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Veterinary Therapeutics Fall 2010 (Vol 11, No 3)

In Vitro Comparison of Staphylococcus pseudintermedius Susceptibility to Common Cephalosporins Used in Dogs

by Rebekah R. Westermeyer, MM, DVM, DACVD, Alma F. Roy, MS, PhD, Maria S. Mitchell, DVM, Sarah R. Merchant, DVM, DACVD

    Clinical Relevance

    The in vitro activity of 10 cephalosporin antimicrobial agents against 75 isolates of methicillin-susceptible Staphylococcus pseudintermedius derived from dogs was assessed. The lowest minimal inhibitory concentration for 90% of strains (MIC90) values obtained were for cephalothin, cefovecin, and cefazolin (0.12 µg/mL), followed by ceftiofur and cefoxitin (0.25 µg/mL), cefpodoxime (0.5 µg/mL), and cefaclor and cefadroxil (1 µg/mL). The highest MIC90 values were found for cephalexin and cefixime (2 µg/mL). In this in vitro study, sensitivity to cephalothin was indicative of cephalexin susceptibility, although there were marked differences in MICs. Cephalothin susceptibility was not indicative of susceptibility to all tested cephalosporins, nor was there a clear trend in susceptibility based on cephalosporin generation.

    Introduction

    Cephalosporins are bactericidal β-lactam antibiotics commonly used in veterinary dermatology for the treatment of canine pyoderma.1 Recent phylogenetic studies have shown that the commensal staphylococcal species of dogs, which is also the most common bacterial species isolated from canine pyoderma, is Staphylococcus pseudintermedius.2 Cephalosporins are often recommended as the first choice for treatment of S. pseudintermedius infections1; however, with the increasing prevalence of resistant bacterial pathogens, culture and susceptibility testing are becoming more important for the selection of appropriate antibiotic therapy.

    Cephalosporins have been categorized into generations based on their antimicrobial properties. First-generation cephalosporins generally have antimicrobial activity against most gram-positive cocci, with Pseudomonas and Proteus spp being resistant. Second-generation cephalosporins have increased activity against gram-negative bacteria, and third-generation cephalosporins have even greater efficacy against gram-negative bacteria (including some with activity against Pseudomonas aeruginosa). However, it has been reported that with the increased gram-negative spectrum of activity, the efficacy against gram-positive cocci is decreased.3,4

    Bacterial culture and susceptibility reports are provided by most diagnostic laboratories using one or two antibiotics per class of antimicrobial drug. It is well established that differences in susceptibility to the various fluoroquinolones used in veterinary medicine5 exist and that susceptibility to one fluoroquinolone does not guarantee susceptibility to another.6 However, many culture and susceptibility panels list susceptibility to only one or two cephalosporins. Moreover, the most commonly available cephalosporin for disk-diffusion susceptibility testing is cephalothin, a first-generation parenteral cephalosporin that has greater efficacy against gram-positive cocci7–9 (particularly staphylococci) than later-generation cephalosporins. Because antimicrobial susceptibilities vary among cephalosporins and generations of cephalosporins, the purpose of this study was to characterize susceptibility patterns of S. pseudintermedius isolates from dogs to commonly available, orally administered cephalosporins, as well as veterinary parenteral cephalosporins.

    It was hypothesized that the values for the minimal inhibitory concentration (MIC) for 50% of strains (MIC50) and 90% of strains (MIC90) would be different for the cephalosporins tested but that there would be similarities within generations of cephalosporins. It was also hypothesized that susceptibility to one cephalosporin would not be indicative of susceptibility to all cephalosporins.

    Materials and Methods

    Isolates of non–methicillin-resistant S. pseudintermedius from dogs were obtained using clinical samples submitted to the Louisiana Animal Disease Diagnostic Laboratory from April to November of 2009. Isolates were identified as S. pseudintermedius using standard microbiologic procedures, including pigmentation of colonies, coagulase, catalase, hemolysis, urease production, mannitol fermentation, acetoin production, maltose fermentation, and the API Staph test (bioMerieux, Durham, NC). Randomly selected isolates were verified as S. pseudintermedius by a 16S bacterial identification polymerase chain reaction procedure.10 Methicillin susceptibility was determined by oxacillin disk-diffusion susceptibility testing using BD Sensi-Disc Antimicrobial Susceptibility Test Discs (BBL, Cockeysville, MD) according to the 2008 Clinical and Laboratory Standards Institute (CLSI) guidelines.11

    MIC testing was performed using custom-designed frozen Sensititre plates (TREK Diagnostic Systems, Cleveland, OH) according to the manufacturer’s instructions. Strains were subcultured on blood agar before MIC testing. Microdilution wells were inoculated with approximately 1.5 × 108 CFU/mL. Plates were incubated for 18 hours at 37°C. Individual MIC runs were validated by concurrent quality-control testing with Staphylococcus aureus ATCC strain # 29213. All plates had 96 wells with increasing concentrations of the 10 antimicrobial agents selected for testing (cefaclor, cefadroxil, cefazolin, cefixime, cefovecin, cefoxitin, cefpodoxime, ceftiofur, cephalexin, cephalothin). Positive controls were present on each plate. MIC testing was performed in groups of 5 to 10 isolates every 1 to 2 weeks as available. When available, breakpoints were determined by the MIC guidelines stipulated by the CLSI.11 All other breakpoints were determined based on previously reported or recommended breakpoints. For cefoxitin, breakpoints established for predicting oxacillin resistance in S. aureus were used.12

    Results

    Seventy-five isolates of methicillin-susceptible S. pseudintermedius were available for testing during this study. Sixty-five isolates came directly from cutaneous or mucocutaneous sources (e.g., wounds, pustules, nasal swabs, vaginal swabs, periocular sites). Seven isolates were from urine, one from a urinary bladder wall, one from an infected implant, and one from joint fluid. The distributions and ranges of MICs for the 75 S. pseudintermedius isolates are listed in Table 1 .7–9,11–18

    All strains tested were susceptible to cephalexin (MIC90 of 2 µg/mL, range of 0.5 to 8 µg/mL), cefazolin (MIC90 of 0.12 µg/mL, range of ≤0.06 to 0.25 µg/mL), cefaclor (MIC90 of 1 µg/mL, range of 0.12 to 4 µg/mL), cefoxitin (MIC90 of 0.25 µg/mL, range of 0.12 to 1 µg/mL), ceftiofur (MIC90 of 0.25 µg/mL, range of 0.12 to 2 µg/mL), cephalothin (MIC90 of 0.12 µg/mL, range of ≤0.06 to 0.25 µg/mL), cefovecin (MIC90 of 0.12 µg/mL, range of 0.06 to 2 µg/mL), and cefadroxil (MIC90 of 1 µg/mL, range of 0.25 to 8 µg/mL). For cefpodoxime, one strain showed intermediate susceptibility and one strain was resistant (MIC90 of 0.5 µg/mL, range of 0.25 to 8 µg/mL). For cefixime (MIC90 of 2 µg/mL, range of ≤1 to 32 µg/mL), 55 strains were intermediate and seven were resistant.

    Discussion

    β-Lactamase production is a major mechanism of resistance in staphylococci and has been reported for S. pseudintermedius in at least 62% of cases.8 Therefore, the use of penicillinase-stable antimicrobial agents such as cephalosporins is indicated when treating infections caused by S. pseudintermedius. In general, compared with first-generation cephalosporins, third-generation cephalosporins are considered to be less active against gram-positive cocci. However, in this study, the MIC90 values for cefovecin, ceftiofur, and cefpodoxime (0.12, 0.25, and 0.5 µg/mL, respectively) were lower than the MIC90 for cephalexin (2 µg/mL), a commonly used oral first-generation cephalosporin. Similar results have previously been reported for cefquinome (a fourth-generation cephalosporin) and ceftiofur.8

    Cephalexin is a first-generation cephalosporin available for oral administration that is commonly used for the treatment of staphylococcal pyoderma in dogs.1 Cephalexin has been most frequently recommended for twice-daily administration based on achieved skin concentrations of >1 µg/mL when dogs were treated with 25 mg/kg1 twice daily, although some researchers have demonstrated that once-daily administration of higher doses (30 to 60 mg/kg) may also be effective.19 Other studies have recommended administration of 20 mg/kg every 6 to 8 hours to achieve steady-state serum concentrations >5 µg/mL.20 In this study, all isolates were susceptible to cephalexin, but one isolate had an MIC of 8 µg/mL, which is much higher than the demonstrated skin concentrations of cephalexin achieved with twice-daily 25-mg/kg administration or steady-state concentrations achieved with dosing three or four times daily. In addition, four isolates had MIC values of 4 µg/mL, which might render these isolates resistant to standard cephalexin administration.

    Cephalothin was the first cephalosporin to be introduced. It is still used frequently in laboratories for disk-diffusion testing to determine susceptibility to cephalexin. In this study, all isolates were susceptible to both antibiotics, but the MICs for cephalothin (≤0.06 to 0.25 µg/mL) were markedly lower than for cephalexin (0.5 to 8 µg/mL). Given that the breakpoints are considered to be the same for both antimicrobial agents,11,13 it is possible that S. pseudintermedius could be intermediate or resistant to cephalexin but susceptible to cephalothin, although this was not demonstrated by the results of this study. In a previous study by Barry and Jones,13 12 of 19 methicillin-resistant staphylococci showed susceptibility to cephalothin but not cefadroxil. This underscores the importance of properly identifying methicillin-resistant isolates and not using cephalosporins based on cephalothin susceptibility alone.

    Cefadroxil is another first-generation cephalosporin and is a parahydroxy derivative of cephalexin. It is available for oral administration and has been shown to have similar pharmacokinetics, MIC values,21 and disk-diffusion zone size13 to those of cephalexin. In this study, the MIC50 values were the same, but the MIC90 was slightly higher for cephalexin. The difference between cefadroxil and cephalexin is small, as 89% of isolates (67 of 75) had an MIC of ≤1 for cephalexin, and 92% of isolates (69 of 75) had an MIC of ≤1 for cefadroxil.

    Cefazolin, a first-generation cephalosporin, has the addition of a tetrazole ring at the 7-carbon atom of the nucleus, which has been suggested as the reason for its increased activity against gram-negative microbes, specifically Escherichia coli. It has previously been shown to have very similar MIC90 values to cephalothin for staphylococci.9 In this study, the MIC50 and MIC90 values for both antimicrobial agents were the same (≤0.06 µg/mL and 0.12 µg/mL, respectively). Cefazolin is commonly used in surgical procedures because it is available for intravenous (IV) and intramuscular (IM) administration. This makes it inconvenient for use in canine pyoderma but very effective for perioperative use. It has been shown that a perioperative dose of 20 mg/kg, followed 6 hours later by the same dose given subcutaneously (SC), resulted in serum levels remaining >4 µg/mL for >12 hours,22 a dose that would be very effective for all strains tested in the study.

    Cefaclor is a second-generation oral cephalosporin that is structurally related to cephalexin. Unlike in humans, cefaclor is extensively metabolized in dogs.23 Therefore, in dogs, higher concentrations of cefaclor are found in bile and urine than in serum and tissues.24 Thus, it is recommended that cefaclor be given to dogs three times daily to maintain tissue concentrations. In this study, all isolates were susceptible to cefaclor. Cefaclor had lower MIC50 and MIC90 values (0.5 µg/mL and 1 µg/mL, respectively) than the other orally available first-generation cephalosporins tested (i.e., cephalexin [1 µg/mL and 2 µg/mL] and cefadroxil [1 µg/mL and 1 µg/mL]).

    Cefoxitin is a second-generation parenteral cephalosporin that has been recommended for IV or SC administration. In 2004, the CLSI recommended use of cefoxitin disk testing for predicting mecA-mediated resistance in staphylococci.25 While this has been shown to be very accurate for S. aureus, Bemis et al26 showed these criteria to significantly underestimate mecA-mediated resistance in S. pseudintermedius isolates. In this study, all isolates were susceptible to cefoxitin, with MIC values well below the designated breakpoint of ≤4 µg/mL (range: 0.12 to 1 µg/mL).

    Cefovecin is a relatively new third-generation cephalosporin available for SC administration. It has a very long half-life of 5.5 days in dogs, with serum concentrations of approximately 10 µg/mL at 14 days after a single recommended dose.27 The previously reported MIC90 value for cefovecin is 0.25 µg/mL. In this study, the MIC90 was 0.12 µg/mL. With the currently recommended US breakpoint for cefovecin of 2 µg/mL,17 all isolates were susceptible to cefovecin. In Australia, the breakpoint has been set at 1 µg/mL, with 2 µg/mL designated as “intermediate susceptible.”28 By these standards, two isolates would have been determined to be intermediate susceptible.

    Ceftiofur is also a third-generation parenteral cephalosporin approved for use in dogs in the United States. Due to its relatively long half-life of 6 hours, ceftiofur is recommended for once-daily administration. A previously reported MIC90 for S. aureus was 1 µg/mL.29 In this study, for S. pseudintermedius, the MIC90 was 0.25 µg/mL. Studies have shown that ceftiofur reaches serum concentrations of 0.556 µg/mL at 24 hours after SC administration of 2.2 mg/kg,30 so once-daily SC injection would be effective for most S. pseudintermedius isolates.

    Cefixime is a third-generation oral cephalosporin. It has a relatively long half-life of 6 hours due to renal tubular resorption and protein binding. However, cefixime has been shown in previous studies to have relatively poor activity against S. aureus and S. (pseud)intermedius. Although serum concentrations remained above 2 µg/mL at 24 hours after administration of 6.25 mg/kg, previous MIC values for S. (pseud)intermedius ranged from 1.56 to 6.25 µg/mL.31 Therefore, twice-daily administration has been recommended.32 In this study, the MIC90 was 2 µg/mL, but the formerly recommended susceptibility breakpoint is 1 µg/mL,18 making only 13 of the 75 isolates tested susceptible to cefixime.

    Cefpodoxime is another third-generation cephalosporin available in an oral formulation and approved for use in dogs. As a third-generation cephalosporin, cefpodoxime has been considered to be slightly less effective for treatment of staphylococci than first-generation cephalosporins.14 In the present study, the MIC90 value for cefpodoxime was 0.5 µg/mL, whereas the MIC90 for cephalexin was 2 µg/mL. However, one isolate had an MIC of 8 µg/mL, which (according to the CLSI11) is resistant to cefpodoxime. A previous study has shown serum concentrations of approximately 0.9 µg/mL after a single 10-mg/kg dose.33 Steady-state serum concentrations after 6 days were much higher at 16.4 ± 11.8 µg/mL.33 Given the variability in steady-state serum concentrations, although cefpodoxime has a lower MIC90 than cephalexin, some isolates would be susceptible to cephalexin and resistant to cefpodoxime, as previously indicated (Table 2).9,21,24,27,30,33–35

    Conclusion

    In vitro, cephalosporins in general remain highly effective for treatment of methicillin-susceptible S. pseudintermedius. However, cephalothin susceptibility was not indicative of susceptibility for all cephalosporins tested in this study, and there was no clear trend in susceptibility based on cephalosporin generation. Although cephalothin sensitivity was indicative of cephalexin susceptibility for methicillin-susceptible isolates, there was a marked difference in MICs.

    Acknowledgments

    We thank Veronica McCall, Louisiana Animal Disease Diagnostic Laboratory, for all her help with MIC testing.

    Downloadable PDF

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    References »

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