Antibiotic susceptibility of Brachyspira hyodysenteriae isolates from Czech swine farms: a 10-year follow-up study

Brachyspira hyodysenteriae is the causative agent of swine dysentery. Loss of clinical efficacy of some antimicrobial agents authorized for treating swine dysentery was observed on certain Czech pig farms. The aim of the present study was to evaluate the antimicrobial sensitivity of six antibiotics using a set of 202 randomly selected B. hyodysenteriae isolates obtained from farms in the Czech Republic between years 1997 and 2006. Minimum inhibitory concentration of antibiotics tylosin, lincomycin, tylvalosin, chlortetracyclin, tiamulin and valnemulin were tested, using an agar dilution method. All antibiotics tested showed an increase in minimal inhibitory concentrations. Continual decrease in susceptibility of B. hyodysenteriae isolates to tiamulin and valnemulin was observed. Multiresistant B. hyodysenteriae were isolated more frequently in the past years. Only a careful use of antibiotics can ensure their efficacy, especially in case of pleuromutilins, in the strategic therapy of swine dysentery. This rare study demonstrates the minimal inhibitory concentration changes of selected antidysenterics among Czech isolates of Brachyspira hyodysenteriae during a ten-year period. Pigs, swine dysentery, therapy, minimal inhibition concentration, antimicrobial resistance Brachyspira hyodysenteriae is an intestinal spirochete which colonizes the large intestine of pigs after being ingested, and induces diarrhoeal disease – swine dysentery (SD) (Hampson et al. 2006) which is spread worldwide. For the therapy and control of swine dysentery various antimicrobials have been used. The use of antimicrobials has been reduced in the past decade both due to a ban on nitroimidazoles and olaquindox, and the development of an acquired resistance of B. hyodysenteriae against formerly used antibiotics such as tylosin and lincomycin (Čížek et al. 2002). The situation resulted in an increased occurrence of clinical SD cases and spreading of the disease to other pig farms due to uncontrolled pig transports. Because of the gradual reduction of clinical efficacy of drugs of choice on swine farms with SD, the agent isolation and laboratory in vitro sensitivity tests were increasingly required. Pleuromutilins (tiamulin and valnemulin) became the last choice antibiotics for SD therapy on most affected farms. In the late 1990s, an increased use of pleuromutilins resulted in the rise of minimal inhibitory concentration (MIC) values (Lobová et al. 2004). Afterwards, a complete loss of clinical efficacy of one but sometimes even of both pleuromutilins was observed on some farms. When B. hyodysenteriae lost its sensitivity to most registered antimicrobials, there was a risk of spreading multiresistant clones of B. hyodysenteriae to other farms within integrations or even outside them. The aim of the study was to evaluate the MICs for six antibiotics most frequently used for the treatment of SD, using a set of randomly selected Brachyspira hyodysenteriae isolated from pigs in the Czech Republic during years 1997 and 2006. Materials and Methods Bacterial isolates and growth conditions In the period 1997–2006, the diagnostic service of the Institute of Infectious Diseases and Microbiology, UVPS Brno obtained 2 035 isolates of Brachyspira hyodysenteriae. Of those, 202 were randomly selected for ACTA VET. BRNO 2014, 83: 003–007; doi:10.2754/avb201483010003 Address for correspondence: Josef Prášek Institute of Infectious Diseases and Microbiology, Faculty of Veterinary Medicine University of Veterinary and Pharmaceutical Sciences Palackého tř. 1/3, 612 42 Brno, Czech Republic Phone: +420 775 956 986 Fax: +420 541 562 274 E-mail: [email protected] http://actavet.vfu.cz/ the 10-year follow-up study. Only one isolate per farm and year was chosen. Isolates of B. hyodysenteriae were confirmed by the assessment of strong haemolysis, microscopy and species specific PCR (La et al. 2003). The cultures were stored at -80 °C in cryoprotective medium. For susceptibility testing, thawed isolates were grown on Trypticase Soy agar (TSA) (BD BBL, USA) with 5% ovine blood for up to 3–4 days in anaerobic jars (Oxoid, UK) with AnaeroGen sachets (Oxoid, UK) at 37 °C. The purity of all cultures was confirmed by microscopy of crystal violet stained smears. Control strains of Brachyspira hyodysenteriae B78T (ATCC 27164T), Streptococcus pneumoniae ATCC 49619 and Staphylococcus aureus ATCC 29213 were included in the analyses as controls. Their MICs for pleuromutilin were published by Odland et al. (2000) and Karlsson et al. (2003). Susceptibility testing The antimicrobial susceptibility of Brachyspira hyodysenteriae isolates was determined by the agar dilution method based on Clinical and Laboratory Standards Institute (CLSI) guidelines for susceptibility testing of anaerobic bacteria (CLSI 2004). Wilkins-Chalgren anaerobe agar (CM 619, Oxoid) with 5% ovine blood (WCABA) was used to determine MICs. The antimicrobial agents tested were tylosin (Elanco, USA), acetylisovaleryltylosin (Eco, UK), lincomycin (Sigma, Czech Republic), chlortetracycline (Sigma, Czech Republic), tiamulin fumarate and valnemulin (Novartis AH, Switzerland). The concentrations of tested antibiotics were 0.031, 0.062, 0.125, 0.25, 0.5, 1, 2, 4, 8 and 16 μg/ml of tylosin, lincomycin, chlortetracyklin, tiamulin and valnemulin in this order. The concentrations of acetylisovaleryltylosin were 3.125, 6.25,12.5, 25, 50, 100, 200 μg/ml. Pure Brachyspira hyodysenteriae cultures were scraped from Trypticase Soy agar with sterile cotton swabs and suspended in 2 ml sterile phosphate buffered saline. The turbidity was adjusted with a photometer (Densi-La-Meter, LIAP, Latvia) to 1.0 McFarland standard. The suspension was added to the wells of a sterile microplate (Piove di Sacco, Italy) and multipoint inoculator (Trios, Czech Republic) was used to inoculate the agar surface. The final inoculum (7–10 μl) on the agar surface provided approximately 105 CFU per spot. After 3–4 day incubation at 37 °C, the result was read as MIC, i.e. the lowest concentration of the drug tested that prevented growth and haemolysis of the isolate on the inoculated spot. The growth of B. hyodysenteriae isolates was evaluated in parallel with controls on WCABA with no antimicrobial agents added. Three independent examinations of each of the B. hyodysenteriae isolates were made on different occasions with this test procedure. Statistical analysis Recorded MIC values were processed using MS Excel®. When a difference of one dilution was found in a set of three repeated tests, the value that was obtained twice was used for further computations. In the case of a two-dilution difference, the mean value was used. Results with differences greater than two dilutions were not evaluated, and repeated MIC tests were made. The new results were evaluated in the same way. The values obtained for each of the drugs tested were used for the computation of MIC50, MIC90 and the range of MICs.