Evaluating the in vitro susceptibility of bovine mastitis pathogens to a combination of kanamycin and cefalexin: Recommendations for a disk diffusion test

Abstract

Cows suffering from bovine mastitis have markedly reduced milk production because of inflammation within the udder subsequent to infection and damage from bacterial toxins. Antibiotic treatment is commonly used as a preventative and therapeutic measure for bovine mastitis. The most common pathogens include Staphylococcus aureus, various streptococci (Streptococcus dysgalactiae, Streptococcus uberis), and coliforms (Escherichia coli), which can be contracted from other infected cows or from the environment. A combination of kanamycin and cefalexin (1:1.5 wt/wt) is currently used therapeutically in Europe for the treatment of bovine mastitis, although standardized methods for the in vitro determination of the susceptibility of target pathogens have not been developed. This study evaluates the appropriate broth microdilution testing criteria for kanamycin and cefalexin administered in combination and reports the development of a disk diffusion test. At a ratio of kanamycin:cefalexin relevant to that observed in milk postadministration (10:1 wt/wt), the minimum inhibitory concentrations were determined against 307 isolates of target mastitis pathogens (staphylococci, streptococci, and E. coli). Based on achievable concentrations in milk and the resulting distribution of minimum inhibitory concentrations, preliminary broth breakpoints for kanamycin/cefalexin (10:1 fixed ratio) of <or=8/0.8 microg/mL susceptible, 16/1.6 microg/mL intermediate, and >or=32/3.2 microg/mL resistant were applied to evaluated staphylococci, streptococci, and E. coli. Parallel testing by disk diffusion and resulting error-rate bounded analysis using a combined disk concentration of 30 microg of kanamycin and 15 microg of cefalexin resulted in the establishment of preliminary disk interpretive breakpoints of >or=20 mm susceptible, 18 to 19 mm intermediate, and <or=17 mm resistant for staphylococci, streptococci (Strep. uberis and Strep. dysgalactiae only), and E. coli.

In vitro susceptibility of bovine mastitis pathogens to a combination of penicillin and framycetin: Development of interpretive criteria for testing by broth microdilution and disk diffusion

“…aureus veterinary isolates were determined to be framycetin-susceptible. The high degree of susceptibility of staphylococci to aminoglycosides observed with mastitis isolates was consistent with other veterinary surveillance studies (Gentilini et al, 2000;Erskine et al, 2002;Makovec and Ruegg, 2003;Aarestrup and Schwarz, 2006;Pillar et al, 2009;Silley et al, 2012). In contrast, penicillin resistance among these isolates ranged from 26.4% with CNS to 31.2% with Staph.…”

“…Using an ECOFF of 4 μg/mL for framycetin as determined by the overall MIC distribution ( Figure 2C), 98.2% of isolates were determined to be framycetin-susceptible. The levels of aminoglycoside susceptibility observed with veterinary isolates of E. coli is consistent with that observed in other veterinary surveillance studies (Erskine et al, 2002;Lehtolainen et al, 2003;Makovec and Ruegg, 2003;Wallmann et al, 2003;White, 2006;Pillar et al, 2009). As expected, penicillin had little to no activity alone against E. coli.…”

Kill rate of mastitis pathogens by a combination of cefalexin and kanamycin

“…For this study, cefalexin : kanamycin ratios and concentrations were derived from available PK data. Following infusion into the mammary gland the cefalexin : kanamycin concentration ratio changes in vivo from ratio 1·5 : 1 (original concentrations in the intramammary syringe) at time of drug administration, to 1·25 : 1 after 3 h, further to 1 : 2·3 after 6 h and c. 1 : 10 after 12 h (Pillar et al. 2009).…”

“…For this study, cefalexin : kanamycin ratios and concentrations were derived from available PK data. Following infusion into the mammary gland the cefalexin : kanamycin concentration ratio changes in vivo from ratio 1AE5 : 1 (original concentrations in the intramammary syringe) at time of drug administration, to 1AE25 : 1 after 3 h, further to 1 : 2AE3 after 6 h and c. 1 : 10 after 12 h (Pillar et al 2009). The study from Ganiere and Denuault (2009), which investigated the in vitro bactericidal activity of the combination at a fixed ratio of 1AE5 : 1, indicated that the combination mainly exhibits a concentration-dependent bactericidal activity and that complete kill of major mastitis pathogens could be achieved within 4-6 h. Ganiere et al, however, did not show whether the same killing rates could be achieved even if the ratio of the two antibiotics is changing.…”

Plant Essential Oils and Mastitis Disease: Their Potential Inhibitory Effects on Pro-inflammatory Cytokine Production in Response to Bacteria Related Inflammation

“…Although stress and physical injuries may be other causes of inflammation of the udders, infections by bacteria or other microorganisms remain the major cause [51,52]. The most common aetiological agents include Staphylococcus aureus, various streptococci (Streptococcus dysgalactiae, Streptococcus uberis), and coliforms (Escherichia coli), which can be contracted from other infected cows or directly from the environment [53,54]. In response to bacterial infiltration in dairy cows, there are two major forms of inflammation of the mammary glands: clinical and subclinical mastitis.…”