Bacterial strains from bovine and swine respiratory infections were used in the present study and came from the Zoetis surveillance program isolated from clinical cases of BRD and SRD. Bovine isolates used in the studies (P. multocida, M. haemolytica, H. somni) were part of the 2008 Zoetis surveillance program and swine isolates (P. multocida, A. pleuropneumoniae, S. suis) were part of the 2009 surveillance program. Additionally, mycoplasmas were recovered from tulathromycin clinical trials from 2008 to 2010. All isolates were identified by clinical diagnostic laboratories using biochemical methods. The isolates were frozen in trypticase soy broth containing 10% glycerol and maintained at -70°C as part of the Zoetis culture collection. Isolates were plated and grown on trypticase soy agar plates containing 5% sheep blood. The quality control strains Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922, Streptococcus pneumoniae ATCC 49619, and Mannheimia haemolytica ATCC 33396 were originally acquired from the American Type Culture Collection (Manassas, VA, USA).

The antibiotic powders used in the experiments were obtained from the indicated sources: danofloxacin, enrofloxacin (Riedel-de Haen); clinafloxacin, ceftiofur (Zoetis).

Cation-adjusted Müller-Hinton broth (MHB; Difco Laboratories, Detroit, MI, USA) was used for susceptibility testing of P. multocida and M. haemolytica. Cation-adjusted MHB supplemented with 2.5% lysed horse blood was used for susceptibility testing of S. suis. Veterinary fastidious medium (VFM) broth (TREK Diagnostic Systems) was used for susceptibility testing of A. pleuropneumoniae and H. somni. The minimum inhibitory concentration (MIC) values were determined using the broth microdilution protocol according to the standards of the [Clinical and Laboratory Standards Institute (CLSI), 2008]. The MIC was the lowest concentration of drug which prevented macroscopically visible growth under the conditions of the test. Assay control was monitored with ATCC quality control strains. MICs were done in duplicate on two separate days. When MICs for all compounds tested were within one doubling dilution, the original results were used for analysis and reporting. On those occasions where results differed by more than one doubling dilution, the isolate was tested a third time and those results were used for analysis unless the results were inconsistent with either of the previous two sets of MIC results. When an isolate tested differently all three times it was determined to be too inconsistent in testing to be included in the analysis. In those rare instances, a new bacterial isolate was selected for MIC testing. Determination of MICs against all mycoplasma isolates were conducted at Microbial Research, Inc. (MRI) in Fort Collins, CO, USA. There is no standardized procedure for the susceptibility testing of veterinary Mycoplasma listed in CLSI document M31-A3 (CLSI, 2008). MIC plates were prepared by MRI using Modified Hayflick’s broth with alamarBlue for isolates except M. hyopneumoniae which required Friis broth. Each well contained 50 μl of the appropriate diluted drug prepared at 2× the final concentration and each well was inoculated with 50 μl of each isolate. Plates were incubated aerobically at 36°C for 16–24 h (M. bovis and bovirhinis) and 2–14 days (M. hyopneumoniae). There are no published quality control ranges available for the testing of Mycoplasma species, however, M. hyopneumoniae ATCC 25934 was included as part of the test isolates. Positive growth control wells for each tested isolate were monitored and sterility checks of plates by sterile diluent inoculation of the negative control well was performed. MIC results were the lowest concentration of antimicrobial agent that completely inhibited growth of the organism as detected by color change of the broth when color change was noted in the positive control well.

The bactericidal effects of clinafloxacin and comparator fluoroquinolones against 10 bacterial isolates were assessed by a time-kill assay. For M. haemolytica and P. multocida, cultures were grown overnight at 35°C on Müller-Hinton agar and for H. somni and A. pleuropneumoniae cultures were grown on chocolate Müller-Hinton agar. Assay tubes were prepared to contain 8.5 ml of the appropriate medium (cation-adjusted MHB for M. haemolytica and P. multocida, and VFM for H. somni and A. pleuropneumoniae), 0.5 ml of the antibacterial agent, and 1.0 ml of the McFarland-equivalent culture in appropriate medium which resulted in a cell concentration of approximately 10⁷ CFU/ml for most cultures. The final drug concentrations tested were 1× and 4× the MIC for each test strain. An identical assay tube that contained broth and inoculum, but no antibacterial agent, served as the culture growth control. The assay tubes were incubated at 35°C and samples were removed for viable counts at 0, 2, 6, and 24 h for all cultures. A 100-μl sample was removed from the assay tube at each timepoint and serial dilutions (10^-1 to 10^-8) were prepared in appropriate media. A 100-μl volume of each dilution was applied to duplicate blood agar plates (BAPs), spread evenly over the plate surface, and incubated overnight at 35°C with or without CO₂. Colonies were manually counted and plates at a dilution yielding a count of 20–200 were utilized.

Methods for mutant prevention concentrations (MPCs) were determined following the method of Blondeau et al. (2001). Briefly, two bacterial strains, S. suis and P. multocida from SRD and BRD, respectively, were selected for testing and subcultured to multiple agar plates (five each) and incubated for 18–24 h under proper incubating conditions for the microorganisms. The contents of the inoculated agar plates were removed with a sterile swab and transferred to cation-adjusted MHB or cation-adjusted MHB containing 2.5% lysed horse blood (250 ml). The inoculated broth was incubated for 18–24 h under proper incubating conditions. Broth cultures were centrifuged and the pellet resuspended in a lower volume of fresh broth media. Once the bacterial density was adjusted, d10⁹ CFUs were inoculated to agar plates containing clinafloxacin, danofloxacin, or enrofloxacin at twofold concentrations ranging from 256 to 0.007 μg/ml and incubated under ambient conditions at 35°C. Cultures were read at 24 and 48 h and the lowest drug concentration that prevented growth was determined as the MPC. Based on parent MIC, an MPC/MIC ratio was determined for evaluation. Data for time-kill and MPC analyses were collected once and results were consistent with data from previous journal reports.

Female CD-1 mice were acquired from Charles River Labs (Portage, MI, USA) at a weight of 13–14 g, and acclimated for 3–6 days. Mice were randomized when delivered on-site and housed 10/large shoebox cage, on litter with nestlets for environmental enrichment. On Study Day 1, approximately 10% of the mice were weighed to provide an average weight for calculation of drug concentrations to be administered. On Study Day 0, all mice received a challenge inoculation (induced infection). Compounds tested included clinafloxacin, enrofloxacin, and ceftiofur. All mice received one to three subcutaneous (SC) injections of stated compounds starting at 1 or 18 h post-challenge (PC) depending on the model. SC injections were administered in the left and/or right inguinal region, using a 28 gage X½ inch insulin needle-syringe to deliver 0.1 ml per injection. Serial two- or threefold dilutions of test compounds were dosed to different groups of mice, and mice were monitored for 6 or 8 days. Efficacy of test compounds was calculated as ED₅₀ values (efficacious dose-50%) by use of GraphPad Prism^®. All animal studies were conducted under the guidelines, and with the approval of, the Pfizer Animal Health Institutional Animal Care and Use Committee.

For the Mannheimia haemolytica systemic infection model, 25 colonies from overnight growth on BAP were aseptically picked and inoculated into 50 ml of room temperature brain-heart infusion broth (BHIB; BBL^TM, Becton, Dickinson and Company) and placed at 35–37°C for 3.5–4 h of incubation. Appropriate dilutions in cold BHIB with 4% brewer’s yeast were made to achieve ~2.5 × 10⁸ CFU/ml. A volume of 0.2 ml of this adjusted challenge material was administered to mice by the intraperitoneal (IP) route using a 25 gage X 5/8 inch needle on a 1-ml tuberculin syringe. Placement of the IP challenge was in the mouse’s lower right abdominal quadrant. Drugs were administered via SC injection as described above at either 1 and 3 h PC or at 1, 24, and 48 h PC. Mice were monitored for 6 days.

For the Pasteurella multocida intranasal (IN) infection model, five colonies from the overnight growth on BAP were picked and inoculated into 10 ml of room temperature BHIB. Appropriate dilutions in cold BHIB were made to achieve ~1.25 × 10⁵ CFU/ml. Mice were anesthetized either by an IP injection of 100 mg/kg ketamine + 10 mg/kg xylazine, or by isoflurane inhalation via the Compac 5 Anesthesia Center. A volume of 0.04 ml (40 μl) of the adjusted challenge material was slowly applied to the nares of anesthetized mice, using a Rainin L100 Pipet Lite manual pipettor with sterile tips. At 18 h PC, mice received one SC injection of 0.1 ml of appropriate dilutions of drug prepared in phosphate buffered saline (PBS). Mice were monitored for 8 days.

For the Pasteurella multocida systemic infection model, dilutions in cold PBS were made to achieve ~1.5 × 10⁴ CFU/ml. A volume of 0.1 ml of this adjusted challenge material was administered to mice by the IP route as described above. Drugs to be tested were administered via SC injection as described above at 1 and 3 h PC. Mice were monitored for 6 days.

For the A. pleuropneumoniae systemic infection model, fresh supplement C at 2% concentration was added to 30 ml of room temperature BHIB. Fifteen A. pleuropneumoniae colonies were transferred to the BHIB with supplement C, then incubated for ~4 h until the A₆₀₀ of undiluted growth approximated a pre-determined optical density known to yield ~2 × 10⁸ CFU/ml. Mice were challenged with a volume of 0.2 ml/mouse, by IP administration, as described above. Drugs to be tested were administered via SC injection as described above at 1, 24, and 48 h PC. Mice were monitored for 6 days.

The MICs of clinafloxacin and comparator compounds for 480 bacterial clinical isolates are summarized in Table 1. Clinafloxacin demonstrated strong activity against all tested bovine and swine isolates with MIC₉₀ values lower for all isolate groups compared to danofloxacin and enrofloxacin (MIC₉₀ values bovine P. multocida, M. haemolytica, H. somni, and M. bovis were 0.125, 0.5, 0.125, and 1 μg/ml, respectively, and the MIC₉₀ values against swine P. multocida, A. pleuropneumoniae, S. suis, and M. hyopneumoniae were ≤0.03, ≤0.03, 0.125, and ≤0.008 μg/ml, respectively). Clinafloxacin was also very active against mycoplasma isolates recovered from cattle and pigs. Clinafloxacin MICs obtained against ATCC cultures in this study were similar to what is listed as acceptable QC parameters in CLSI (formerly National Committee for Clinical Laboratory Standards, NCCLS; CLSI, 2005; data not shown).

The results of the time-kill kinetic studies are summarized in Table 2 when tested at drug concentrations that were four times the MIC. The data are presented in terms of the log CFU change in which cidality is defined as a ≥3.0 log CFU reduction in the initial inoculum at 24 h. All of the test agents produced a similar degree of killing (reduction of 3.8–5.0 log CFU/ml) and were defined as being cidal. The greatest decrease in CFUs occurred between 2 and 6 h for all compounds (data not shown). Clinafloxacin had a log decrease range of 4.4–5.0 against all organisms tested.

Determination of MPC/MIC ratios was determined for test compounds using one strain of S. suis and P. multocida (Table 3). For clinafloxacin and S. suis, the parent MIC shifted from 0.125 to 0.25 μg/ml for a MPC/MIC ratio of 2 whereas the MIC did not shift with P. multocida resulting in a ratio of 1. MPC/MIC ratios using S. suis were 16 and 8 for danofloxacin and enrofloxacin, respectively; using P. multocida the ratio was 4 for danofloxacin and enrofloxacin.

Clinafloxacin and comparator compounds were efficacious with low ED₅₀ values when tested against livestock pathogens in mouse IP or IN models (Table 4). ED₅₀ values for clinafloxacin were determined when dosed three times at 1, 24, and 48 h and 1 and 3 h after an IP challenge with a bovine strain of M. haemolytica (0.019 and 0.008 mg/kg, respectively) while an ED₅₀ value of 0.55 mg/kg was obtained when bovine P. multocida was administered intranasally and 0.08 mg/kg when administered intraperitoneally. The ED₅₀ value was slightly higher at 0.7 mg/kg with the IP A. pleuropneumoniae model. No evidence of hypoglycemia or photosensitivity was observed in any mice during these efficacy studies.