In Vitro Activities of Tulathromycin and Ceftiofur Combined with Other Antimicrobial Agents Using Bovine Pasteurella multocida and Mannheimia haemolytica Isolates* Michael T. Sweeney, MS Gordon W. Brumbaugh, DVM, PhD Jeffrey L. Watts, PhD VMRD Discovery and Laboratory Sciences Pfizer Animal Health 333 Portage Street Kalamazoo, MI 49007 CLI NI CA L RE L E VA N C E The purpose of this study was to determine the activities of two antibacterial agents used in the treatment of bovine respiratory infections tulathromycin, a macrolide, and ceftiofur, a third-generation cephalosporin alone, in combination with each other, and in combination with each of seven additional antibiotics (tilmicosin, florfenicol, enrofloxacin, danofloxacin, ampicillin, tetracycline, and penicillin G) against bovine Pasteurella multocida (n = 60) and Mannheimia haemolytica (n = 10) isolates for determination of synergy, antagonism, or indifference. Of 458 organism drug combinations, 160 combinations of tulathromycin and 209 combinations of ceftiofur with eight antimicrobial drugs were indifferent. One combination was antagonistic (ceftiofur + florfenicol against one isolate of P. multocida). Time kill studies showed loss of cidality for ceftiofur when combined with florfenicol at 1 the minimal inhibitory concentration. Overall, the in vitro data demonstrated that tulathromycin and ceftiofur, in combination with each other or seven other antimicrobial agents, primarily produce an indifferent response with no occurrences of synergism and rare occurrences of antagonism. INTRODUCTION Combination antimicrobial therapy may provide a complementary spectrum that increases the likelihood of activity against multiple pathogens in potentially mixed infections, which in turn may allow for reduced dosages of *This study was funded by Pfizer Animal Health, Kalamazoo, Michigan. one or both agents and decrease the probability of induced resistance in the pathogens. 1 Antimicrobial combinations are considered synergistic if the effect of the combination is greater than that of either agent alone or greater than the sum of the effects of the individual agents, an- 1
M. T. Sweeney, G. W. Brumbaugh, and J. L. Watts tagonistic if the combination provides an effect less than that of either agent alone or less than the sum of the effects of the individual agents, and indifferent if the combination provides an effect equal to that of either agent alone. A common in vitro laboratory method to determine whether antimicrobial combinations are synergistic, antagonistic, or indifferent uses fractional inhibitory concentrations (FICs), and the checkerboard technique has been one of the traditional methods to determine FIC indices for synergism, antagonism, or indifference. 1, Examples of clinically useful synergistic antimicrobial drug combinations in veterinary medicine include penicillin streptomycin and ampicillin clavulanic acid for bovine Staphylococcus aureus mastitis, erythromycin rifampin for Rhodococcus equi pneumonia in foals, minocycline streptomycin for Brucella canis infections in dogs, and trimethoprim sulfamethoxazole for coliform meningitis. 3 Concurrent use of more than one antimicrobial agent may prove beneficial in the treatment of some diseases, but evidence from controlled clinical investigations to support such use in treating bovine respiratory disease (BRD) is lacking. 4 6 Currently, there is no guidance for clinical veterinarians in the field regarding the administration of antibiotic combinations or sequential antimicrobial therapy in the treatment of BRD, especially for longer-acting drugs, even though the latter is used in animals that do not respond successfully to the first course of treatment. With sequential therapy, subsequent medications may be administered before the previous compound has been completely eliminated by the animal. Thus, it is important to know whether any residual drug from the previous dosage regimen will have a positive synergistic effect, a negative antagonistic effect, or no effect on a subsequent medication. Tulathromycin is a macrolide antimicrobial agent characterized by a 15-membered ring with three amine groups and therefore belongs to the subclass of macrolides termed triamilides. This agent is currently approved for the treatment of BRD associated with Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, and Mycoplasma bovis and for the control of respiratory disease in cattle at high risk of developing BRD associated with M. haemolytica, P. multocida, and H. somni. Ceftiofur is a broad-spectrum third-generation cephalosporin currently approved for treatment of BRD associated with M. haemolytica, P. multocida, and H. somni and for the control of respiratory disease in cattle at high risk of developing BRD associated with the same organisms. Tulathromycin and ceftiofur were tested in vitro alone, in combination with each other, and in combination with each of seven additional antibiotics using checkerboard broth microdilution plates prepared by a semiautomated method. The objective of this study was to determine the interactions of tulathromycin and ceftiofur with each other and with other antimicrobial agents by generating and analyzing FIC indices for synergism, antagonism, or indifference using isolates of P. multocida and M. haemolytica from animals with BRD. Combinations that showed an effect other than indifference were further evaluated by time kill and sequential drug-exposure studies. MATERIALS AND METHODS Bacterial Strains The bacterial isolates used in this study were from the Pfizer Animal Health surveillance program and were isolated from clinical cases of BRD. All isolates were identified to the species level by clinical sites using biochemical methods. The isolates were frozen in trypticase soy broth containing 10% glycerol and maintained at 70 C as part of the Pfizer Animal Health Development and Research culture collection. Isolates were plated and grown on trypticase soy 13
Log 10 CFU/ml 10 8 6 4 2 Results of Time Kill Assay to Further Assess Antagonism of Ceftiofur + Florfenicol Control Flor 1 Cef 1 Flor 1 + Cef 1 Flor 0.5 + Cef 1 Flor 0.25 + Cef 1 Flor 0.125 + Cef 1 0 0 4 6 24 Hours Figure 1. Time kill curves for P. multocida 30105 exposed to florfenicol (Flor) alone, ceftiofur (Cef) alone, and florfenicol at 1, 0.5, 0.25, and 0.125 MIC combined with ceftiofur at 1 MIC. Time 0 is the point at which cells were exposed to drug. The starting cell concentration was approximately 1 10 6. agar plates containing 5% sheep blood. The quality-control strain S. aureus ATCC 913 was originally acquired from the American Type Culture Collection (Manassas, VA). Antimicrobial Agents The following antibiotic powders were used in the experiments: amoxicillin, potassium clavulanate, florfenicol, tetracycline, tilmicosin, penicillin G, sparfloxacin, and chloramphenicol (Sigma Chemical, St. Louis, MO); ampicillin (Gibco, Grand Island, NY); danofloxacin, enrofloxacin, and ceftiofur (Riedel-de Haen, Seelze, Germany); and tulathromycin (Pfizer Animal Health). Susceptibility and Fractional Inhibitory Concentration Testing Mueller Hinton broth (MHB; Difco Laboratories, Sparks, MD) at ph 7.3 was used for susceptibility testing of all isolates. The minimal inhibitory concentration (MIC) of each drug was determined by broth microdilution according to methods described by the Clinical and Laboratory Standards Institute (CLSI; formerly National Committee for Clinical Laboratory Standards). 7 Robotics were used to create and inoculate microdilution checkerboard plates. MICs were determined for each drug against each strain, and drug concentrations used for combination testing ranged from below to above the MIC. For each drug combination tested, a 96-well deep-well plate (Beckman Coulter, Fullerton, CA) was filled with MHB containing 1% alamarblue (Trek Diagnostic Systems, Cleveland, OH) by the Multidrop instrument (Labsystems, Helsinki, Finland). According to the package insert, alamarblue is a colorimetric redox indicator used to aid visual reading of checkerboard plates; it had no effect on organism growth. 8 The MIC for each isolate against each drug was defined as the well that had the lowest concentration of drug that produced no organism growth (i.e., the blue well). A test drug was diluted twofold from column 1 to column 7 of each deep-well mother plate using the Biomek 000 instrument (Beckman Coulter). Column 8 contained no test drug. Twofold dilutions of the second drug were then added to rows 1 to 7 of each deep-well plate. Row 8 did not contain the second drug. A total of 50 µl was transferred by the Multimek 96 instrument (Beckman Coulter) from each well of each mother plate to a daughter plate. The Biomek instrument was used to inoculate daughter plates with 50 µl of a 1:100-diluted culture 14
M. T. Sweeney, G. W. Brumbaugh, and J. L. Watts equal to a 0.5 McFarland standard (approximately 1 10 8 CFU/ml) from bacterial growth on 18- to 4-hour-old blood agar plates for a final organism concentration of approximately 5 10 5 CFU/ml. The daughter plates were incubated at 35 C for 18 ± hours. Plates were read visually by observing a reduction (a color change from blue to purple or pink, indicating organism growth) or no reduction (a blue color, indicating no growth of organism as a result of inhibition by drugs). The MIC of each drug could be determined; for wells along the growth no growth interface, FICs were determined using the following formula: MIC of Drug 1 in Combination + MIC of Drug 1 Alone MIC of Drug in Combination MIC of Drug Alone Assay reproducibility for synergism was monitored using a combination of amoxicillin and clavulanic acid with S. aureus ATCC 913. 1 Assay reproducibility for antagonism was monitored using a combination of sparfloxacin and chloramphenicol with S. aureus ATCC 913. 9 Time Kill Assay Isolates were streaked on a 5% sheep blood agar plate and incubated at 35 C for 4 hours. Colonies were selected and placed in MHB to equal a 0.5 McFarland standard (approximately 1 10 8 CFU/ml) and diluted 1:10 in fresh MHB. Diluted cell suspension (0.5 ml) was added to 4.5 ml MHB for a final volume in each tube of 5 ml containing approximately 1 10 6 CFU/ml cells exposed to ceftiofur alone at 1 MIC, florfenicol alone at 1 MIC, and ceftiofur (1 MIC) plus florfenicol at 1, 0.5, 0.5, and 0.15 MIC. A tube containing MHB without drugs and with diluted culture It is important to know whether any residual drug from a previous dosage regimen will have a positive synergistic effect, a negative antagonistic effect, or no effect on a subsequent medication. The average FIC index was calculated from individual FICs by the formula (FIC 1 + FIC + FIC n )/n, where n is the total number of individual wells per plate for which FICs were calculated. Synergism was defined as an average FIC index 0.5, antagonism as an average FIC index >4.0, and indifference as an average FIC index of 0.51 to 4.0. 1,8 Ten strains each of P. multocida and M. haemolytica from BRD infections were used for each drug combination test. If synergism or antagonism was observed for any drug combination, an additional 50 isolates of P. multocida or M. haemolytica were tested to further determine the extent of the synergistic or antagonistic combination. was used as a growth control. A 0-hour time point measurement was obtained by removing an aliquot of broth from each tube, diluting in sterile saline, plating on 5% sheep blood agar plates, and incubating at 35 C for 4 hours. Tubes were then stored in a 35 C incubator, and aliquots were collected at 4, 6, and 4 hours; at each collection time, an aliquot of broth from each tube was diluted in sterile saline, plated on 5% sheep blood agar plates, and incubated at 35 C for 4 hours. CFUs grown on plates were recorded and graphed 15
Log 10 CFU/ml 10 8 6 4 2 Time Kill Curves for Cells Exposed to Ceftiofur at 1 MIC for 24 Hr and Then Florfenicol Flor 0 Flor 0.5 Flor 1 Flor 2 Flor 20 (Figure 1), and the limit of detection was defined as log 10 CFU/ml. For analyzing kill curves, synergy was defined as a 3 log 10 decrease in bacterial CFU/ml between the drug combination and the most active single drug after 4 hours. Antagonism was defined as a 3 log 10 increase in bacterial CFU/ml between the drug combination and the most active single drug after 4 hours. 0 0 1 2 3 6 Days Figure 2. Time kill curves for P. multocida 30105 exposed to ceftiofur at 1 MIC for 24 hours followed by florfenicol (Flor) exposure at 0, 0.5 1, 2, and 20 MIC for 6 days. Time 0 is 24 hours after treatment. Log 10 CFU/ml 10 8 6 4 2 0 Time Kill Curves for Cells Exposed to Ceftiofur at 10 MIC for 24 Hr and Then Florfenicol Flor 0 Flor 0.5 Flor 1 Flor 2 Flor 20 0 1 2 3 6 Days Figure 3. Time kill curves for P. multocida 30105 exposed to ceftiofur at 10 MIC for 24 hours followed by florfenicol (Flor) exposure at 0, 0.5, 1, 2, and 20 MIC for 6 days. Time 0 is 24 hours after treatment. Drug-Exposure Assays Isolates were streaked on a 5% sheep blood agar plate and incubated at 35 C for 4 hours. Colonies were selected and placed in MHB to equal a 0.5 McFarland standard (approximately 1 10 8 CFU/ml). A final concentration of approximately 1 10 7 CFU/ml cells was exposed to 1 MIC ceftiofur (0.00 µg/ml), 10 MIC ceftiofur (0. µg/ml), 1 MIC florfenicol (4 µg/ml), and 10 MIC florfenicol (40 µg/ml) for 4 hours at 35 C. Cells were then placed in centrifuge tubes and centrifuged at 5,000 rpm for 10 minutes. The supernatant was poured off, and MHB was added to the tubes, which were then vortexed and spun again at 5,000 rpm for 10 minutes to remove and wash away antibiotic. Cells were then resuspended in fresh MHB and vortexed. Ceftiofur-exposed cells were added to tubes containing florfenicol at 0, 0.5, 1,, and 0 MIC (Figures and 3), 16
M. T. Sweeney, G. W. Brumbaugh, and J. L. Watts TABLE 1. MICs of the Antibacterials for Organisms Used in This Study MIC (µg/ml) P. multocida (n = 10) M. haemolytica (n = 10) DRUG Range MIC 50 MIC 90 Range MIC 50 MIC 90 Tulathromycin 1 3 4 16 4 4 Ceftiofur 0.001875 0.03 0.00375 0.03 0.00375 0.015 0.00375 0.0075 Tilmicosin 3 16 1 3 8 Florfenicol 4 4 4 3 4 8 Danofloxacin 0.00375 0.15 0.015 0.15 0.015 0.15 0.03 0.15 Enrofloxacin 0.00375 0.15 0.015 0.06 0.007 0.15 0.015 0.15 Ampicillin 0.03 0.15 0.06 0.15 0.06 0.15 0.06 0.15 Tetracycline 0.5 3 0.5 3 0.5 3 4 3 Penicillin G 0.03 0.5 0.03 0.5 0.03 0.5 0.5 0.5 and florfenicol-exposed cells were added to tubes containing ceftiofur at 0, 0.5, 1,, and 0 MIC. A 0-hour time point measurement was obtained by removing an aliquot of broth from each tube, diluting in sterile saline, plating on 5% sheep blood agar plates, and incubating at 35 C for 4 hours. Tubes were stored in a 35 C incubator, and aliquots were collected at 1,, 3, and 6 days; at each collection time, an aliquot of broth from each tube was diluted in sterile saline, plated on 5% sheep blood agar plates, and incubated at 35 C for 4 hours. CFUs grown on plates were recorded and graphed, and the limit of detection was defined as log 10 CFU/ml. After plotting the number of recovered CFUs at each time point, a 3 log 10 decrease in bacterial CFU/ml between the drug and control curves on each day was determined. RESULTS Susceptibility Results MIC ranges and the MIC at which 50% and 90% of isolates are inhibited (MIC 50 and MIC 90, respectively) were determined for this study, and susceptibilities were determined based on veterinary-specific or CLSI-approved human interpretive criteria 7 (Table 1). The MIC 90 for tulathromycin was 4 µg/ml for both P. multocida and M. haemolytica. The MIC 90 for ceftiofur was <0.06 µg/ml for both organisms. Fractional Inhibitory Concentrations The combined activities of tulathromycin, ceftiofur, and other antimicrobial agents from the in vitro checkerboard interactions against bovine isolates of P. multocida and M. haemolytica are summarized in Tables through 4. Average minimum and maximum FICs (average Σ FIC index range) and interpretations for the activities of tulathromycin in combination with eight of eight antimicrobials showed indifference for 160 organism drug combinations; synergism or antagonism was not observed (Table ). Minimum and maximum FICs and interpretations for the activities of ceftiofur in combination with seven of eight antimicrobials showed indifference for 09 organism drug combinations; synergism was not observed. 17
TABLE 2. Results of Checkerboard Testing of the Activities of Tulathromycin Drug Combinations against Organisms Used in This Study* Average Σ FIC Index Range Drug Used in Combination P. multocida M. haemolytica with Tulathromycin (n = 10) (n = 10) Ceftiofur 1.07 1.37 0.67 1.9 Tilmicosin 0.53 1.15 0.76 1.54 Florfenicol 0.58 1.10 0.73 1.33 Danofloxacin 0.99 1.10 0.95 1.33 Enrofloxacin 0.98 1.33 0.99 1.94 Ampicillin 1.9 1.9 1.9 Tetracycline 0.55 1.9 0.57 1.9 Penicillin G 0.71 1.98 1.9 1.34 *Results for fractional inhibitory concentration (FIC) indices for 160 drug isolate combinations tested in this study. FIC indices of 0.5 are synergistic, FIC indices of >0.5 4.0 are indifferent, and FIC indices >4.0 are antagonistic. TABLE 3. Results of Checkerboard Testing of the Activities of Ceftiofur Drug Combinations against Organisms Used in This Study* Average Σ FIC Index Range Drug Used in Combination P. multocida M. haemolytica with Ceftiofur (n = 10) (n = 10) Tulathromycin 1.07 1.37 0.67 1.9 Tilmicosin 0.84.37 0.94 1.71 Florfenicol 0.98 6.91 0.99 1.38 Danofloxacin 0.99 1.31 0.78 1.1 Enrofloxacin 0.76 1.33 0.99 1.7 Ampicillin 1.13 1.79 0.71 1.9 Tetracycline 0.71.16 0.73 1.9 Penicillin G 0.76 1.1 0.98 1.94 *Results for fractional inhibitory concentration (FIC) indices for 10 drug isolate combinations tested in this study. FIC indices of 0.5 are synergistic, FIC indices of >0.5 4.0 are indifferent, and FIC indices >4.0 are antagonistic. The total number of isolates tested with the ceftiofur + florfenicol combination was 60. This combination resulted in antagonism for 1 strain of the 60 tested. Ceftiofur combined with florfenicol was antagonistic with one isolate of P. multocida (FIC = 6.91; Table 3). Additional drug combinations tested included tilmicosin with enrofloxacin, tilmicosin with florfenicol, florfenicol with enrofloxacin, and tetracycline with penicillin G. For these 80 organism drug combinations, all showed indifference (Table 4). Inherent assay variability appeared to be kept at a minimum, as determined from the results for the control plates for synergism (100% synergism with amoxicillin clavulanic acid plates using S. aureus ATCC 913 in four separate runs) and the control plates for antagonism (100% antagonism with sparfloxacin chloramphenicol plates using S. aureus ATCC 913 in four separate runs; Table 5). These control plates, which were made during each drug combination experiment, were included to ensure the accuracy of the assay. Time Kill Assay A single isolate of P. multicoda that exhibited antagonism when tested with ceftiofur + florfenicol in the checkerboard assay (FIC = 6.91) was retested in the checkerboard assay to confirm antagonism and then further tested in a time kill assay (Figure 1). Growth curves for this isolate at the organism s MIC was bactericidal with ceftiofur (MIC = 0.00 µg/ml) and bacteriostatic with florfenicol (MIC = 4 µg/ml; Figure 1). Cidality of ceftiofur at 1 MIC was suppressed when combined with florfenicol at 1 MIC. As the concentration of florfenicol was lowered to 0.15 MIC, cidality of ceftiofur at 1 MIC was restored at 6 and 4 hours. 18
M. T. Sweeney, G. W. Brumbaugh, and J. L. Watts Drug-Exposure Assay This single isolate was further tested in an in vitro drug-exposure assay to represent a treatment setting in which an animal is given one antibiotic followed by another antibiotic rather than given both agents concurrently. Cells at approximately 1 10 7 CFU/ml were exposed to ceftiofur at 1 and 10 MIC for 4 hours before being exposed to florfenicol for 6 days. Because ceftiofur is a cidal drug, this higher cell concentration was chosen to ensure recovery of cells after 4 hours of pre-exposure to ceftiofur. Cells exposed to ceftiofur at 1 MIC for 4 hours, in which approximately 1 10 4 CFU/ml were recovered and then exposed to florfenicol, showed inhibition at 1 and higher florfenicol concentrations through day 6 and did not show resistance (Figure ). Cells exposed to ceftiofur at 10 MIC for 4 hours, in which approximately 1 10 3 CFU/ml were recovered and then exposed to florfenicol, showed inhibition at all florfenicol concentrations through day 6 and did not show resistance (Figure 3). Thus, exposure of cells first to ceftiofur for 4 hours and then to florfenicol for 6 days did not show evidence of any antagonistic effect. Cells exposed to florfenicol for 4 hours followed by ceftiofur for 6 days showed similar growth inhibition at all concentrations of ceftiofur (data not shown). DISCUSSION Tulathromycin is a relatively new antimicrobial agent belonging to the macrolide class and is primarily active against gram-negative pathogens associated with BRD. Ceftiofur is a broad-spectrum third-generation cephalosporin that is also active against gram-negative pathogens associated with BRD. Studies have found instances of improved efficacies of certain antibiotics when they are combined with antibiotics of other classes and used for the treatment of specific diseases. 3,5,6 None of those studies involved BRD. An extended literature search has found that to date there are no journal articles related to studies of the activities of tulathromycin or ceftiofur in combination with each other or with other drugs. Thus, it is important to know if the drug remaining from the previous dosage regimen will have a positive synergistic effect, a negative antagonistic effect, or no effect on the medication subsequently administered. These studies with tulathromycin and ceftiofur in combination with each other and with other antimicrobial agents were performed to determine whether synergism, antagonism, or indifference would be the primary response when the combinations were tested against isolates of bovine P. multocida and M. haemolytica. Combinations that showed an effect other than indifference were further evaluated by time kill and sequential drugexposure studies. The results from this study predominantly showed indifference when tulathromycin or ceftiofur was combined with each other or any of seven other antimicrobial agents and tested against isolates of bovine P. multocida and M. haemolytica. In the evaluation of the activities of 458 organism drug combinations, 449 combinations (98%) were indifferent. Of the four determinations of synergism (0.9%), all resulted from synergy controls using amoxicillin + clavulanic acid. Of the five determinations of antagonism (1.1%), four resulted from antagonism controls using sparfloxacin + chloramphenicol and one from ceftiofur + florfenicol. For the isolate exhibiting antagonism in the ceftiofur + florfenicol combination, the MIC of ceftiofur increased by three twofold dilutions (0.00 µg/ml to 0.016 µg/ml in the presence of 1 MIC florfenicol) and the florfenicol MIC (4 µg/ml) remained unchanged in any concentration of ceftiofur. A time kill study showed the antagonistic effect of florfenicol, which was measured to be bacteriostatic in this study, on 19
TABLE 4. Results of Checkerboard Testing of the Activities of Other Drug Combinations against Organisms Used in This Study* Average Σ FIC Index Range P. multocida M. haemolytica Drug Combination (n = 10) (n = 10) Tilmicosin Enrofloxacin 1.04 1.47 1.1 1.65 Tilmicosin Florfenicol 0.58 0.94 0.88 1.34 Florfenicol Enrofloxacin 1.34 1.78 1.3 1.36 Tetracycline Penicillin G 1.1 1.77 1.3 1.46 *Results for fractional inhibitory concentration (FIC) indices for 80 drug isolate combinations tested in this study. FIC indices of 0.5 are synergistic, FIC indices of >0.5 4.0 are indifferent, and FIC indices >4.0 are antagonistic. TABLE 5. Results of Quality-Control Checkerboard Testing for Synergism and Antagonism Using S. aureus ATCC 29213* Drug Combination Average Σ FIC Index Range Amoxicillin Clavulanic acid 0.34 0.38 Sparfloxacin Chloramphenicol 6.47 6.49 *Results for fractional inhibitory concentration (FIC) indices for eight drug isolate combinations tested in this study. FIC indices of 0.5 are synergistic, FIC indices of >0.5 4.0 are indifferent, and FIC indices >4.0 are antagonistic. Drug combination resulted in synergism for each of four tests. Drug combination resulted in antagonism for each of four tests. the cidality of ceftiofur (Figure 1). However, a sequential drug-exposure study in which the isolate was exposed to MIC and supra-mic levels of ceftiofur followed by exposure to florfenicol showed no antagonistic effects (Figures and 3). Because only one of 60 isolates of P. multocida displayed an antagonistic reaction with this combination, we feel that the result is a rare occurrence and would not be seen in a clinical setting. Differences in the results of checkerboard assays for the detection of in vitro interactions between antimicrobial agents have been demonstrated. Mackay et al 10 tested the same checkerboard on 3 separate days to test reproducibility and found no statistically significant differences; however, they did conclude that all combinations that showed synergism based on FIC also showed synergism by time kill assays. In this study, the one isolated result of antagonism was reproduced in the checkerboard format and also determined in time kill studies at 4 hours. Problems have been associated with the checkerboard microdilution method itself, such as variability in the inoculum as a potential source of error as well as the selection of inappropriate antibiotic concentrations. 11 The antimicrobial drugs chosen to be tested in combination with ceftiofur and tulathromycin in this study are commonly known to be used for the treatment of BRD-infected cattle in the clinical setting. 1,13 Robotic instruments used in the experiments described here and elsewhere 14 alleviated the laborious and time-consuming constraints associated with checkerboard assays. In our experiments, the results for each synergistic or antagonistic combination were successfully reproduced upon retesting, whereas combinations that showed indifference were not further evaluated. Assay variability appeared minimal as determined by amoxicillin clavulanic acid synergy and sparfloxacin chloramphenicol antagonism control combinations (Table 5). There is no gold standard for testing antimicrobial drug combinations. Numerous definitions have been assigned for synergism, antagonism, and indifference. Cappelletty and Rybak 15 defined synergy as having an FIC index 0.5, whereas antagonism has been defined as an FIC index >1, 1, >, or >4. From these different values, a result may be defined as indifferent or antagonistic depending on the 0
M. T. Sweeney, G. W. Brumbaugh, and J. L. Watts FIC index chosen. In addition, conclusions from drug combination studies that define interactions as indifferent or showing partial synergy based on FIC indices just above or below the synergy cutoff of 0.5 that seem to put a positive aspect on findings are being urged to be reported as no interaction between agents. 16,17 In our experiments, synergism was defined as an FIC index 0.5 and antagonism as an FIC index >4.0. On the basis of information available in the literature and recommended FIC interpretations, the FIC interpretations applied in this study adequately categorized the tulathromycin and ceftiofur drug interactions. 1,18 Even with the variabilities known to be inherently associated with drug combination studies, many studies nonetheless indicate that certain combinations of antibiotics are more useful than others against specific organisms.,19 CONCLUSION The combination of tulathromycin or ceftiofur with a particular antibiotic used in treating cattle with BRD infections caused by P. multocida or M. haemolytica predominantly results in indifference and rarely results in antagonism. These results should not be interpreted as supporting the concurrent administration of antimicrobial medication for treatment of BRD. However, these results do indicate that antimicrobials administered sequentially after tulathromycin or ceftiofur should not be adversely affected by concentrations remaining in the animal from the previous regimen. In vivo animal studies are needed to establish clinical correlation with these in vitro results. REFERENCES 1. Eliopoulos GM, Moellering RC Jr. Laboratory methods used to assess the activity of antimicrobial combinations. In: Lorian V, ed. Antibiotics in Laboratory Medicine. 3rd ed. Baltimore: Williams & Wilkins; 1991:43-49.. Washington JA II. Susceptibility tests. In: Kagan BM, ed. Antimicrobial Therapy. 3rd ed. Philadelphia: WB Saunders; 1980:11-19. 3. Prescott JF, Walker RD. Principles of antimicrobial drug selection and use. In: Prescott JF, Baggot JD, Walker RD, eds. Antimicrobial Therapy in Laboratory Medicine. 3rd ed. Ames, IA: Blackwell Publishing; 000:88-104. 4. Ames TR, Baker JC, Wikse SE. The bronchopneumonias (respiratory disease complex of cattle, sheep, and goats). In: Smith BP, ed. Large Animal Internal Medicine. 3rd ed. St. Louis: Mosby; 00:551-570. 5. Brumbaugh GW, Langston VC. Principles of antimicrobial therapy. In: Smith BP, ed. Large Animal Internal Medicine. 3rd ed. St. Louis: Mosby; 00:1349-1360. 6. Chambers HF, Sande MA. Antimicrobial agents: general considerations. In: Hardman JG, Limbird LE, Molinoff PB, et al, eds. Goodman & Gilman s The Pharmacological Basis of Therapeutics. 9th ed. New York: The McGraw-Hill Companies; 1996:109-1056. 7. Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals; Approved Standard M31-A3. 3rd ed. Wayne, PA: Clinical and Laboratory Standards Institute; 008. 8. Baker CN, Banerjee SN, Tenover FC. Evaluation of alamar colorimetric MIC method for antimicrobial susceptibility testing of gram-negative bacteria. J Clin Microbiol 1994;3:161-167. 9. Neu HC. Synergy of fluoroquinolones with other antimicrobial agents. Rev Infect Dis 1989;11:S105- S1035. 10. Mackay ML, Milne K, Gold IM. Comparison of methods for assessing synergistic antibiotic interactions. Int J Antimicrob Agents 000;15:15-19. 11. Eliopoulos GM, Eliopoulos CT. Antibiotic combinations: should they be tested? Clin Microbiol Rev 1988; 1:139. 1. Evans NA. Tulathromycin: an overview of a new triamilide antimicrobial for livestock respiratory disease. Vet Ther 005;6():83-95. 13. Washburn K, Johnson R, Clarke CR, et al. Penetration of ceftiofur into sterile vs. Mannheimia haemolytica infected tissue chambers in beef calves after subcutaneous administration of ceftiofur crystalline free acid sterile suspension in the ear pinna. J Vet Pharmacol Ther 005;8:47-51. 14. Sweeney MT, Zurenko GE. In vitro activities of linezolid combined with other antimicrobial agents against staphylococci, enterococci, pneumococci, and selected gram-negative organisms. Antimicrob Agents 1
Chemother 003;47:190-1906. 15. Cappelletty DM, Rybak MJ. Comparison of methodologies for synergism testing of drug combinations against resistant strains of Pseudomonas aeruginosa. Antimicrob Agents Chemother 1996;40:677. 16. Johnson MD, MacDougall C, Ostrosky-Zeichner L, et al. Combination antifungal therapy. Antimicrob Agents Chemother 004;48:693-715. 17. Odds FC. Synergy, antagonism, and what the chequerboard puts between them. J Antimicrob Chemother 003;5:1. 18. American Society for Microbiology. Instructions to authors. Antimicrob Agents Chemother 007;51:1-. 19. Domaracki BE, Evans AM, Venezia RA. Vancomycin and oxacillin synergy for methicillin-resistant staphylococci. Antimicrob Agents Chemother 000;44:1394-1396. 0. Ednie LM, Credito ML, Khantipong M, et al. Synergic activity, for anaerobes, of trovafloxacin with clindamycin or metronidazole: chequerboard and timekill methods. J Antimicrob Chemother 000;45:633-638. 1. Eliopoulos GM, Wennersten CB. Antimicrobial activity of quinupristin-dalfopristin combined with other antibiotics against vancomycin-resistant enterococci. Antimicrob Agents Chemother 00;46:1319-134.. Kiffer CRV, Sampaio JLM, Sinto S, et al. In vitro synergy test of meropenem and sulbactam against clinical isolates of Acinetobacter baumannii. Diag Microbiol Infect Dis 005;5:317-3.