325 Veterinaria Italiana 2022, 58 (3), 325-329. doi: 10.12834/VetIt.2241.16176.1 Accepted: 09.09.2021 | Available on line: 31.12.2022 1Department of Pharmacology, Faculty of Veterinary Medicine, University of Murcia, Campus de Espinardo, 30.071‑Murcia, Spain. 2Escuela de Medicina Veterinaria, Universidad San Francisco de Quito, EC 170157, Cumbayá, Ecuador. *Corresponding author at: Department of Pharmacology, Faculty of Veterinary Medicine, University of Murcia, Campus de Espinardo, 30.071‑Murcia, Spain Tel.: +34 868 888404, Fax: +34 868 884147, e‑mail: pmarin@um.es. Elena Badillo1, Elisa Escudero1, Juan Sebastian Galecio1,2, Verónica Hernandis1 and Pedro Marín1* Keywords Cephalosporins, Staphylococcus aureus, Minimum inhibitory concentration (MIC), Goats, Rabbits. Summary Antimicrobial drug resistance is an important problem that challenges veterinary clinicians to provide effective treatments without further spreading this resistance to other animals and people. The most commonly used pharmacodynamic parameter to define potency of antimicrobial drugs is minimum inhibitory concentration (MIC). The aim of this study was to evaluate the antibiotic susceptibility of thirty-six strains of Staphylococcus aureus isolated from dairy goats with mastitis and rabbits with chronic staphylococcosis. Four cephalosporins were tested: cephalexin, cephalotin, cefonicid and ceftiofur. MIC tests were performed according to the microdilution broth method. The calculated values of sensitivity in goats and rabbits were 66.67% and 72.22% for cephalexin, 72.22 % and 94.44% for cefonicid, 77.78% and 94.44% for cephalotin and 77.78% and 100% for ceftiofur, respectively. For all antibiotics, MIC 90 of S. aureus from rabbits were lower than MIC 90 from goats. These data suggest that more antibiotics are used in goat milk production than in rabbit farming. According to MIC values obtained in this study, ceftiofur and cephalotin may be the best option for treating S. aureus infections in lactating goats. For rabbits, ceftiofur showed lowest MIC values, but cephalosporins can produce fatal diarrhoea in this species, therefore additional studies are needed to evaluate the effects of repeated ceftiofur administration on microflora of rabbits before recommending the use of this antibiotic in this species. Cephalosporin susceptibility of Staphylococcus aureus strains isolated from commercial rabbit and goat farms in Spain cephalothin) second (cefonicid), third (ceftiofur) and fourth generation compounds displaying a variable activity in  vitro, structural similarities and the time of their introduction into the market (Caprile 1988). From first to fourth generation, the spectrum of activity against gram negative organisms and the stability against β-lactamase increase commonly together with the same or reduced spectrum of activity against gram positive organisms, except for fourth generation agents, which have enhanced activity against gram positive organisms (Papich and Lindeman 2018). Cephalotin (CL) and cefonicid (CD) are developed for use in human medicine, cephalexin (CX) for use in human and veterinary medicine and ceftiofur (CR) for exclusive use in animals and approved for use in food animals in Europe. Staphylococcus aureus is one of the most important pathogenic Staphylococcus species in veterinary medicine. In rabbits, it mainly causes skin infections, pododermatitis and mastitis with subsequent economic losses in industrial rabbitries (Hermans et  al. 1999). In goats, Staphylococcus aureus is probably the most important infectious agent (Mørk et al. 2005), since it causes a chronic and deep infection in the mammary glands which implies an evident economic loss in relation to the decrease in milk production and health status of animals (Seegers et al. 2003). Cephalosporins are a broad group of antimicrobial agents with a bactericidal mechanism of action similar to all β-lactam antibiotics inhibiting cell-wall synthesis. In accordance with their antimicrobial spectrum, they are divided into first (cephalexin, SHORT COMMUNICATION 326 Veterinaria Italiana 2022, 58 (3), 325-329. doi: 10.12834/VetIt.2241.16176.1 Cephalosporin susceptibility of Staphylococcus aureus strains Badillo et al. The antibiotics selected for the study were provided by manufacturers in this way: CR and CL by Sigma-Aldrich (ST. Louis, USA), CX by Tokyo Chemical Industry (Tokyo, Japan) and CD by Santa Cruz Biotechnology (Heidelberg, Germany). According to the Clinical and Laboratory Standards Institute’s recommendations, antibiotic stock solutions were prepared at concentrations of 1,280 mg/L by dissolution in suitable solvents and then diluted in sterile distilled water (CLSI 2015). Minimum inhibitory concentration (MIC) tests were performed according to the microdilution broth method recommended by the CLSI (CLSI 2015) using U-bottomed, 96-well microtiter plates. Serial two-fold dilutions of the antimicrobial agents were prepared starting from the stock solution of each drug (concentrations ranging from 0.03 to 128 mg/l). Inocula were prepared by diluting an overnight Mueller-Hinton Broth culture in buffered saline solution to a density of 0.5 McFarland turbidity scale and finally diluting again 40-fold before testing. The MIC was defined as the lowest concentration at which bacterial growth was completely inhibited, that is the lowest concentration of an antimicrobial agent that prevents visible growth of a microorganism in a broth dilution susceptibility test (CLSI 2015). The MIC values of these drugs that inhibited 50% and 90% of the isolates were expressed as MIC 50 and MIC 90 , respectively. In relation to breakpoints of susceptibility and resistance, a strain was considered resistant when the MIC was ≥ 8 mg/L for CD, CX, CL and CR (CLSI 2018). Reference strains of S. aureus (ATCC 29213) and E. coli (ATCC 25922) were used as controls for each plate. In this case, all results for S. aureus ATCC 29213 and E.  coli ATCC 25922 were within the accepted range (CLSI 2018). The specific MIC values for each selected antimicrobial agent are showed in Table I, where also MIC 50 and MIC 90 , and the minimum and maximum MIC values (range) are included. In goats, it was According to the European Medicines Agency (EMA), antibiotics are classified based on the potential consequences for public health of increased antimicrobial resistance due to their use in animals and depending on the necessity to use them in veterinary medicine. In the EMA categorization, there are four categories, A (Avoid), B (Restrict), C  (Caution), and D (Prudence), with the latter as the lowest risk category (EMA 2019). Antibiotics from the A category are reserved for human treatment only, and their use is not allowed in food-producing animals. B category antibiotics (as third and fourth-generation cephalosporins) are critically important antimicrobials, and should be used only as a last option after susceptibility testing has been conducted when no other antibiotic would be clinically effective. C category antibiotics (as first and second-generation cephalosporins) should be considered only when there are no antibiotics in D Category that could be clinically effective. Because of this, the antibiotic consumption in animal production should be progressively reduced in order to decrease the antimicrobial resistance against the antibiotics to which the bacteria have been exposed. Thus, the purpose of this study was designed to evaluate the degree of in  vitro activity of CD, CX, CL and CR against 36 S.  aureus strains, isolated from rabbits with chronic staphylococcosis and goats with clinical mastitis in Spain. A number of 18 S.  aureus strains were isolated from rabbit farms with mastitis, subcutaneous abscesses and pododermatitis in the southeast region of Spain. Similarly, 18 S.  aureus strains were isolated from milk of goats with clinical mastitis. The plates were incubated aerobically at 37 °C to be examined at 24, 48, and 72 h. Bacteria with target hemolysis were identified (presumptive) by catalase test and Gram staining. Commercial latex agglutination kit (Staphytec Plus, Oxoid) was used by specific identification of S. aureus. The strains were stored at - 80 °C in a nutrient broth enriched with 15% glycerol. Table I. Frequency of Staphylococcus aureus strains in relation to their minimal inhibitory concentrations and MIC 50 , MIC 90 , and MIC range values of cefonicid, ceftiofur, cephalotin and cephalexin for rabbits (n = 18) and goats (n = 18). Antibiotics Animals Number of strains with MIC (mg/L) MIC 50 (mg/L) MIC 90 (mg/L) MIC range (mg/L) 0.03 0.06 0.12 0.25 0.5 1 2 4 8 16 32 64 128 Cefonicid Goats - - - - 1 3 3 6 1 - - - 4 4 128 0.5-128 Rabbits - - - 1 - 3 5 8 - - - - 1 2 4 0.25-128 Ceftiofur Goats - - - - 3 8 3 - - 2 1 1 - 1 32 0.5-64 Rabbits - - - 1 3 11 2 1 - - - - - 1 2 0.25-4 Cephalotin Goats - - 3 7 4 - - - - 1 2 - 1 0.25 32 0.12-128 Rabbits - - 2 10 4 - - 1 - 1 - - - 0.25 4 0.12-16 Cephalexin Goats - - - - - 1 3 8 2 - - 2 2 4 128 1-128 Rabbits - - 1 - - - 2 10 4 - - 1 - 4 8 0.12-64 327Veterinaria Italiana 2022, 58 (3), 325-329. doi: 10.12834/VetIt.2241.16176.1 Badillo et al. Cephalosporin susceptibility of Staphylococcus aureus strains caused by Gram-positive bacteria is becoming a difficult problem. The situation is particularly critical in infections caused by S.  aureus which is also frequently resistant to multiple groups of antibiotics (Livermore 2000). One of the strategies proposed for the control of S. aureus infections is the re-evaluation of older antimicrobial agents and develop them for using against this type of microorganisms with high degrees of resistance (Chopra et  al. 1997). Therefore, first and second generation of cephalosporins and ceftiofur have become a good option for the treatment of infectious diseases in veterinary medicine. The ratio T > MIC is the best parameter for predicting the antimicrobial effect of time-dependent antimicrobial agents as β-lactam antibiotics. It has been previously determined that the maximal bactericidal action for cephalosporins is reached when plasma concentrations are greater than the MIC of the pathogen for 60-70% of the dosing interval and a bacteriostatic effect is observed when T > MIC is 30-40% of the dosing interval (Drusano 2004). Distribution of cephalosporins to the mammary gland and the consequent access to milk is limited. Pharmacokinetic parameters of these four cephalosphorins in lactating goats (Lacchini et  al. 2007, Doré et  al. 2011, Fernández-Varón et  al. 2016, Badillo et  al. 2020) after different routes of administration showed a low elimination through the milk. Therefore, the treatment of mastitis in goats with these cephalosporins should be performed by intramammary administrations. According to MIC values obtained in the present study, CR and CL may be the best options for treating S. aureus infections in these species due to their lowest MIC values. Considering MIC values obtained in this study and pharmacokinetic parameters in rabbits (Dzierzanowska et  al. 1982, Meneses et  al. 2013, Gardhouse et  al. 2017) after different routes of administration, CR could be an option for treating S.  aureus infections on account to the lowest MIC value, high plasma levels and long half-life showed in this species. However, CR (B category) should be used only as a last option after susceptibility testing has been conducted when no other antibiotic would be clinically effective. Moreover, cephalosporins can disrupt the caecal microflora in rabbits (because of biliary elimination of some members of this group) resulting sometimes in fatal diarrhoea. Although single-dose parenteral administration of CR resulted in minimal adverse effects (Gardhouse et  al. 2017), additional studies are needed to evaluate the effects of repeated CR administration in rabbits and to develop and implement appropriate specific dosing regimens that can maximize its clinical efficacy for use in production animals and reduce the risk of selection of resistant pathogens. observed four resistant strains for CR and CL each, five for CD and six for CX. In contrast, the number of resistant strains in rabbits was considerably lower, one resistant strain for CD and CL each, zero for CR and five resistant strains for CX were found. The calculated values of sensitivity in goats and rabbits were 66.6% and 72.2% for CX, 72.2 % and 94.4% for CD, 77.7% and 94.4% for CL and 77.7% and 100% for CR, respectively. The lowest MIC 90 were obtained in goats for CR and CL (MIC 90 = 32 mg/L). Instead in rabbits, CR showed a lower value (MIC 90 = 2 mg/L) than CD (MIC 90 = 4 mg/L), CL (MIC 90 = 4 mg/L) and CX (MIC 90 = 8 mg/L). Indeed, for all antibiotics, MIC 90 of S.  aureus from rabbits were lower than MIC 90 from goats. These evidences suggest that more antibiotics are used in goat milk production than in rabbit farming. There are reduced reported data on cephalosporin susceptibility of S.  aureus isolated from milk of goats with subclinical or clinical mastitis. For CL, our data were higher than those obtained in a previous study (MIC 50 = 0.25 mg/l, MIC 90 = 0.25 mg/l, range 0.06-128 mg/l, Virdis et  al. 2010). There are no reported data with CD, CR and CX in S.  aureus isolated from goats. However, published research papers on cephalosporin susceptibility of S.  aureus isolated from bovine milk are numerous. In these studies, reported MIC values were lower than those obtained in this research for CR (MIC 50 = 1 mg/L, MIC 90 = 1 mg/L, range 0.06-64 mg/L, de  Oliveira et  al. 2000; MIC 50 = 1 mg/L, MIC 90 = 1 mg/L range 0.25-8 mg/L, De Jong et al. 2015), CX (MIC 50  =2 mg/L, MIC 90 = 4 mg/L, range 0.25-64 mg/L, De Jong et  al. 2015) and CL (MIC 50 = 0.25 mg/L, MIC 90 = 0.5 mg/L, Lindeman et  al. 2013). Cefonicid has not been studied in veterinary medicine, but susceptibility tests for S.  aureus from human isolates showed values of MIC 50 = 4 mg/l and MIC 90 = 8 mg/l (Barry et  al. 1986). There are no reported MICs for four cephalosporins tested in this study against isolated bacteria from rabbits. However, the obtained values of MIC 90 were high compared to the cefiofur and cephalexin breakpoints [≤  2 μg/mL (susceptible), 4  μg/mL (intermediate), and ≥ 8 μg/mL (resistant)] reported for other bacterial species isolated from other animals (De Jong et  al. 2014, Papich and Lindeman 2018). S.  aureus affects rabbits producing important dermal lesions and even invading subcutaneous tissues, resulting in suppurative dermatitis, mastitis, multisystemic abscessation and pododermatitis (Segura et al. 2007). In addition, S. aureus is probably the most significant infectious agent that can cause mastitis in cows, goats, and sheep, because it produces a chronic and deep infection in the mammary glands that is extremely difficult to treat (Mørk et  al. 2005, Aires de Sousa et  al. 2007). Due to antibiotic resistance, treatment of infections 328 Veterinaria Italiana 2022, 58 (3), 325-329. doi: 10.12834/VetIt.2241.16176.1 Cephalosporin susceptibility of Staphylococcus aureus strains Badillo et al. Aires-de-Sousa M., Parente C.E., Vieira-da-Motta O., Bonna I.C., Silva D.A. & de Lencastre H. 2007. Characterization of Staphylococcus aureus isolates from buffalo, bovine, ovine, and caprine milk samples collected in Rio de Janeiro State, Brazil. Appl Environ Microbiol, 73, 3845-3849. Badillo E., Escudero E., Hernandis V., Galecio J.S. & Marín P. 2020. Pharmacokinetics of cefonicid in lactating goats after intravenous, intramuscular and subcutaneous administration, and after a long-acting formulation for subcutaneous administration. J Vet Pharmacol Therap, 43 (1), 50-56. Barry A.L., Jones R. & Packer R.R. 1986. Antistaphylococcal activity of ceforanide and cefonicid in the presence of human serum. Antimicrob Agents Chemother, 29 (1), 147-149. Caprile K.A. 1988. The cephalosporin antimicrobial agents: a comprehensive review. J Vet Pharmacol Therap, 11, 1-32. Chopra I., Hodgson J., Metcalf B. & Poste G. 1997. The search for antimicrobial agents effective against bacteria resistant to multiple antibiotics. Antimicrob Agents Chemother, 41 (3), 497-503. Clinical and Laboratory Standards Institute (CLSI). 2015. Methods for dilution antimicrobial susceptibility test for bacteria that grow aerobically. 10th Edition. NCCLS document M07-A10. Clinical and Laboratory Standards Institute, Pennsylvania. Clinical and Laboratory Standards Institute (CLSI). 2018. Performance Standards for Antimicrobial Susceptibility Testing. 28th Edition. NCCLS document M100. Clinical and Laboratory Standards Institute, Pennsylvania. De Jong A., Moyaert H., Simjee S., El Garch F., Haag-Diergarten S., Klein U., Ludwig C., Butty P., Richard-Mazet A., Rigaut D., Thiry J. & Thomas V. 2015. Antimicrobial susceptibility monitoring of mastitis pathogens isolated from acute cases of clinical mastitis in dairy cows across Europe: VetPath results. Int J Antimicrob Agents, 46, 13-20. De Jong A., Thomas V., Simjee S., Moyaert H., El Garch F., Maher K., Morrissey I., Butty P., Klein U., Marion H., Rigaut D. & Vallé M. 2014. Antimicrobial susceptibility monitoring of respiratory tractpathogens isolated from diseased cattle and pigs acrossEurope: The VetPath study. Vet Microbiol, 172, 202-215. De Oliveira A.P., Watts J.L., Salmon S.A. & Aarestrup F.M. 2000. Antimicrobial Susceptibility of Staphylococcus aureus isolated from bovine mastitis in Europe and the United States. J Dairy Sci, 83 (4), 855-862. Doré E., Angelos J.A., Rowe J.D., Carlson J.L., Weizlich S.E., Kieu H.T. & Tell L.A. 2011. Pharmacokinetics of ceftiofur crystalline free acid after single subcutaneous administration in lactating and nonlactating domestic goats (Capra aegagrus hircus). J Vet Pharmacol Therap, 34 (1), 25-30. Drusano G.L. 2004. Antimicrobial pharmacodynamics: critical interactions of “bug and drugs”. Nat Rev Microbiol, 2 (4), 289-300. References Dzierzanowska D., Patzer J. & Jeljaszewicz J. 1982. Pharmacokinetic studies of azlocillin, mezlocillin, cephalothin and sisomicin in rabbits. Infection, 10 (3), S217-20. European Medicines Agency (EMA). 2019. Categorisation of antibiotics in the European Union. Answer to the request from the European Commission for updating the scientific advice on the impact on Public Health and Animal Health of the use of antibiotics in animals. European Medicines Agency, Amsterdam, The Netherlands. Fernández-Varón E., Cárceles-García C., Serrano-Rodríguez J.M. & Cárceles-Rodríguez C.M. 2016. Pharmacokinetics (PK), pharmacodynamics (PD), and PK-PD integration of ceftiofur after a single intravenous, subcutaneous and subcutaneous-LA administration in lactating goats. BMC Vet Res, 12 (1), 232. Gardhouse S., Guzman D.S., Cox S., Kass P.H., Drazenovich T.L., Byrne B.A. & Hawkins M.G. 2017. Pharmacokinetics and safety of ceftiofur crystalline free acid in New Zealand White rabbits (Oryctolagus cuniculus). Am J Vet Res, 78 (7), 796-803. Hermans K., De Herdt P., Devriese L.A., Hendrickx W., Godard C. & Haesebrouck F. 1999. Colonisation of rabbits with Staphylococcus aureus in flocks with and without chronic staphylococcosis. Vet Microbiol, 67, 37-46. Lacchini R., Rule R., García-Román A., Antonini A. & de Buschiazzo P. 2007. Influence of feed type on the pharmacokinetics of cephalothin administered to lactating goats. Arch Zootec, 56, 807-815. Lindeman C.J., Portis E., Johansen L., Mullins L.M. & Stoltman G.A. 2013. Susceptibility to antimicrobial agents among bovine mastitis pathogens isolated from North American dairy cattle, 2002-2010. J Vet Diagn Invest, 25 (5), 581-591. Livermore D.M. 2000. Antibiotic resistance in staphylococci. Int J Antimicrob Agents, 16, S3-10. Meneses M.L., Albarellos G. & Landoni M.F. 2013. Pharmacokinetics of cephalexin after intravenous and single and multiple intramuscular administration to rabbit. Int J Vet Med: Res Rep, 2013, 898594. Mørk T., Tollersrud T., Kvitle B., Jørgensen H.J. & Waage S. 2005. Comparison of Staphylococcus aureus genotypes recovered from cases of bovine, ovine, and caprine mastitis. J Clin Microbiol, 43, 3979-3984. Papich M.G. & Lindeman C. 2018. Cephalexin susceptibility breakpoint for veterinary isoletes: Clinical Laboratory Standars Institute revision. J Vet Diagn Invest, 30 (1), 113-120. Seegers H., Fourichon C. & Beaudeau F. 2003. Production effects related to mastitis and mastitis economics in dairy cattle herds. Vet Res, 34, 475-491. Segura P., Martínez J., Peris B., Selva L., Viana D., Penades J.R. & Corpa J.M. 2007. Staphylococcal infections in rabbits does on two industrial farms. Vet Record, 160 (25), 869-872. 329Veterinaria Italiana 2022, 58 (3), 325-329. doi: 10.12834/VetIt.2241.16176.1 Badillo et al. Cephalosporin susceptibility of Staphylococcus aureus strains Virdis S., Scarano C., Cossu F., Spanu V., Spanu C. & De Santis E.P.L. 2010. Antibiotic resistance in Staphylococcus aureus and coagulase negative staphylococci isolated from goats with subclinical mastitis. Vet Med Int, 2010 (2), 517060.