Determination of the minimum inhibitory concentration (MIC) and mutant prevention concentration (MPC) of selected antimicrobials in bovine and swine Pasteurella multocida, Escherichia coli, and Staphylococcus aureus isolates

We compared the values of the minimum inhibitory concentration (MIC) and mutant prevention concentration (MPC) values of three antimicrobial agents for 72 bovine isolates of Pasteurella multocida, 80 swine isolates of P. multocida, 80 bovine isolates of Escherichia coli, 80 swine isolates of E. coli, and 80 isolates of Staphylococcus aureus from bovine mastitis. The ratio of MIC90 /MPC90 which limited mutant selection window (MSW) was ≤ 0.12/4 mg/l for enrofloxacin, 0.5/≥ 64 mg/l for florfenicol and 4/≥ 128 mg/l for tulathromycin in bovine P. multocida isolates, ≤ 0.12/2 mg/l for enrofloxacin, 0.5/≥ 64 mg/l for florfenicol and 4/≥ 128 mg/l for tulathromycin in swine P. multocida isolates, 1/16 mg/l for enrofloxacin, 8/≥ 64 mg/l for florfenicol and 8/≥ 128 mg/l for tulathromycin in bovine E. coli isolates, 0.5/16 mg/l for enrofloxacin, ≥ 64/≥ 64 mg/l for florfenicol and 8/≥ 128 mg/l for tulathromycin in swine E. coli isolates, and 0.25/16 mg/l for enrofloxacin, 4/≥ 64 mg/l for florfenicol and 4/≥ 128 mg/l for tulathromycin in S. aureus isolates. These findings indicate that the dosage of antimicrobial agents to achieve serum concentration equal to or higher than MPC could reduce selection of resistant bacterial subpopulation. Bacteria, antibiotics, resistance, MIC, MPC, MSW, cattle, pigs Effective antimicrobial therapy is compromised by spreading bacterial resistance which has been detected worldwide at different levels also in breeding animals. The reduction of bacterial resistance over time is mostly associated with a reduction in the consumption of antimicrobials (Aarestrup et al. 2008). However, there are a number of bacteria with mutations that prevent the action of antimicrobial substances and cause the selection of a resistant mutant during clinical treatment in the bacterial population susceptible to antimicrobials (Canton and Morosini 2011). The antimicrobial agents are used for therapy on the basis of the dosage strategy for antimicrobials according to their pharmacokinetic (PK) and pharmacodynamic (PD) properties and determination of minimum inhibitory concentration (MIC) for the pathogen. A new concept in the testing of antimicrobial resistance with the potential to reduce bacterial resistance is the dosing of antimicrobials based on their PK/PD properties and determination of the mutant prevention concentration (MPC) for the pathogen (Drlica 2001). The MPC is defined as the concentration of antimicrobial substances able to prevent the growth of resistant mutants with a density of inoculum ≥ 109 CFU/ml. The MPC is an indicator of susceptibility designed for a more accurate assessment of the potential of selection of a resistant mutant to antimicrobial substances than traditional methods for the determination of susceptibility (Zhao and Drl ica 2008). It mostly better corresponds to the concentration of bacteria in the blood or in the target organ during acute infection of animals and people than MIC determination for inoculum with a density of 105 CFU/ml (Croisier et al. 2004; Zhao and Drl ica 2008). The concentration range between the MIC and the MPC is called the mutant selection window (MSW). When this window is wider, the risk of selection of resistant strains is also greater (Drlica 2001; Tam et al. 2005; Blondeau 2009). ACTA VET. BRNO 2015, 84: 83–89; doi:10.2754/avb201584020083 Address for correspondence: MVDr. Katerina Nedbalcova, Ph.D. Veterinary Research Institute, Department of Immunology Hudcova 70, 621 00 Brno, Czech Republic Phone: + 420 533 331 217 Fax: +420 541 211 229 E-mail: [email protected]; [email protected] http://actavet.vfu.cz/ The objective of this study was to compare the MIC and MPC values for enrofloxacin, florfenicol, and tulathromycin in E. coli, P. multocida, and S. aureus isolated from clinical cases of bovine or swine infections in the Czech Republic during the period from 2008 to 2012. Material and Methods Isolates Isolates of enterotoxigenic E. coli were obtained from enteric infections of post-weaned calves (80 isolates) and piglets (80 isolates) with diarrhoea, P. multocida isolates from the lungs after necropsy of fattening cattle (72 isolates) and nursery or grower-finisher pigs (80 isolates) with respiratory disorders, and 80 S. aureus isolates from milk of dairy cows suffering from mastitis over the period 2008–2012. All E. coli isolates were positive in testing for the presence of the gene coding the production of enterotoxin STa, according to Cheng et al. (2006). Pasteurella multocida isolates were identified by PCR (Townsend et al. 1998) a S. aureus isolates were identified biochemically using the commercial kit STAPHYTEST (Erba Lachema, Czech Republic). The samples originated from animals without previous antimicrobial therapy during the last 3 weeks and the isolates from the same herds were included in the monitoring of MICs and MPCs of antimicrobials, with a minimum interval of 6 months from the last collection. MIC, MPC and MSW determination Sussceptibility to antimicrobial substances was determined by assignment of MIC according to the standardised dilution micromethod of the Clinical and Laboratory Standards Institute (CLSI 2013a; 2013b) using commercial kits (Trek Diagnostics Systems Inc., England). The kit quality was controlled by the reference strains of E. coli ATCC 25922 and S. aureus ATCC 29213. The MIC values were defined as the lowest concentration of an antimicrobial agent inhibiting visible bacterial growth of culture with the density of 105 CFU/ml. Breakpoints of resistances were defined according to the CLSI and European Committee on Antimicrobial Susceptibility Testing (EUCAST 2013). Determination of the MPC was made in duplicate by the dilution method according to CLSI (2013a; 2013b), using a modification of the previously published method (Blondeau 2001; Randall et al. 2004). Quality control of the experiment was performed with the reference strains of E. coli ATCC 25922 and S. aureus ATCC 29213. The MPC was determined as the lowest concentration of an antimicrobial agent inhibiting visible growth of bacterial culture with density of 109 CFU/ml. The MSW was determined as the ranges of concentration between MPC90 and MIC90. Results Distribution of MICs for enrofloxacin, florfenicol, and tulathromycin in bovine and swine P. multocida isolates, bovine and swine E. coli isolates, and S. aureus isolates from dairy cows mastitis and calculation of the MIC50 and MIC90 values are in Table 1. There are also comparative MPC distribution data for the isolates tested against the 3 antimicrobial agents and calculation of the MPC50 and MPC90 values. Table 2 shows the percentages of susceptible, intermediately susceptible, and resistant P. multocida, E. coli, and S. aureus isolates to enrofloxacin, florfenicol, and tulathromycin. The ranges between MIC90 and MPC90 that present the MSW for enrofloxacin, florfenicol, and tulathromycin are also shown. As detected by the CLSI (2013a; 2013b) and EUCAST (2013) methods for MIC determination the majority of the tested bovine and swine P. multocida isolates were susceptible to the tested antimicrobial substances with MIC90 values below the breakpoint of resistance. On the other hand, bovine and swine E. coli isolates and S. aureus isolates from dairy cow mastitis were mostly resistant or intermediately susceptible to florfenicol. The MIC90 values for all three antimicrobials were the same in bovine and swine P. multocida isolates and S. aureus isolates, but differences were found between bovine and swine E. coli, except for tulathromycin. The MIC90 value for enrofloxacin was slightly higher in bovine E. coli isolates (bovine isolates 1 mg/l and swine isolates 0.5 mg/l) and marked difference (bovine isolates 8 mg/l and swine isolates ≥ 64 mg/l) was detected for florfenicol. The MPC values were above the susceptibility/resistance breakpoints of enrofloxacin, florfenicol, and tulathromycin in all cases. Even for florfenicol and tulathromycin, the MPC50 values 84