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Decreased susceptibility to tiamulin and valnemulin among Czech isolates of Brachyspira hyodysenteriae

Institute of Microbiology and Immunology, Section of Pathobiology, Faculty of Veterinary Medicine, University of Veterinary and Pharmaceutical Sciences, Palackého 1-3, 612 42 Brno, Czech Republic

Correspondence Alois Cizek cizeka{at}vfu.cz

Received July 30, 2003
Accepted January 15, 2004

The agar dilution method was used to investigate the sensitivity to pleuromutilins of 100 isolates of Brachyspira hyodysenteriae isolated from 63 pig farms between 1997 and 2001. In the period under investigation, MICs to both tiamulin and valnemulin increased, with differences between the periods 1997–98 and 1999–2001 being statistically significant (P < 0.001 for tiamulin and P < 0.0001 for valnemulin). Between 1997 and 2001, the MIC₅₀ and MIC₉₀ of tiamulin increased from 0.062 and 0.25 µg ml^-1, respectively, to 1.0 and 4.0 µg ml^-1. Valnemulin MIC₅₀ and MIC₉₀ were 0.031 µg ml^-1 in 1997 and by 2001 were respectively, 2.0 and 8.0 µg ml^-1. The increase in MICs of tiamulin and valnemulin demonstrated in this study reflect the intensity of pleuromutilin use in the treatment of swine dysentery in the Czech Republic.

Abbreviations: SD, swine dysentery; TSBA, trypticase soy agar with 5 % ovine blood; WCABA, Wilkins–Chalgren anaerobe agar with 5 % ovine blood.

	INTRODUCTION

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
The intestinal spirochaete Brachyspira hyodysenteriae is the causative agent of swine dysentery (SD), which is manifested by severe mucohaemorrhagic colitis in pigs mainly during the growing–finishing period (Harris & Lysons, 1992). SD is a cause of considerable financial loss (Hampson et al., 1997), particularly when the infection spreads from herds with endemic disease to disease-free ones. Historically, the incidence of SD in the Czech Republic has been monitored by the State Veterinary Authority. The data from the 1980s (Fig. 1), when an obligation to report SD was still in force, show that there were between four and 20 clinically active SD sites identified per year. Following the liberalization of the trade in pigs, and the withdrawal in 1995 of SD from the list of contagious diseases subject to obligatory reporting, the number of farms with clinical forms of SD increased significantly during the mid-1990s. Another increase in the number of pig farms with clinical cases of SD in the Czech Republic was observed after 1998, when nitroimidazoles and the growth promoter olaquindox (which has anti-dysenteric effects) were banned for the treatment of SD. Up to that time, tylosin, lincomycin and tiamulin were also used in the treatment of SD in the Czech Republic, but as in other countries, B. hyodysenteriae isolates resistant to tylosin and lincomycin were frequently reported (Cizek et al., 1998). The situation worsened in 1999, leading to the introduction of targeted treatment with pleuromutilins on farms with endemic SD. At the time, it was also common to use feed containing preventive medication in at-risk herds. Increasingly frequent field reports of reduced clinical efficiency of pleuromutilins in the treatment of SD highlighted the need for a laboratory assessment of pleuromutilin efficacy. Pleuromutilins (tiamulin and valnemulin) are semi-synthetic derivatives of the naturally occurring diterpene antibiotic pleuromutilin. Both have outstanding activity against anaerobic bacteria and are used exclusively in animals, largely in swine.

View larger version (20K): [in this window] [in a new window]   Fig. 1. Number of Czech pig farms with clinical cases of swine dysentery.

In the past, dilution methods with various blood agars were used in studies of B. hyodysenteriae sensitivity to antimicrobials (Kitai et al., 1987; Molnar, 1996; Hommez et al., 1998; Fossi et al., 1999), and attempts were also made to verify the broth dilution procedure (Weber & Earley, 1991; Buller & Hampson, 1994). In recent years, attention has focused on the standardization of these methods and a better understanding of the mechanism that triggers B. hyodysenteriae antimicrobial resistance, including its genetic background (Karlsson et al., 1999, 2001, 2003).

The aim of this study was to evaluate tiamulin and valnemulin MICs using a set of randomly selected isolates of B. hyodysenteriae obtained from farms in the Czech Republic between 1997 and 2001, and to assess any changes in sensitivity to pleuromutilins.

	METHODS

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Bacterial cultures.

The study included a total of 100 B. hyodysenteriae isolates from clinical cases of SD from 63 pig farms in 28 districts of the Czech Republic between 1997 and 2001. For study purposes, 20 isolates representing different years were chosen at random. The only restriction was that no more than two isolates from individual farms, from the period under investigation (1997–2001), were selected. The isolates, stored in tryptic soy broth (BBL) containing 10 % fetal bovine serum at -80 °C, were thawed, cultivated and confirmed by strong ß-haemolysis on trypticase soy agar with 5 % ovine blood (TSBA), biochemical activity (Fellström & Gunnarsson, 1995) and a species-specific PCR (Elder et al., 1994). The type strains B. hyodysenteriae B78^T (ATCC 27164^T), Brachyspira innocens B256^T (ATCC 29796^T), Brachyspira pilosicoli P43/6/78 (ATCC 51139^T) and Brachyspira murdochii 56-150^T (ATCC 51284^T) were used to test the identification process.

Antimicrobial agents.

Tiamulin and valnemulin powders (Novartis) were dissolved and diluted in accordance with the instructions of the drug manufacturer and of the National Committee for Clinical Laboratory Standards (NCCLS, 2001). Stock solutions were stored in polystyrene vials at -20 °C for 5 months. When needed, they were thawed and used the same day.

Agar dilution method.

Wilkins–Chalgren anaerobe agar (CM 619, Oxoid) with 5 % ovine blood (WCABA) was used to determine MICs. After thawing the antibacterial substance to be tested, a series of twofold dilutions was prepared and 2 ml of the dilutions were pipetted into sterile Petri dishes 90 mm in diameter, where they were mixed with 18 ml WCABA. In this way, the following concentrations of active substances were obtained: 0.031, 0.062, 0.125, 0.25, 0.5, 1, 2, 4, 8 and 16 µg ml^-1. The dishes were pre-dried and used the same day. Frozen isolates of B. hyodysenteriae were inoculated on TSBA and incubated in an anaerobic jar using a gas generating kit (BR38, Oxoid) for 3–4 days at 37 °C. Purity of the cultures was checked microscopically using culture smears stained with 1 % crystal violet solution. Pure B. hyodysenteriae cultures were scraped from TSBA using sterile cotton swabs and suspended in 2 ml sterile PBS. The turbidity was adjusted with a photometer (Densi-La-Meter, LIAP, Latvia) to 1 McFarland standard. Twenty microlitres of the working suspension, which was prepared by a hundredfold dilution, was applied to the agar surface, which brought the final inoculum on the agar surface to approximately 10⁴ c.f.u. per spot. The isolates of B. hyodysenteriae to be tested were pipetted onto the surface of pre-dried WCABA with twofold dilutions of the individual antimicrobial agent. Each dish was inoculated with six isolates in spots distributed evenly over the surface in a rosette-like pattern. After 3–4 days incubation at 37 °C, the result was read as the 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. To check the quality of the agar dilution tests, the strains B. hyodysenteriae B78^T (ATCC 27164^T), Staphylococcus aureus ATCC 29213 and Streptococcus pneumoniae ATCC 49619 (Odland et al., 2000; NCCLS, 2001) were used. Each batch of WCABA plates was tested with the control strains listed above.

Evaluation of results.

MICs obtained were documented and statistically evaluated by chi-square tests using MS Excel with the Analyse-it module. 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 MIC tests were repeated. A total of seven isolates (four for tiamulin and three for valnemulin) were retested. 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.

	RESULTS AND DISCUSSION

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
The aim of the present study was to evaluate MICs of tiamulin and valnemulin, using a set of randomly selected isolates of B. hyodysenteriae isolated from farms in the Czech Republic during a time when there was a growing number of farms with clinical cases of SD (Fig. 1). This chart represents the Czech Republic as a whole. Estimates of the number of pig farms are only available for 2000, and figures for the total number of pig herds over time do not exist. There were 405 pig farms, of which 120 had populations of 10 000 pigs or more. An estimated 1400 further farms were mixed producers.

The distribution of MICs for tiamulin and valnemulin for the B. hyodysenteriae isolates over the period investigated is given in Table 1. Between 1997 and 1998, MICs of the two pleuromutilins remained practically unchanged. Tiamulin MICs were around 0.062 and 0.125 µg ml^-1, and valnemulin MICs for an absolute majority of isolates were 0.031 µg ml^-1. Even in those years, however, a few B. hyodysenteriae isolates with MICs of 1 and 2 µg ml^-1 were found (Fig. 2, Table 1). The upward trend in MICs observed between 1999 and 2001 was reflected in gradually decreasing B. hyodysenteriae isolate susceptibility. In 1999, 50 % of the isolates tested still had an MIC of < 0.125 µg valnemulin ml^-1. In the 2000–2001 period, almost no MICs below 0.125 µg ml^-1 were found for either of the pleuromutilins (Fig. 2, Table 1). MICs of tiamulin and valnemulin were significantly different in the period 1997–98 compared with 1999–2001 (P < 0.001 for tiamulin and P < 0.0001 for valnemulin). Similar decreases in the susceptibility of B. hyodysenteriae isolates to tiamulin were also reported in Poland, Hungary, Finland and Germany (Binek et al., 1994; Molnár, 1996; Fossi et al., 1999; Karlsson et al., 2002b).

View larger version (11K): [in this window] [in a new window]   Fig. 2. MICs for tiamulin (open bars) and valnemulin (filled bars) of 100 Czech B. hyodysenteriae isolates from 1997 to 2001.

A comparison between tiamulin and valnemulin MICs proved interesting. In 1997 and 1998, before valnemulin was registered for the therapy of SD in the Czech Republic, MICs of the two drugs differed by only one or two dilutions. This difference is not considered significant (Odland et al., 2000; NCCLS, 2001). B. hyodysenteriae isolates whose tiamulin MICs averaged 0.125 µg ml^-1 had valnemulin MICs 0.031 µg ml^-1. This relationship was also demonstrable in B. hyodysenteriae isolates from the years 1999 to 2001. In addition, B. hyodysenteriae isolates with tiamulin MICs 0.5 µg ml^-1 showed increased valnemulin MICs (Fig. 2). In 1997, before valnemulin was registered, a B. hyodysenteriae isolate with an increased valnemulin MIC was found on a farm. Such findings suggest the existence of cross resistance between the two pleuromutilins.

A summary of MIC₅₀, MIC₉₀ and MIC ranges is presented in Table 2. The gradual increase of tiamulin MIC₅₀ values in the first 3 years (from 0.062 to 0.125 µg ml^-1), accelerated in 2000 and 2001 (from 0.5 to 1.0 µg ml^-1). Tiamulin MIC₉₀ values also increased gradually from 0.25 µg ml^-1 in 1997 and 1998 to 1.0 µg ml^-1 in 1999, and showed a twofold annual increase in the period which followed. Valnemulin MIC₉₀ values followed the same pattern of gradual increases. In 2001, there were no tiamulin MICs below 0.125 µg ml^-1 or valnemulin MICs below 0.062 µg ml^-1. In most cases when tiamulin MICs for B. hyodysenteriae isolates were between 1.0 and 4.0 µg ml^-1, the effectiveness of the drug was reduced or absent in pig herds when medicated feeds were used for therapy. The failure of the drugs may have, however, been caused by a number of factors related either to the host, technology or the environment. The proposed clinical breakpoint of tiamulin resistance (Rønne & Szancer, 1990), particularly with regard to peroral administration, seems to be in need of some adjustment. Karlsson et al. (2003) suggested that a lower microbiological breakpoint for resistance evaluation should be used. It is important that there be increased awareness of these problematic isolates of B. hyodysenteriae.

This study records the development of decreased sensitivity of B. hyodysenteriae isolates to pleuromutilins in the Czech Republic. However, the study may have been influenced by the choice of B. hyodysenteriae isolates, because B. hyodysenteriae isolates from the last 2 years were mainly from farms experiencing problems with a lack of clinical efficiency of pleuromutilins, and by the testing methods used. On the other hand, MICs obtained in tests with control strains (Table 3) corresponded to the acceptable quality-control range (NCCLS, 2002; Karlsson et al., 2003). A comparison between the agar dilution procedure used here and the broth dilution procedure (Karlsson et al., 2002a) has demonstrated certain differences between the two, in that some B. hyodysenteriae isolates showed lower MICs for tiamulin, tylosin and lincomycin when tested by the broth dilution procedure. This fact should be borne in mind in a critical assessment of the higher MICs reported in the present study. In a more general sense, however, results obtained by the two methods have shown good agreement (Karlsson et al., 2002a). This, however, does not change the fact that a significant increase in MIC for both pleuromutilins was demonstrated amongst Czech B. hyodysenteriae isolates during the reported period.

Initially, the occurrence of B. hyodysenteriae isolates with reduced sensitivity to pleuromutilins was limited to pig farms with enzootic SD, where antimicrobial agents were not rotated systematically, and, at the same time, inadequate doses of the drug were administered due to poor mixing of the active substance with the feed. In the last 2 years of the study period, B. hyodysenteriae isolates with reduced sensitivity were being spread by unregulated shipments of pigs.

The increasing number of B. hyodysenteriae isolates with decreased susceptibility demonstrated in this present study should alert veterinary surgeons and pig farmers to the need for a very responsible approach to the selection and use of antimicrobial agents. Only a prudent policy of antibiotic use can help to maintain the effectiveness of pleuromutilins in the treatment of SD by strategic medication in pig production.

	ACKNOWLEDGEMENTS

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
The technical assistance of Mrs M. Slavíková is acknowledged. This study was supported by the Ministry of Education of the Czech Republic research project MSM 16170 0001. The authors thank Novartis for supplying the pleuromutilins tested.

	REFERENCES

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES