The determination of minimum inhibitory concentrations of selected antimicrobials for porcine Haemophilus parasuis isolates from the Czech Republic

Haemophilus parasuis isolates obtained from pigs in the Czech Republic were tested for their susceptibility to amoxicillin, penicillin, ceftiofur, enrofloxacin, tetracycline, and tulathromycin by determination of minimum inhibitory concentrations using the broth microdilution method. The H. parasuis isolates were mostly susceptible to majority of tested antimicrobials (amoxicillin 90%, penicillin 73.3%, enrofloxacin 83.3%, and tulathromycin 83.3%). All isolates were susceptible to ceftiofur. On the other hand, no isolate was susceptible to tetracycline, 30% of tested isolates were intermediately susceptible, and 70% were resistant. These findings indicate that tested antimicrobials with the exception of tetracycline should be the preferred option used for the treatment of infection caused by H. parasuis but due to the potential transmission of resistance from animals to humans, the use of ceftiofur is considered as a last resort option in antimicrobial treatment of animals. Bacterial infection, susceptibility, resistance, pigs Haemophilus parasuis may be the cause of great economic loss for breeders due to the cost of antibiotic therapy and piglet death in the acute forms of the disease (Oliveira et al. 2001). In conventional herds, H. parasuis is predominantly involved in the respiratory syndrome but can also cause acute septicaemia or Glässer’s disease, systemic illness characterized by fibrinous polyserositis, polyarthritis, and meningitis (Amano et al. 1994). Infections caused by H. parasuis can be enzootic and may be acute or chronic, depending on the immunological situation of the breeding herd. If H. parasuis entered into farms with immunologically naive animals, it can cause serious acute illness and the main affected age categories are early weaned piglets about the age of 4–6 weeks (Nicolet 1992). A high antigenic heterogeneity exists among H. parasuis strains. According to the currently worldwide accepted classification, 15 serovars of H. parasuis (1–15) have been defined. However, it is necessary to say that a large number of non-typeable H. parasuis isolates also exist (Kielstein and Rapp-Gabrielson 1992). Antibiotic treatment is one of the commonly used measures for the control of H. parasuis infections. However, the use of antimicrobial agents may lead to both selection and increase of resistance (Schwarz et al. 2001). Correct use of antimicrobial agents for treatment of bacterial infections requires the knowledge of the susceptibility of the infecting strain to antimicrobial agents to enhance efficacy and to prevent the emergence of resistance among other organisms, which may be causing respiratory diseases in pigs (De la Fuente et al. 2007). Moreover, continuous monitoring of the prevalence of drug resistance of bacterial pathogens to antimicrobials provides essential data on the state and development of the susceptibility in pathogens causing bacterial infections. These data are necessary for the prediction of an antimicrobial’s efficacy in the initial treatment and to determine the position of individual substances as ‘drugs of choice’ or as alternative drugs in the therapy of infections. Susceptibility tests are therefore the basis of the formulation of objective antimicrobial treatment and prevention strategies (Nedbalcova et al. 2013). ACTA VET. BRNO 2017, 86: 175–181; https://doi.org/10.2754/avb201786020175 Address for correspondence: Dr. Kateřina Nedbalcová Veterinary Research Institute Hudcova 70, 621 32 Brno, Czech Republic Phone: 420 533 331 217 Fax: 420 541 211 229 E-mail: nedbalcova@vri.cz http://actavet.vfu. cz/ The objective of this study was to determine the minimum inhibitory concentrations (MICs) for selected antimicrobials (amoxicillin, penicillin, ceftiofur, enrofloxacin, tetracycline, and tulathromycin) in the Czech field strains of Haemophilus parasuis according to the latest guidelines of the Clinical and Laboratory Standards Institute (CLSI 2013a; CLSI 2013b). Materials and Methods Isolates Thirty isolates of H. parasuis (1–30) were obtained from diseased pigs from pig herds in the Czech Republic from 2014 to 2015. Haemophilus parasuis isolates were isolated on Columbia blood agar (OXOID, England) plates with 5% sheep blood with a nurse strain of Staphylococcus aureus and then on chocolate blood agar (OXOID, England) plates with 5% sheep blood for β-nikotinamid adenin dinucleotide growth dependence of H. parasuis for 24–48 h at 37 °C, were confirmed by polymerase chain reaction test (Oliveira et al. 2001), and serotyped by modified co-agglutination test originally described for Actinobacillus pleuropneumoniae (Mittal et al. 1983). All isolates were stored at –80 °C in vials containing 0.25 ml Foetal Bovine Serum Gold (PAA Laboratories GmbH, Austria) and 0.25 ml of Cation Adjusted Mueller Hinton Broth II (CAMHB) (Becton, Dickinson and Company, USA). All isolates originated from animals without previous antimicrobial therapy during the last 3 weeks and only individual isolates from individual herds were included in this study. MIC determination The MICs of selected antimicrobials for the isolates of H. parasuis were determined by the standardized dilution micro-method for Actinobacillus pleuropneumoniae and Histophilus somni (CLSI 2013a; CLSI 2013b). Preparation of microdilution trays According to these documents, special microdilution trays were prepared. These trays included dilutions of tested antimicrobial agents (mg/l) in Veterinary Fastidious Medium (VFM) prepared according to CLSI (2013a). The tested antimicrobials and their concentrations are shown in Table 1. Inoculum preparation, inoculation and incubation of trays The culture of H. parasuis grown on blood agar was re-suspended in 5 ml CAMHB (Becton, Dickinson and Company, USA) and the density of the suspension was adjusted to a turbidity equivalent to that of a 0.5 McFarland standard and 5 μl of inoculum with the density of 0.5 McFarland standard were transferred into all of the wells of the micro-titre plates containing 100 ml of medium. The trays were incubated at 35°C in CO2 for 24 h. Interpretation of results The MICs values were read at the lowest concentration of an antimicrobial agent that inhibited the visible bacterial growth in the wells. The break-points of tested antimicrobials for H. parasuis are derived from breakpoints for A. pleuropneumoniae and Pasteurella multocida according to CLSI (2013b) and they are shown in Table 2. Quality control Quality control of the results was performed with reference strains of Actinobacillus pleuropneumoniae (ATCC 27090). The acceptable quality control ranges for tested antimicrobials are in CLSI document Vet 01-S2 (2013b). 176 Table 1. Microplate tray with concentrations of tested antimicrobials (mg/l). PNC penicillin; AMX amoxicillin; EFT ceftiofur; TUL tulathromycine; TET tetracycline; ENR enrofloxacin; PC positive growth control. PNC AMX EFT TUL TET ENR PNC AMX EFT TUL TET ENR 8 4 16 64 64 4 8 4 16 64 64 PC 4 2 8 32 32 2 4 2 8 32 32 2 2 1 4 16 16 1 2 1 4 16 16 1 1 0.5 2 8 8 0.5 1 0.5 2 8 8 0.5 0.5 0.25 1 4 4 0.25 0.5 0.25 1 4 4 0.25 0.25 0.125 0.5 2 2 0.125 0.25 0.125 0.5 2 2 0.125 0.125 0.06 0.25 1 1 0.06 0.125 0.06 0.25 1 1 0.06 0.06 0.03 0.125 0.5 0.5 0.03 0.06 0.03 0.125 0.5 0.5 0.03