteria and reinforced the need for improved standards to establish effective susceptibility breakpoints in ocular tissue.
Characteristics of macrolides
Macrolides and fluoroquinolones are two classes of antibiotic that achieve high concentrations in the tissues. Both drugs have developed some resistance since their introduction. Fluoroquinolones are bactericidal and have been shown to be effective in eradicating both gram-negative and gram-positive bacteria.7
As concentration-dependent antibiotics, fluoroquinolones in increased concentration offer increased efficacy. Fluoroquinolones eradicate bacteria by attacking DNA polymerase, thus inhibiting bacterial replication. Macrolides are safe, effective bacteriostatic medications that have also been found to be bactericidal in certain isolates when dosed at high concentrations. 5,8,9 Macrolides are effective medications for treating gram-positive and gram-negative bacteria. Azithromycin is especially effective against atypical infections, including atypical mycobacteria, mycoplasma, and Legionella pneumophila. A unique property of macrolides and azithromycin is that there may be anti-inflammatory effects of the class, according to some clinical studies. Further, because the mechanism of action is the inhibition of protein synthesis, some evidence suggests that macrolides may reduce virulence factor formation in bacteria such as H influenzae.10
Conclusion
Although MICs can be useful in helping to determine antimicrobial efficacy, they offer more conclusive data when applied to systemic medications vs topical medications. Azithromycin in DuraSite is a broad-spectrum antibiotic that is more effective for ocular infections when used as a topical eye drop than as a systemic medication. Therefore, ophthalmologists cannot rely solely on MICs when predicting antibiotic efficacy because frequent dosing to obtain high ocular tissue concentrations can inhibit intermediate levels of bacterial resistance regardless of the class of antibiotic. A determination of antimicrobial efficacy should include in vitro data such as MICs, review of in vivo results from animal studies, and evidence-based practice such as clinical trials and clinical practice.
References
- Romanowski EG, Mah FS, Yates KA, Kowalski RP, Gordon YJ. The successful treatment of gatifloxacin-resistant Staphylococcus aureus keratitis with Zymar (gatifloxacin 0.3%) in a NZW rabbit model. Am J Ophthalmol. 2005;139:867-877.
- Yates KA, Romanowski EG, Kowalski RP, Mah FS, Gordon YJ. Frequent dosing with topical 1% azithromycin is effective in reducing azithromycin-resistant Psuedomonas aeruginosa colony counts in NZW rabbit model. E-abstract 2677. Presented at: The Association for Research in Vision and Ophthalmology Annual Meeting. May 6-10, 2007; Fort Lauderdale, Fla.
- Lode H. The pharmacokinetics of azithromycin and their clinical significance. Eur J Clin Microbiol Infect Dis. 1991;10:807-812.
- Zithromax. [package insert 70-5179-00-4] (azithromycin tablets and oral suspension). New York, NY:Pfizer, Inc. 2004.
- Retsema JA, Girard AE, Girard D, Milisen WB. Relationship of high tissue concentrations of azithromycin to bactericidal activity and efficacy in vivo. J. Antimicrob Chemother. 1990 Jan; (25 suppl A):83-89.
- Foulds G, Shepard RM, Johnson RB. The pharmokinetics of azithromycin in human serum and tissues. J Antimicrob Chemother.1990 Jan; (25 suppl A):73-82.
- Mah FS. New antibiotics for bacterial infections. Ophthalmol Clin North Am. 2003;16:11-27.
- Imamura Y, Higashiyama Y, Tomono K, et al. Azithromycin exhibits bactericidal effects on Pseudomonas aeruginosa through interaction with the outer membrane. Antimicrob Agents Chemother. 2005;49(4):1377-1380.
- Van Bambeke F, Tulkens PM. Macrolides: Pharmacokinetics and pharmacodynamics. Int J Antimicrob Agents. 2001;18(suppl)S17-S23.
- Tateda K, Comte R, Pechere JC, et al. Azithromycin inhibits quorum sensing in Pseudomonas aeruginosa. Antimicrob Agents Chemother. 2001;456:1930-1933.