Introduction
Limited information is available regarding anti-microbial administration and related pharmacokinetic properties in amphibians. Consequently, current antimicrobial dosing strategies are based primarily on cross-species extrapolations from reptiles or mammals. Most of the published amphibian antimicrobial data pertain to a single study,1 which demonstrated that skin bacterial loads decreased over time when Northern leopard frogs (Lithobates pipiens) were immersed in baths of various antimicrobial solutions. Route of administration is an important factor to consider because amphibian skin is highly permeable, allowing for most chemicals to be absorbed systemically when applied topically.2–4 This unique adaptation has led to the exploration of medications applied topically or via immersion, but few pharmacokinetic studies have been performed to demonstrate that medications applied topically will reach effective plasma concentrations in amphibians.3
Ceftazidime is a third generation cephalosporin that has broad-spectrum coverage and a particular affinity for gram-negative bacteria, including Pseudomonas spp.5 Ceftazidime has a wide range of tissue dispersion in mammals (including bone and CSF) and is exclusively excreted, unchanged, by the kidneys.5 The method of action is time dependent, and efficacy is defined by the time the serum or plasma concentration remains above the MIC for a specific pathogen.6 Although ceftazidime is a commonly used antimicrobial in reptiles and is widely used in amphibians, few pharmacokinetic studies7–9 have been performed to inform that use, none of which involved amphibian species. The most commonly isolated bacteria from amphibian skin are gram-negative organisms (ie, Pseudomonas spp, Aeromonas spp, Citrobacter spp, and Klebsiella spp), making ceftazidime an increasingly popular antimicrobial choice in amphibian medicine during the last decade.2,10 Published anecdotal information recommends ceftazidime administration at 20 mg/kg SC, IM, or IV every 48 to 72 hours in a variety of reptile species.4,10–12
The primary objective of the study reported here was to determine whether ceftazidime administration to Northern leopard frogs at anecdotally recommended amphibian doses would result in effective plasma concentrations and detectable kidney and skin concentrations. A secondary objective was to evaluate the effect of 2 different doses administered SC and 2 routes of administration (SC and TC) on plasma, renal, and cutaneous ceftazidime concentrations. Our hypotheses were that a higher dose of ceftazidime would result in higher plasma concentrations, regardless of route, and that TC administration would result in higher skin ceftazidime concentrations than achieved with SC administration.
Materials and Methods
Animals
The study protocol was approved by the University of Wisconsin-Madison School of Veterinary Medicine Institutional Animal Care and Use Committee (protocol No. V006145-V02). Northern leopard frogs (n = 44) of unknown sex were obtained from a commercial supplier.a Body weight ranged from 23 to 74 g. At least 2 weeks before the study began and during the study period, frogs were housed in groups of 2 or 3 in 10-gallon glass terrariums on a 12-hour light and 12-hour dark schedule. The enclosures were lined with recycled polymer paperb and contained a large water dish, which allowed for partial submersion.c Distilled water was used to mist enclosures and refill water dishes daily. Humidity was maintained above 80%, and ambient room temperature was between 23.9°C and 25.6°C. Each frog was fed 3 superwormsd daily. The superworms were fed a complete insect diete and dusted with calcium powderf immediately before they were fed to the frogs.
Frogs were treated with fenbendazoleg (100 mg/kg, PO) and praziquantelh (20 mg/kg, SC) on intake at least 2 weeks before the study began. Each frog received a physical examination on arrival and before the study began. Ten frogs were replaced after evidence of disease was found during the study period. This evidence included ventral erythema, gastrointestinal and pulmonary parasitism, and grossly discolored organs.
Study design and procedures
Frogs were randomly assigned by means of a randomization programi to a control group (n = 4; no ceftazidime), SC20 group (10; ceftazidime administered once at 20 mg/kg, SC), TC20 group (10; ceftazidime administered once at 20 mg/kg, TC), or SC40 group (10; ceftazidime administered once at 40 mg/kg, SC). Less than 24 hours prior to administration, ceftazidimej was reconstituted to 20 mg/mL by a veterinary pharmacist at the School of Veterinary Medicine, University of Wisconsin-Madison. The reconstituted product was then frozen and thawed immediately prior to use. Subcutaneous ceftazidime administration was performed in the right forelimb at the level of the humerus. For TC administration, the dorsal skin surface was first wiped with sterile gauze to remove any surface debris and then the ceftazidime was applied on mid dorsum immediately caudal to the eyes.
Frogs in the control group were euthanized prior to the study period to validate ceftazidime assays for skin, plasma, and renal tissue. For the SC20, TC20, and SC40 groups, 2 frogs each were euthanized 12, 24, 48, 72, and 96 hours after ceftazidime administration. Euthanasia was performed following a 3-step protocol in accordance with AVMA guidelines for amphibians with focus on tissue preservation.13 Frogs were first immobilized with 5% isoflurane anesthesia by means of chamber induction until no response was perceived following hind limb toe-pinch stimulation and no gular movements were seen for at least 60 seconds. Between 0.3 and 0.7 mL of blood was collected via cardiocentesis with a 28-gauge, 0.5-inch needle attached to a 1-mL syringek and transferred to a tube containing lithium heparin.l The frogs were then decapitated at the level of the atlas with a No. 10 scalpel blade and pithed in the foramen magnum with a 22-gauge needle.
Within 30 minutes after collection, blood samples were centrifuged at 200 × g for 10 minutes and plasma was harvested. After euthanasia, a rectangle of dorsal skin tissue was removed from each frog by use of iris scissors from between the eyes to the level of the pelvis and extending laterally approximately 3 cm from the vertebral column. Both kidneys were removed in their entirety. All samples (plasma, skin, and renal tissue) were frozen at −80°C and shipped frozen to the University of Tennessee College of Veterinary Medicine for analysis.
Sample analysis
Measurement of ceftazidime concentration in plasma and tissue samples was conducted by use of a reversed-phase HPLC method. A standard HPLC systemm and computer equipped with chromatography softwaren were used to analyze the samples. Compounds were separated on a C18 column (4.6 × 250 mm; 5 μm) with a 5-μm guard column.o The mobile phase was a mixture of 10mM potassium phosphate monobasic (pH of 2.5 adjusted with 1M phosphoric acid) and an acetonitrile-water mixture (90:10 ratio). Absorbance was measured at 260 nm with a flow rate of 0.9 mL/min.
Plasma samples— Ceftazidime was extracted from plasma samples with a centrifugation method. Previously frozen plasma samples were thawed and vortexed, and 100 μL of plasma was transferred to a 13 × 100-mm glass tube followed by 10 μL of cefotaxime (internal standard, 100 μg/mL) and 140 μL of distilled water. The tubes were vortexed for 30 seconds and then loaded into a centrifugation filter unitq and centrifuged for 20 minutes at 16,060 × g. The resulting solution (approx 200 μL) was transferred into HPLC vials, and 100 μL was injected into the chromatography system.
A partial validation of the plasma assay was performed because very little blank plasma was available from the control frogs. It was ensured that the same matrix was used as the incurred samples for preparation of the calibration curve, the curve was linear (R2 value for calibration curve, > 0.99), and samples for the calibration curve could be back-calculated to within 15% of the true value. The calibration curve range was 0.1 to 100 μg/mL. The lower limit of quantification during validation was 0.1 μg/mL. Mean recovery for ceftazidime was 94%.
Tissue samples— Tissue was weighed and placed in plastic homogenizer tubes with a stainless steel grinding ball. Ten microliters of cefotaxime (internal standard, 100 μg/mL) was added, followed by 100 μL of 0.1 N HCl and 500 μL of methanol. The tubes were capped and placed in a homogenizerq for 7 minutes (speed set to 6). The solution was transferred to a screw-top tube, another 500 μL of methanol was added to the tube used for homogenization, and the homogenization process was repeated. The solution was removed, added to the same screw-top tube as before, and mixed for 15 minutes, then centrifuged for 20 minutes at 1,060 × g. The supernatant was removed to a clean tube and evaporated with nitrogen. The residue was redissolved in 250 μL of mobile phase and loaded into a centrifugal filter.p Samples were centrifuged for 20 minutes at 16,060 × g, and the solution (approx 220 μL) was transferred into HPLC vials and 100 μL was injected into the chromatography system.
A partial validation of the tissue assay was performed because very little blank tissue was available from the control frogs. The same conditions as described for plasma samples were ensured. The calibration curve range was 0.1 to 100 μg/g. The lower limit of quantification during validation was 0.1 μg/g. Mean recovery for ceftazidime was 91%.
Pharmacokinetic analysis
Pharmacokinetic parameters for ceftazidime were calculated with specialized software.r Values for the elimination rate constant, plasma t1/2, Cmax, AUC from time 0 to the last measured concentration, AUC0–∞, percentage of the AUC0–∞ that was extrapolated, and mean residence time were calculated by means of noncompartmental analysis. The AUC was calculated with the log-linear trapezoidal rule. Variability in pharmacokinetic parameters was expressed as the SD. For plasma t1/2, harmonic mean and pseudo-SD were used.
Statistical analysis
Data on ceftazidime concentrations were analyzed with statistical software.s Normality of data distribution and equality of group variance were tested with the Shapiro-Wilk test and Brown-Forsythe test, respectively. For each type of sample (plasma, renal, or skin), comparison among treatment groups was performed with the paired t test if data were normally distributed and the Mann-Whitney rank sum test if not. Values of P < 0.05 were considered significant. Mean and SD values for ceftazidime concentration were calculated for each time point.
Results
Median time to onset of isoflurane anesthesia with lack of hind limb withdrawal was 43 minutes (range, 22 to 65 minutes) for the study frogs. Pharmacokinetic data for plasma ceftazidime concentrations were summarized (Table 1). Plasma, renal, and skin ceftazidime concentrations were tabulated on the basis of dose and route of administration (Table 2).
Mean ± SD values of plasma pharmacokinetic parameters for Northern leopard frogs (Lithobates pipiens) following administration of a single ceftazidime dose at 20 mg/kg, SC (n = 10); 40 mg/kg, SC (10); and 20 mg/kg, TC (10).
| Parameter | 20 mg/kg, SC | 40 mg/kg, SC | 20 mg/kg, TC |
|---|---|---|---|
| t1/2* (h) | 9.01 ± 0.27 | 14.49 ± 0.77 | NC |
| lz (1/h) | 0.080 ± 0.002 | 0.050 ± 0.003 | NC |
| tmax (h) | 12.0 ± 0.0 | 18.0 ± 8.5 | 36.0 ± 16.9 |
| Cmax (^g/mL) | 92.9 ± 6.4 | 96.0 ±, 7.2 | 1.3 ± 0.1 |
| AUC0–∞ (h·μ/mL) | 1,455 ± 136 | 1,993 ± 315 | NC |
| AUC0-last (h·μ/mL) | 1,453 ± 136 | 1,902 ± 387 | 38 ± 10 |
| AUCextrap (%) | 0.16 ± 0.0003 | 4.94 ± 4.36 | NC |
| MRT (h) | 16.9 ± 0.4 | 24.3 ± 1.6 | NC |
Harmonic mean reported.
lz = Elimination rate constant. AUCextrap = Percentage of the AUC0–∞ that was extrapolated. AUC0-last = AUC from time 0 to the last measured concentration. MRT = Mean residence time. NC = Not calculated owing to insufficient data.
Mean ± SD ceftazidime concentrations in plasma (mL/mL) and tissue (mg/g) samples at various time points (n = 2 frogs/time point) for the 3 groups in Table 1.
| Group | 12 hours | 24 hours | 48 hours | 72 hours | 96 hours |
|---|---|---|---|---|---|
| 20 mg/kg, SC | |||||
| Plasma | 92.9 ± 3.2 | 12.4 ± 1.1 | 4.5 ± 0.5 | 0.2 ± 0.0 | 0.2 ± 0.0 |
| Kidney | 30.1 ± 1.1 | 22.7 ± 0.6 | 14.4 ± 0.5 | 2.1 ± 1.1 | 5.8 ± 0.7 |
| Skin | 11.0 ± 1.0 | 2.6 ± 0.4 | 2.0 ± 1.0 | 0.5 ± 0.1 | 0.5 ± 0.3 |
| 40 mg/kg, SC | |||||
| Plasma | 76.4 ± 22.5 | 48.4 ± 25.1 | 1.2 ± 0.5 | 4.8 ± 1.6 | 0.9 ± 0.7 |
| Kidney | 28.2 ± 0.9 | 38.6 ± 6.2 | 39.6 ± 27.6 | 32.4 ± 0.7 | 40.3 ± 24.7 |
| Skin | 14.3 ± 5.6 | 9.1 ± 2.5 | 2.5 ± 0.1 | 1.3 ± 0.1 | 1.2 ± 0.9 |
| 20 mg/kg, TC | |||||
| Plasma | 0.4 ± 0.2 | 0.7 ± 0.4 | 0.8 ± 0.4 | 0.2 ± 0.0 | 0.0 ± 0.0 |
| Kidney | 0.4 ± 0.0 | 2.2 ± 1.5 | 1.6 ± 0.9 | 3.5 ± 2.4 | 0.3 ± 0.2 |
| Skin | 2.7 ± 1.4 | 5.8 ± 3.6 | 6.8 ± 1.0 | 0.8 ± 0.2 | 1.2 ± 0.9 |
A significant difference in ceftazidime concentrations was identified between groups SC20 and TC20 for plasma samples (P = 0.045) and between groups SC20 and SC40 (P = 0.03) and groups SC20 and TC20 (P = 0.002) for renal samples. Plasma ceftazidime concentration in the SC20 and SC40 groups exceeded a previously reported9 MIC for a Pseudomonas isolate recovered from loggerhead sea turtles (Caretta caretta; ie, 8 μg/mL) for 24 hours but was detectable for up to 96 hours (Figure 1). Transcutaneous ceftazidime administration at 20 mg/kg yielded no plasma ceftazidime concentrations above this MIC at any time point. Renal and skin ceftazidime concentrations were detectable at all time points for both doses and routes of administration; however, skin concentrations were significantly lower than both plasma and renal concentrations for both doses and both routes of administration.
Mean ± SD ceftazidime concentrations in Northern leopard frogs (Lithobates pipiens) following administration of a single ceftazidime dose at 20 mg/ kg, SC (n = 10; circles); 40 mg/kg, SC (10; squares); and 20 mg/kg, TC (10; triangles). The dashed horizontal line indicates the effective serum ceftazidime concentration against Pseudomonas spp in loggerhead sea turtles9 (Caretta caretta) and small mammals14 (8 μg/mL).
Citation: American Journal of Veterinary Research 82, 7; 10.2460/ajvr.82.7.560
Mean ± SD ceftazidime concentrations in Northern leopard frogs (Lithobates pipiens) following administration of a single ceftazidime dose at 20 mg/ kg, SC (n = 10; circles); 40 mg/kg, SC (10; squares); and 20 mg/kg, TC (10; triangles). The dashed horizontal line indicates the effective serum ceftazidime concentration against Pseudomonas spp in loggerhead sea turtles9 (Caretta caretta) and small mammals14 (8 μg/mL).
Citation: American Journal of Veterinary Research 82, 7; 10.2460/ajvr.82.7.560
Mean ± SD ceftazidime concentrations in Northern leopard frogs (Lithobates pipiens) following administration of a single ceftazidime dose at 20 mg/ kg, SC (n = 10; circles); 40 mg/kg, SC (10; squares); and 20 mg/kg, TC (10; triangles). The dashed horizontal line indicates the effective serum ceftazidime concentration against Pseudomonas spp in loggerhead sea turtles9 (Caretta caretta) and small mammals14 (8 μg/mL).
Citation: American Journal of Veterinary Research 82, 7; 10.2460/ajvr.82.7.560
Discussion
One goal of the present study was to determine whether SC and TC ceftazidime administration at 2 different doses would result in plasma concentrations exceeding 8 μg/mL, which represents the MIC of ceftazidime for a loggerhead sea turtle Pseudomonas isolate9 and Pseudomonas aeruginosa in dogs and cats.14,15 Another goal was to investigate whether both the SC and TC routes of administration would result in measurable skin and renal ceftazidime concentrations. Pseudomonas spp was chosen as the organism of interest with regard to MIC because this genus is one of the most commonly identified bacteria on amphibian skin.2,10 Ceftazidime appeared to have a particular affinity for renal tissue in the present study, specifically when administered SC at 40 mg/kg, as shown by concentrations that remained high for the entire 96-hour follow-up period. Similarly, the highest concentration of ceftazidime in dogs, rabbits, rats, and monkeys has been identified in renal tissue versus plasma or other tissues.16
Our study revealed a surprising lack of plasma or tissue concentration efficacy following TC ceftazidime administration, which was in contrast to our hypothesis regarding this route of administration. Although ceftazidime had not previously been evaluated in amphibians, topical administration has been shown to be clinically effective for other antimicrobials.17–19 Percutaneous drug absorption in amphibians is affected by several factors, including the natural thickness (which differs by species), integrity, and vascularization of the skin; duration of drug application; and molecular components of the drug.3 Frog skin is more permeable than human and mammalian skin; however, ceftazidime in the injectable form may not have the appropriate molecular density, lipophilicity, or ionization to be readily absorbed through the dermis.3 Length of cutaneous exposure to a medication can affect systemic absorption as well, as shown with long-term bath exposure of Panamanian golden frogs (Atelopus zeteki) to itraconazole.20 On the basis of our findings for Northern leopard frogs, we do not recommend TC administration of injectable ceftazidime in frogs because it did not result in theoretically therapeutic plasma concentrations at any time point.
The MIC of ceftazidime is dependent on the targeted bacterial isolate. Reported MICs include 4 to 8 μg/mL for Pseudomonas spp in loggerhead sea turtles9; 1 μg/mL for Aeromonas spp, Klebsiella spp, and Pseudomonas spp in snakes21,22; and between 0.06 and 128 μg/mL for Escherichia coli, P aeruginosa, and Staphylococcus spp in dogs and cats.14,15 Ceftazidime is frequently prescribed for reptiles and amphibians with bacterial infections because of the infrequent dosing required, broad-spectrum coverage, and enhanced activity against gram-negative microbes.1 In contrast, a retrospective study22 of antimicrobial susceptibility patterns for aerobic bacterial isolates from reptiles revealed that all gram-positive isolates were resistant to ceftazidime, although gram-negative isolates were still susceptible. The most common bacterial isolates in that study22 were gram-negative organisms and included Pseudomonas spp, Morganella spp, and Escherichia spp. All of the isolates specifically evaluated for ceftazidime susceptibility (Pseudomonas spp, Morganella spp, and Escherichia spp) were found to be susceptible. Those results highlighted the need for bacterial culture and antimicrobial susceptibility testing when treating bacterial infections.
The plasma pharmacokinetics of ceftazidime have been reported for a single snake and several chelonian species, and all related studies7–9,22 involved a dose of 20 mg/kg. Plasma concentrations of ceftazidime administered at 20 mg/kg, IM in clinically ill snakes of various species resulted in therapeutic plasma concentrations for 120 hours.22 In loggerhead sea turtles, a single ceftazidime injection at 20 mg/ kg, IM or IV resulted in plasma concentrations > 8 μg/mL for 60 hours for both administration routes. Similarly, a single 22-mg/kg, IM injection of ceftazidime in Kemp's ridley sea turtles (Lepidochelys kempii) resulted in therapeutic plasma concentrations for at least 72 hours.7 In contrast, our study in leopard frogs resulted in potentially therapeutic plasma concentrations for only 24 hours at both SC doses, indicating the need for more frequent administration of ceftazidime in this species. Although a significant difference in plasma ceftazidime concentration was found between the 20- and 40-mg/kg doses, that difference was unlikely to be clinically relevant given that neither dose resulted in potentially therapeutic concentrations lasting > 24 hours.
Limitations of the study reported here included the inability to perform crossover trials, the small number of frogs used for each time point, and the need to extrapolate doses and MIC data from reptile and small mammal studies. The tissue ceftazidime concentrations reported here should be interpreted cautiously given that human pharmacokinetic study guidelines suggest that tissue drug concentrations should not be used in evaluations of pharmacokinetic-pharmacodynamic parameters because all such parameters are based on unbound plasma concentrations.23 We are aware of no previous amphibian pharmacokinetic studies of tissue drug dispersion, so it remains unclear whether the same principle would apply to other species, such as frogs. Nevertheless, we believe our preliminary findings might inspire and inform future amphibian antimicrobial studies. Although a ceftazidime MIC of 8 μg/mL has been used in reptile ceftazidime pharmacokinetics research9 and is the clinical breakpoint regularly applied for Pseudomonas spp,10,14,22 such an MIC has not been established in frogs. We therefore recommend bacterial culture and antimicrobial susceptibility testing before starting ceftazidime treatment in any amphibian species to ensure appropriate antimicrobial selection.
Overall, results of the present study suggested that SC administration of ceftazidime to leopard frogs at 20 mg/kg will result in effective plasma concentrations for 24 hours. Although TC antimicrobial administration has been previously shown to result in therapeutic plasma concentrations in amphibians, plasma ceftazidime concentrations were minimal following TC administration and this method as described is not recommended.
Abbreviations
| AUC | Area under the concentration-versus-time curve |
| AUC0–∞ | Area under the concentration-versus-time curve from time 0 to infinity |
| Cmax | Maximum plasma concentration |
| HPLC | High-performance liquid chromatography |
| MIC | Minimum inhibitory concentration |
| t1/2 | Half-life |
| TC | Transcutaneous |
| tmax | Time to maximum plasma concentration |
Footnotes
Niles Biological Inc, Sacramento, Calif.
WyPall wipes, Kimberly-Clark Worldwide Inc, Irving, Tex.
Reptile ramp bowl, Zoo Med Laboratories Inc, San Luis Obispo, Calif.
Fluker's, Port Allen, La.
Orange Cube complete cricket diet, Fluker's, Port Allen, La.
Repta calcium with vitamin D3 and phosphorous free, Fluker's, Port Allen, La.
Panacur granules 22.2% compounded to 100 mg/mL, Merck Animal Health USA, Madison, NJ.
Praziquantel 56.8 mg/mL, Bimeda Inc, Le Sueur, Minn.
Research Randomizer, version 4.0, Urbaniak GC, Plous S, Middletown, Conn. Available at: www.randomizer.org. Accessed Dec 9, 2019.
Ceftazidime 1-g vial, WG Critical Care LLC, Paramus, NJ.
U-100 insulin syringe, Becton, Dickinson and Co, Franklin Lakes, NJ.
BD microtainer blood collection tubes, Becton, Dickinson and Co, Franklin Lakes, NJ.
2695 HPLC system with 2487 dual absorbance detector, Waters Corp, Milford, Mass.
Empower chromatography data system, Waters Corp, Milford, Mass.
Symmetry Shield C18 column, Waters Corp, Milford, Mass.
Millipore Amicon Ultra 0.5mL Ultracel 30k filters, Millipore-Sigma, Burlington, Mass.
Powergen high throughput homogenizer, Thermo Fisher Scientific, Waltham, Mass.
Phoenix 64 WinNonlin, version 8.1, Certara Co, Princeton, NJ.
SigmaPlot 13, Systat Software, San Jose, Calif.
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