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Veterinary Therapeutics Winter 2009 (Vol 10, No 4)

Ceftiofur Susceptibility of Streptococcus equi subsp zooepidemicus Isolated From Horses in North America Between 1989 and 2008

by Donald J. Bade, BS, Gary Sibert, DVM, PhD, John Hallberg, DVM, PhD, Ellen Portis, BS, Joseph Boucher, MS, W. Bryson, PhD

    Clinical Relevance

    In vitro activity of ceftiofur and six other antimicrobial agents was assessed for 516 Streptococcus equi subsp zooepidemicus isolates collected from horses with lower respiratory tract infections in North America in 2007 and 2008 and 239 equine S. equi subsp zooepidemicus isolates received from US and Canadian veterinary diagnostic laboratories between 1989 and 2007. The lowest concentration of ceftiofur inhibiting the growth of 90% of the isolates (MIC90) was 0.12 µg/ml for both groups of isolates. The Clinical and Laboratory Standards Institute susceptible breakpoint set for ceftiofur against this organism is a minimal inhibitory concentration value of ≤0.25 µg/ml. The MIC90 values remained consistent for isolates collected over 19 years.

    Introduction

    Lower respiratory tract disease in horses has multiple etiologies, both viral and bacterial. Streptococcus equi subsp zooepidemicus is a primary bacterial pathogen typically isolated in large numbers from transtracheal wash samples collected from horses with lower respiratory tract infections.1,2 Ceftiofur (Naxcel Sterile Powder, Pfizer Animal Health) was approved by the FDA on July 13, 1994, to treat respiratory disease caused by S. equi subsp zooepidemicus in horses. Ceftiofur crystalline free acid (CCFA; Excede Sterile Suspension, Pfizer Animal Health) was introduced later in the form of a ready-to-use, sustained-release injection. As a β-lactam antibiotic, ceftiofur is a bactericidal broad-spectrum cephalosporin that exerts its effect by interrupting cell wall synthesis.

    Ceftiofur is one of the two FDA-approved antibiotics with a veterinary breakpoint from the Clinical and Laboratory Standards Institute (CLSI) for S. equi subsp zooepidemicus—i.e., a minimal inhibitory concentration (MIC) susceptible breakpoint of ≤0.25 µg/ml. From a pharmacokinetic/pharmacodynamic perspective, ceftiofur's effectiveness against any pathogen depends on how long plasma concentrations of ceftiofur are maintained above the MIC of that respective pathogen. Following IM administration of two doses of 6.6 mg/kg CCFA sterile solution 96 hours apart, the plasma concentration remains above 0.2 µg/ml for 10 days. This article presents historical and current ceftiofur MIC data for S. equi subsp zooepidemicus derived from horses with diagnosed lower respiratory tract disease.

    Materials and Methods

    Samples

    Between 1989 and 2007, 239 historical S. equi subsp zooepidemicus samples were collected from veterinary diagnostic laboratories in the United States and Canada. The origins and dates of collection for these isolates are listed in Table 1 . The bacterial isolates were identified by the submitting laboratories. Pfizer Animal Health used a commercially available identification system (Biolog, Hayward, CA) to confirm identification of the isolates when initial identification was questionable. The isolates were stored in 1.0 ml of trypticase soy broth (BD Biosciences, Sparks, MD) supplemented with 10% glycerol and stored at -70°C until tested.

    In the more recent field study of CCFA between November 2007 and May 2008, 516 isolates were collected from horses with lower respiratory tract disease.3 Clinical specimens were collected by transtracheal wash or nasopharyngeal swab during study enrollment, placed in transport medium (5 or 11.5 ml Port-A-Cul tubes, BD Diagnostics, Franklin Lakes, NJ), and sent to Microbial Research Inc. (MRI; Fort Collins, CO). Samples came from seven study sites in the United States and two in Canada. At MRI, the clinical specimens were cultured, and bacterial pathogens were isolated and characterized using standard microbiologic techniques. Definitive classification of the bacterial pathogens was determined using a commercially available identification system (Biolog). Only S. equi subsp zooepidemicus isolates were subjected to susceptibility testing.

    Minimal Inhibitory Concentrations

    MIC values were determined using a broth microdilution system (Trek Diagnostic Systems, Cleveland, OH), which conformed to the current CLSI broth microdilution method. Custom panels with plate code CMP1PEQ manufactured by Trek Diagnostics were used. The MIC panel included ceftiofur, amoxicillin-clavulanic acid, ampicillin, gentamicin, penicillin, tetracycline, and trimethoprim-sulfamethoxazole.

    Bacterial isolates were revived from storage and subcultured onto trypticase soy agar supplemented with 5% sheep's blood and incubated overnight at 36°C ± 2°C in 5% ± 2% carbon dioxide. Purity was confirmed, and an 18- to 24-hour growth period was used for MIC determinations. Bacterial suspensions were made from colonies (direct colony suspensions) and used for all MIC tests. The CLSI-recommended American Type Culture Collection (ATCC) quality control (QC) strains used on each day of testing included Streptococcus pneumoniae ATCC 49619, Staphylococcus aureus ATCC 29213, Escherichia coli ATCC 25922 and ATCC 35218, Pseudomonas aeruginosa ATCC 27853, and Enterococcus faecalis ATCC 29212. The inoculated MIC panels were incubated without the addition of carbon dioxide at 35°C for 20 to 24 hours.

    Before the third edition of the CLSI M31 document Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals, MIC testing of veterinary streptococci was performed using either CAMHB or CAMHB with LHB.4 To provide consistency in testing, the CLSI subcommittee on Veterinary Antimicrobial Susceptibility Testing edited the M31 document to clearly indicate that CAMHB should be supplemented with 2.5 to 5% LHB when testing streptococci.5 For comparing MIC data generated using the current CLSI method with MIC data generated using previous methods, MICs for the 239 historical isolates received from veterinary diagnostic laboratories and 10% of the isolates from the recent field study were ascertained using both CAMHB that was not supplemented with LHB and CAMHB with LHB. The lowest MIC, lowest MIC inhibiting the growth of 50% of the isolates (MIC50), lowest MIC inhibiting the growth of 90% of the isolates (MIC90), and highest MIC were determined for each antimicrobial agent tested.

    Results

    Table 1 lists the MIC90 values for all seven antimicrobial agents tested against the 755 S. equi subsp zooepidemicus isolates collected between 1989 and 2008. The CLSI susceptible breakpoint for ceftiofur against S. equi subsp zooepidemicus is ≤0.25 µg/ml.5 The ceftiofur MIC90 values were the same (0.12 µg/ml) for each group of isolates collected, as well as for all the isolates combined that were collected during the 19-year period.

    Table 2 presents the MIC distributions and frequencies for the 516 S. equi subsp zooepidemicus isolates collected in the 2007 to 2008 field study against all seven antimicrobial agents. Of the tested isolates, 97.3% (502 of 516) were susceptible to ceftiofur. The ceftiofur MIC50 value was 0.06 µg/ml and the MIC90 value was 0.12 µg/ml (range, ≤0.015 to 0.5 µg/ml).

    Comparison of the MIC50 and MIC90 results using CAMHB supplemented with LHB and CAMHB alone appear for all seven antimicrobial agents in Table 3 . Adding LHB to the inoculating broth changed the MIC50 and MIC90 of ceftiofur in both the historical isolates and the field study isolates by several dilutions. Nevertheless, the MIC90 values for ceftiofur remained below the CLSI-defined breakpoint established for S. equi subsp zooepidemicus with both testing methods.

    Discussion

    The CLSI approved only a susceptible breakpoint for ceftiofur against S. equi subsp zooepidemicus of ≤0.25 µg/ml for MIC testing. All isolates reviewed by the CLSI had MIC values ≤0.25 µg/ml; therefore, an intermediate or resistant population was not available. Ceftiofur MIC and agar disk diffusion QC ranges for CLSI susceptibility tests have also been developed and published.5 Recently, QC standards for MIC susceptibility testing of streptococci against ceftiofur using CAMHB with LHB have been approved by the CLSI as well.6

    The 239 historical S. equi subsp zooepidemicus samples collected before November 2007 came from veterinary diagnostic laboratories in 21 US states and three Canadian locations. Exactly how the samples were collected could not be ascertained, although most were likely from nasopharyngeal swabs and necropsy samples. Such samples are typically sent to diagnostic laboratories to confirm a diagnosis or when initial therapy has failed. Although these isolates may represent the most difficult-to-treat cases, >99% were susceptible to ceftiofur regardless of the testing method used.

    MIC testing for all of the historical (diagnostic laboratory) isolates and 10% of the field study isolates was performed using two types of inoculating broth, CAMHB and CAMHB with LHB, to investigate any changes in MIC values resulting from the revised CLSI method for MIC testing of streptococci.

    Table 3 presents the MIC50 and MIC90 results for the 294 isolates tested with both inoculating broths. The addition of LHB to CAMHB affected the MIC values for both ceftiofur and other antimicrobial agents tested against streptococci. The ceftiofur MIC90 values determined with the addition of LHB averaged two to three dilutions higher than those without LHB. At this time, we have not been able to fully explain this change in MIC values caused by the addition of LHB to CAMHB. Because most (286 of 294) of the isolates tested with unsupplemented CAMHB had ceftiofur MIC values lower than the smallest concentration tested (0.015 µg/ml), the apparent difference in effect on the MIC90 between the two methods may be biased downward or present a lower-than-actual difference. Although the MIC90 values for ceftiofur are higher using the current test method (CAMHB with LHB), more than 97% of S. equi subsp zooepidemicus isolates still remained in the CLSI-defined susceptible range for ceftiofur. Thus, the ceftiofur susceptibility results for the more recent field study isolates were consistent with the results of the historical MIC study.

    In 2008, 10 isolates of S. equi subsp zooepidemicus from US, Canadian, and European field studies were tested at Micromyx, LLC (Kalamazoo, MI) with a minimal bactericidal concentration assay and a time-kill kinetic assay for ceftiofur and penicillin G.7 Ceftiofur inhibited the growth of all isolates at a concentration of ≤0.12 µg/ml. The minimal bactericidal concentration assay results showed that ceftiofur was bactericidal for all strains at concentrations ≤2 times the MIC. This finding was generally consistent with the results of the time-kill assays, which demonstrated that bactericidal effects were achieved for 10 of 10 (100%) strains for ceftiofur when the test concentration was two times the MIC or greater. Overall, ceftiofur demonstrated good bactericidal activity against S. equi subsp zooepidemicus.

    Conclusion

    Ceftiofur possesses sufficient in vitro activity against S. equi subsp zooepidemicus, with more than 97% of the isolates from samples collected over a 19-year period found to be susceptible (MIC values ≤0.25 µg/ml) to ceftiofur. The addition of LHB to CAMHB results in higher ceftiofur MIC values than testing with CAMHB alone.

    Downloadable PDF

    Funding for this study was provided by Pfizer Animal Health, Kalamazoo, MI.

    1. Wood JL, Burrell MH, Roberts CA, et al. Streptococci and Pasteurella spp. associated with disease of the equine lower respiratory tract. Equine Vet J 1993;4:314-318.

    2. Clark C, Greenwood S, Boison JO, et al. Bacterial isolates from equine infections in western Canada (1998-2003). Can Vet J 2008;49:153-160.

    3. SR 1153C-60-06-208. Field Dose Confirmation and Safety of Excede® (200 mg/ml) Administered Twice at a Five Day Interval at 6.6 mg/kg BW in Horses for the Treatment of Lower Respiratory Disease Associated with Streptococcus zooepidemicus or Other Relevant Bacterial Pathogens. New York: Pfizer Animal Health; 2009.

    4. National Committee on Clinical Laboratory Standards. Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals: Approved Standard. 2nd ed. Wayne, PA: Clinical and Laboratory Standards Institute; 2002. CLSI document M31-A2.

    5. Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated From Animals: Approved Standard. 3rd ed. Wayne, PA: Clinical and Laboratory Standards Institute; 2008. CLSI document M31-A3.

    6. Clinical and Laboratory Standards Institute Subcommittee on Veterinary Antimicrobial Susceptibility Testing. Summary Minutes. Meeting at the Grand Hyatt Tampa Bay, Tampa, FL, January 24-25, 2008.

    7. SR 8651R-85-08-161. Streptococcus equi subsp. zooepidemicus from US, Canada, and EU Field Studies: MBC and Time-Kill Assessments. New York: Pfizer Animal Health; 2008.

    Correspondence should be sent to Ms. Portis: phone, 269-833-2621; fax, 646-441-4459; email, ellen.s.portis@Pfizer.com.

    References »

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