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Antimicrobial Agents and Chemotherapy, July 2006, p. 2286-2292, Vol. 50, No. 7
0066-4804/06/$08.00+0     doi:10.1128/AAC.00077-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Antimicrobial Activity and Spectrum of Cefovecin, a New Extended- Spectrum Cephalosporin, against Pathogens Collected from Dogs and Cats in Europe and North America

M. R. Stegemann,1* C. A. Passmore,1 J. Sherington,1 C. J. Lindeman,2 G. Papp,2 D. J. Weigel,2 and T. L. Skogerboe2

Pfizer Animal Health, Veterinary Medicine Clinical Development, Sandwich, United Kingdom, and,1 Pfizer Animal Health, Veterinary Medicine Clinical Development, Kalamazoo, Michigan2

Received 18 January 2006/ Returned for modification 21 February 2006/ Accepted 7 March 2006


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ABSTRACT
 
Cefovecin is a new extended-spectrum semisynthetic cephalosporin indicated for the treatment of bacterial infections in dogs and cats. This study evaluated the in vitro activity and spectrum of cefovecin against 2,641 recent clinical isolates (1,660 canine and 981 feline isolates) from Europe and the United States. MIC determinations against cefovecin and other reference antimicrobials were performed by broth microdilution methods recommended by the Clinical and Laboratory Standards Institute (CLSI, formerly NCCLS). Cefovecin demonstrated bactericidal activity against both gram-positive and gram-negative pathogens. Cefovecin exhibited in vitro activity against all major aerobic and anaerobic bacterial pathogens associated with skin, urinary tract, and periodontal infections in dogs and cats. The MIC90 values of cefovecin against Staphylococcus intermedius, Escherichia coli, and Pasteurella multocida were 0.25 µg/ml, 1.0 µg/ml, and 0.06 µg/ml, respectively. No significant differences were observed in terms of the activities of cefovecin against pathogens from different European countries and against pathogens of European and U.S. origin.


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INTRODUCTION
 
Skin and soft tissue infections and urinary tract infections in dogs and cats are among the most common presenting infections in veterinary practices. Skin infection in dogs is mainly represented by pyoderma, a pyogenic bacterial infection. Pyoderma in dogs is manifested in a great diversity of clinical syndromes ranging from mild skin disease (e.g., superficial pyoderma, involving only the epidermis) to life-threatening conditions (e.g., deep pyoderma, a serious condition involving the dermis and the subcutis). The coagulase-positive species Staphylococcus intermedius is considered the main cause for superficial pyoderma infections (8, 12), with Escherichia coli, Proteus spp., and Pseudomonas spp. being secondary invaders, mainly in deep pyoderma. Beta-hemolytic streptococci have been described as pathogens involved in skin and urinary tract infections in dogs and cats (6).

In cats, the most common bacterial skin diseases result from bite wounds and involve Pasteurella multocida and anaerobes such as Porphyromonas spp., Fusobacterium spp., Bacteroides spp., Peptostreptococcus spp., and Clostridium spp. (5).

Urinary tract infections in both dogs and cats are frequently associated with Escherichia coli, Proteus spp., Klebsiella spp., Staphylococcus spp., Streptococcus spp., Enterococcus spp., and (in cats only) Pasteurella multocida (16). Prevotella spp. and Porphyromonas spp. have been identified as key pathogens involved in canine periodontal disease (7).

Antimicrobials play a predominant role in the control of bacterial skin and urinary tract infections. Cefovecin is a newly developed antimicrobial belonging to the cephalosporin family. Cefovecin is formulated for subcutaneous administration; its intrinsic long elimination half-life (dogs, 5.5 days; cats, 6.9 days) will allow 14-day dosing intervals (18).

Within the preclinical and clinical development program for cefovecin, the MICs of 2,641 bacterial pathogens collected in several European countries and in the United States were determined. The in vitro activity of cefovecin was compared to that of other commonly used antimicrobials, including cephalexin, amoxicillin with clavulanic acid, and cefadroxil.


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MATERIALS AND METHODS
 
In vitro activity of cefovecin. Bacterial isolates from clinical cases were collected in two phases: during an epidemiological survey (pathogens collected specifically for this survey at veterinary practices and pathogens banked at diagnostic laboratories) and in the field safety and efficacy studies conducted to register cefovecin for veterinary use. In the epidemiological survey, bacterial pathogens from different canine and feline disease complexes (predominantly skin and soft tissue and urinary tract infections) were collected between 1999 and 2000 in five European countries (Denmark, France, Germany, Italy, and the United Kingdom) and in three geographical areas of the United States (the northeastern United States, the Northwest, and the Midwest). Samples (bacteriological swabs and urine samples, n = 373 from the European Union and n = 285 from the United States) were obtained from dogs and cats presented at veterinary practices with clinical signs indicative of bacterial infections (e.g., skin, wound, abscess, urinary tract, oral cavity, ear, or eye). In addition, 73 bacterial pathogens were obtained from commercial diagnostic laboratories in France, Germany, and the United Kingdom. In the field safety and efficacy studies, canine and feline bacterial pathogens were obtained during 2001 to 2003 in Europe (France, Germany, Spain, and the United Kingdom; n = 845) and in the United States (n = 1,065) from animals with skin and soft tissue infections and urinary tract infections. The animals from which the isolates were collected had not received any antimicrobial treatment within the last 30 days before sampling. The distribution of canine and feline pathogens is depicted in Table 1.


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  		TABLE 1. Sources of canine and feline pathogens used for the in vitro susceptibility determination of cefovecina

All samples were submitted to various diagnostic laboratories (at the national level in the European Union or the regional level in the United States) for pathogen purification and identification. Susceptibility testing (MIC) for all bacterial pathogens was conducted in one European and one U.S. laboratory.

MIC determination. MIC determinations against cefovecin and other reference antimicrobials were performed by broth microdilution methods recommended by the Clinical and Laboratory Standards Institute (CLSI, formerly NCCLS) (9, 11). The susceptibility of each isolate was determined using customized Sensititre microdilution plates (Trek Diagnostic Systems Ltd., East Grinstead, United Kingdom). The concentration ranges tested were as follows: for cefovecin (Pfizer Animal Health, Kalamazoo, Michigan), 0.06 to 32 µg/ml; for cephalexin (GlaxoSmithKline, United Kingdom), 0.5 to 64 µg/ml; for cefadroxil (Sigma-Aldrich, United Kingdom), 0.25 to 32 µg/ml; and for amoxicillin-clavulanic acid (Melfords/GlaxoSmithKline, United Kingdom), 0.5/0.25 to 64/32 µg/ml. Aerobic isolates were prepared for inoculation with cation-adjusted Mueller-Hinton broth, to which 3% lysed horse blood was added for Streptococcus spp. Anaerobic isolates were prepared for inoculation with Wilkins-Chalgren anaerobe broth. Microdilution wells were inoculated with approximately 5 x 105 CFU/ml for aerobes and approximately 1 x 106 CFU/ml for anaerobes. Plates were incubated for at least 16 h at approximately 37°C for aerobes and for at least 46 to 48 h at approximately 37°C in an anaerobic atmosphere for the anaerobes. Individual MIC runs were validated by concurrent testing of the appropriate ATCC strains. The quality control ranges for cefovecin had been determined prior to the testing of the field pathogens in an independent multilaboratory study; results of that study have not yet been published and will be presented to the Subcommittee for Veterinary Antimicrobial Susceptibility Testing. The concentration range chosen for cefovecin accommodated all quality control ranges for cefovecin; due to space limitation, this was not possible for all antimicrobials tested.

Bactericidal activity of cefovecin. A total of 100 clinical bacterial isolates collected within the European safety and efficacy studies were tested. The following pathogens were tested: E. coli (n = 25), P. multocida (n = 15), Proteus mirabilis (n = 10), S. intermedius (n = 30), Streptococcus spp. (n = 10), Fusobacterium spp. (n = 6), and Bacteroides spp. (n = 4). Six ATCC susceptibility quality control strains representing five genera were included for quality assurance purposes. MICs and minimal bactericidal concentrations (MBCs) were determined for cefovecin, cephalexin, and ceftiofur by using broth microdilution methods recommended by the CLSI guidelines M11, M26, and M31 (9-11). MICs were read visually; all wells were plated to proper growth media and incubated. The MBC was defined as a 99.9% reduction in CFU from the starting inoculum after the appropriate incubation interval. The first antimicrobial concentration of the microdilution well that contained fewer colonies than the target bactericidal breakpoint (≥99.9%) was defined as the MBC. Concentration ranges for cefovecin and cephalexin were identical with those employed for the MIC testing (see above); ceftiofur (Pfizer Animal Health, Kalamazoo, Michigan) was tested within a range from 0.002 to 128 µg/ml.


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RESULTS AND DISCUSSION
 
The antimicrobial susceptibilities of a large range of bacterial pathogens (1,660 canine and 981 feline isolates) were tested against cefovecin, cephalexin, cefadroxil, and amoxicillin-clavulanic acid. Clinical breakpoints for cefovecin have not yet been submitted to CLSI; veterinary specific clinical breakpoints for cephalexin and cefadroxil were never established, and CLSI document M31 recommends use of the cephalothin MIC breakpoints established for human medicine instead (11). Consequently, resistance rates are not reported here. The MIC results (mode, MIC50, MIC90, and range) for the most frequently collected bacterial pathogens against cefovecin, cephalexin, amoxicillin-clavulanic acid, and cefadroxil are listed in Table 2 for European and U.S. isolates.


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  		TABLE 2. Antimicrobial susceptibility of canine and feline pathogens collected in the European Union and the United States

A total of 501 S. intermedius isolates were investigated for in vitro susceptibility. Cefovecin exhibited good in vitro activity against both European and U.S. isolates; the MIC90 value of 0.25 µg/ml for U.S. isolates was identical to the Pan-European value. Susceptibilities of S. intermedius to cefovecin were compared by country (Table 3). Overall resistance of S. intermedius isolates against the three cephalosporins and amoxicillin-clavulanic acid was low, an observation consistent with previous publications (14, 15, 17). Five isolates (four were collected in the European Union) had an elevated cefovecin MIC (≥8 µg/ml) and were considered resistant to cefovecin. All five of these isolates were cross-resistant (had an elevated MIC of ≥32 µg/ml) to cephalexin and cefadroxil, while only one had an amoxicillin-clavulanic acid MIC of ≥8/4 µg/ml. The observation made for amoxicillin-clavulanic acid is consistent with the findings of Ganiere et al. (4), who did not observe any resistance for this combination when investigating 50 isolates collected in France. Ganiere et al. (4) reported the highest cephalexin MIC in a collection of French isolates with unknown treatment history to be 2 µg/ml; in our study, although animals were not treated with antimicrobials within 30 days prior to sample collection, 13 out of 270 S. intermedius isolates exhibited MICs higher than 2 µg/ml.


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  		TABLE 3. Antimicrobial susceptibility of canine and feline pathogens—MICs against cefovecin, by country of origin of isolates

The majority (33 out of 36) of Staphylococcus aureus isolates were inhibited at cefovecin concentrations of ≤2.0 µg/ml; no S. aureus was isolated from cats and dogs in the United States during these studies. Three out of 36 S. aureus isolates were inhibited in their growth at cefovecin concentrations of ≥8 µg/ml, and these isolates exhibited MICs of ≥32 µg/ml against cephalexin and cefadroxil. Interestingly, all three isolates were collected during the epidemiological survey in 1999; isolates with elevated cefovecin MICs were not collected during the field efficacy studies conducted in 2001 to 2003. Testing for methicillin susceptibility was not conducted.

Cefovecin demonstrated good activity against coagulase-negative staphylococci (n = 110). As this group includes a wide variety of pathogens (e.g., Staphylococcus schleiferi, Staphylococcus xylosus, and Staphylococcus felis), the MIC range consequently was fairly broad. MIC90 values for European and North American ß-hemolytic streptococci against cefovecin were 0.12 µg/ml and ≤0.06 µg/ml, respectively. According to Biberstein and coworkers (1), ß-hemolytic streptococci of canine origin have an 80% probability of belonging to Lancefield group G (Streptococcus canis), and therefore European isolates were identified to the species level. Both North American and European S. canis isolates were demonstrated to be highly susceptible to cefovecin.

Similarly with the other cephalosporins tested here (cephalexin and cefadroxil), cefovecin was not appreciably active against Enterococcus spp. Amoxicillin-clavulanic acid, however, did show good in vitro activity against Enterococcus spp. isolates (MIC90 of 1/0.5 µg/ml).

Cefovecin exhibited a broad activity against a range of gram-negative pathogens, including P. multocida, E. coli, Proteus spp. (including P. mirabilis), Klebsiella spp. (including Klebsiella pneumoniae), and Enterobacter spp. Like other extended-spectrum cephalosporins (e.g., cefotaxime, ceftiofur, and ceftriaxone), cefovecin was not active in vitro against Pseudomonas aeruginosa. All P. multocida, Proteus spp., and Klebsiella spp. isolates exhibited MICs of ≤2 µg/ml (only one Proteus sp. isolate exhibited an MIC of 8 µg/ml). Regarding Enterobacter spp., 76.9% of all European Union isolates tested (n = 39) were susceptible at concentrations of ≤2 µg/ml; however, the high MIC90 observed (32 µg/ml) indicates a resistant subpopulation. The U.S. isolates (n = 20), however, showed an MIC90 of 2 µg/ml, with only one isolate exhibiting an MIC against cefovecin of >2 µg/ml. The in vitro activity of cefovecin against P. multocida was excellent, with an MIC90 value of 0.12 µg/ml for European Union isolates and ≤0.06 µg/ml for U.S. isolates, compared with both cefadroxil (MIC90 of 4 µg/ml) and cephalexin (MIC90 of 2 µg/ml).

Almost 500 E. coli isolates were investigated within the entire study. An identical MIC90 value (1.0 µg/ml) was observed for European Union and U.S. isolates. MIC90 values were identical across Europe, with only isolates from France exhibiting an MIC90 value that was one dilution step higher. The growth of nine isolates was inhibited at concentrations above 8 µg/ml (or not at all); these isolates were all cross-resistant against cephalexin, cefadroxil, and amoxicillin-clavulanic acid. Applying published clinical breakpoints, the resistance rates for amoxicillin-clavulanic acid, cephalexin, and cefadroxil were 3.7%, 5.0%, and 5.6%, respectively. The rates reported here for amoxicillin-clavulanic acid and cephalexin are considerably lower than those reported earlier (3, 13). However, it has been demonstrated that MICs of pathogens submitted to diagnostic centers tend to be higher than the MICs of pathogens collected from nonreferral cases, which very often are not submitted for antimicrobial susceptibility testing (2).

A total of 286 anaerobic-growing pathogens (Prevotella spp., Porphyromonas spp., Peptostreptococcus spp., Fusobacterium spp., Bacteroides spp., Clostridium spp., and Corynebacterium spp.) were isolated and tested in this study. Overall cefovecin exhibited good in vitro activity against anaerobic pathogens. Only eight isolates (2.8%) were inhibited in their growth at concentrations above 4 µg/ml. The combination of amoxicillin and clavulanic acid exhibited the lowest MICs against anaerobic pathogens with no resistant isolates, considering the CLSI-approved breakpoint of R of ≥32/16 µg/ml.

A summary of the MIC data (MIC50, MIC90, and range) for cefovecin against the three most frequently isolated pathogens (i.e., S. intermedius, E. coli, and P. multocida) is presented, by country of origin, in Table 3. The MIC90 values for European and U.S. pathogens were identical for all three pathogens. Within the European collection, the MIC90 values among countries were almost identical for the three pathogens. Isolates collected in France exhibited an MIC90 value which was one dilution step higher for E. coli and S. intermedius than the MIC90 values of these isolates collected from other European countries.

Bactericidal activity. Cefovecin exhibited bactericidal activity against the majority of tested gram-positive and gram-negative pathogens typical of the ß-lactam (cephalosporin) class. The majority of isolates showed MBC:MIC ratios of ≤2 for cefovecin and cephalexin (Table 4). Ratios for ceftiofur were ≥4 for P. multocida and P. mirabilis in 26% (n = 4) and 80% of investigated cases, respectively. However, the ceftiofur MICs for these four P. multocida isolates were extremely low (≤0.002 µg/ml), with corresponding MBCs at 0.016 µg/ml or 0.031 µg/ml, thus making higher MIC:MBC ratios irrelevant. Ratios for anaerobic-growing pathogens tended to be higher for all three antimicrobials than for aerobic pathogens.


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  		TABLE 4. Ratio of MBC results to reference (CLSI M31-A2) MICs for 100 contemporary isolates of gram-positive and gram-negative pathogens of canine and feline origin

In conclusion, cefovecin exhibited excellent in vitro activity across an extended spectrum of bacteria, encompassing all major pathogens with clinical relevance for common skin, urinary tract, and periodontal infections in dogs and cats. Due to the distinctive pharmacokinetic profile of cefovecin, a single injection provides a complete 14-day course of therapy. The molecule has the potential to be a unique addition to the well-established, safe cephalosporin class.


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FOOTNOTES
 
* Corresponding author. Mailing address: Pfizer Animal Health, Veterinary Medicine Clinical Development, IPC 896, Ramsgate Rd., Sandwich CT13 9NJ, United Kingdom. Phone: 44 1304 646121. Fax: 44 1304 657158. E-mail: michael.stegemann{at}pfizer.com. Back


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Antimicrobial Agents and Chemotherapy, July 2006, p. 2286-2292, Vol. 50, No. 7
0066-4804/06/$08.00+0     doi:10.1128/AAC.00077-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.




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