Further characterization of porcine Brachyspira hyodysenteriae isolates with decreased susceptibility to tiamulin

The severe diarrhoeal disease swine dysentery is caused by an anaerobic spirochaete, Brachyspira hyodysenteriae (Harris et al., 1999). Owing to withdrawal of drugs authorized for use in pigs and reduced susceptibility among B. hyodysenteriae isolates to those that are still available, the antimicrobial arsenal against swine dysentery is diminishing. In many countries, tiamulin, a pleuromutilin, is the drug of choice for treatment of swine dysentery. However, isolates of B. hyodysenteriae with decreased susceptibility to tiamulin have been reported (Cizek et al., 2002; Gresham et al., 1998; Karlsson et al., 2002; Molnar, 1996). The mechanism of tiamulin resistance in B. hyodysenteriae is not known; hence, it is not known whether spread of isolates with decreased susceptibility is clonal or if the resistance trait is transferable between clones. To take preventive measures against the spread of clones with decreased susceptibility, it is necessary to know the impact of different factors on their development and dissemination. Affecting factors could include trade of infected pigs, spread of the agent through vectors, vehicles, etc., and the usage of tiamulin. Besides the total amount of tiamulin used in swine practice, both dose and treatment regimens may also be of importance.

In north-western Germany there has been progressively lower susceptibility to tiamulin among B. hyodysenteriae isolates over the last few years (Karlsson et al., 2002). These isolates originate from clinical submissions to the veterinary university in Hanover, and 16 of these were used in this study. Furthermore, in a report from the UK, seven B. hyodysenteriae isolates from farms with outbreaks of swine dysentery in south-east England had high MICs of tiamulin (Gresham et al., 1998); six of these isolates were included in this study.

PFGE has been shown to be suitable for genotyping of Brachyspira spp. at an adequate discriminatory level for epidemiological studies (Atyeo et al., 1999; Fellström et al., 1999). Our aim was to study the resistance epidemiology of B. hyodysenteriae by further characterization of a set of isolates from Germany and the UK with decreased susceptibility to tiamulin. The relatedness between the isolates was studied by comparing PFGE patterns. Additionally, the in vitro susceptibility to five other antimicrobials was determined.

German (n = 16) and British (n = 6) field isolates of B. hyodysenteriae from clinical submissions in 19912001 were investigated. The isolates originated from different farms in the north-west of Germany and the south-east of England. After transportation to Sweden, the purity of the isolates was checked by phase-contrast microscopy. The isolates were tested for haemolysis on trypticase soy agar with 5% ox blood and for indole reaction. To confirm the biochemical identification and to exclude any mixed cultures with Brachyspira. pilosicoli, both sets of isolates were analysed by a duplex PCR, as described by Råsbäck et al. (2003), based on the tlyA gene (Fellström et al., 2001), which is specific for B. hyodysenteriae, and a B. pilosicoli-specific fragment of the 16S rRNA gene (Fellström et al., 1997). Twenty Swedish and four British tiamulin-susceptible field isolates were included as a control group for the susceptibility testing; these were identified by biochemical tests only.

Antimicrobial-susceptibility testing was performed by broth dilution in brain heart infusion broth with 10 % fetal calf serum as described by Karlsson et al. (2003). In brief, antimicrobial agents were dried in serial twofold dilutions in tissue-culture trays, in which a suspension of the bacteria was dispensed (0.5 ml per well) and incubated. The MIC was read as the lowest concentration of the antimicrobial agent that prevented visible growth. The antimicrobials tested were tiamulin, aivlosin, doxycycline, salinomycin, chloramphenicol and avilamycin. Aivlosin (3-acetyl-4'-isovaleryltylosin) is chemically modified tylosin, and was chosen because it is registered for treatment of swine dysentery in some countries, such as the Czech Republic. The Swedish isolates were also tested for tylosin susceptibility. The B. hyodysenteriae type strain, B78^T (ATCC 27164^T), was included in the analysis as a control.

The PFGE was performed as described by Fellström et al. (1999), except that only one restriction enzyme, MluI, was used. Isolates with identical PFGE patterns were considered to be clonal and given identical upper-case letters (AH). Isolates that differed at one band were given the same letter and numbered suffix (e.g. A and A2).

All isolates had strong haemolysis. The British and Swedish isolates were indole-positive, whereas the indole test reaction varied among the German isolates. All isolates analysed by the duplex PCR were positive for the tlyA gene and negative for the B. pilosicoli-specific fragment of the 16S rRNA gene.

The German isolates (Table 1) had several different PFGE patterns and varying tiamulin MICs (132 µg ml^-1). PFGE pattern C (Fig. 1) was the most common and differed only by one band compared with a German indole-negative, tiamulin-susceptible clone described by Fellström et al. (1999). This indicated a close relatedness between the previously described clone and the group C isolates in this study. The one-band difference between the clones was consistent, which could mean that this difference emerged during the same period as the tiamulin-resistance mechanism was achieved. It was not possible to obtain any information about the distance or possible pig trade between the German farms. However, considering that the emerging decrease of tiamulin susceptibility among B. hyodysenteriae isolates is both novel and sparsely investigated, the aim was to study any available isolates. One conclusion that can be drawn, however, is that selective pressure, most likely tiamulin usage, has been sufficient to promote a number of clones with decreased susceptibility to tiamulin at different farms in Germany.

Table 1. Antimicrobial susceptibility and PFGE patterns for German field isolates of B. hyodysenteriae with decreased susceptibility to tiamulin from 1991 to 2001 Antimicrobials: Tiam., tiamulin; Aivl., aivlosin; Doxy., doxycyline; Salin., salinomycin; Chlor., chloramphenicol; Avil., avilamycin.

(101K): Fig. 1. Different PFGE patterns from German isolates of B. hyodysenteriae with decreased tiamulin susceptibility digested with MluI. Lanes: 1, Lambda Ladder PFG Marker (New England BioLabs); lanes 2 and 5 represent the most common pattern (C).

Among the six British isolates (Table 2) with decreased tiamulin susceptibility, no differences were found. All had a tiamulin MIC of 1632 µg ml^-1, and the other antimicrobials tested also showed similar MICs between the isolates. The PFGE pattern was identical for all six isolates, which indicated that it was a single clone (Fig. 2, lanes 24). These isolates have been studied previously and are known to originate from farms of which at least four are connected by pig movements (Gresham et al., 1998). Considering that tiamulin is one of the few drugs used for treatment of swine dysentery in the UK, as in many other countries, a favourable situation is created for this clone. The extent to which this clone has been spread since 1998 is not known and ought to be studied further. This trait is stable, as frozen storage since 1998, transportation and repeated laboratory culturing has not caused any change in tiamulin susceptibility.

Table 2. Antimicrobial susceptibility and PFGE patterns for British field isolates of B. hyodysenteriae from 1997 to 1998 Antimicrobials: Tiam., tiamulin; Aivl., aivlosin; Doxy., doxycyline; Salin., salinomycin; Chlor., chloramphenicol; Avil., avilamycin. ND, Not determined.

(81K): Fig. 2. PFGE patterns from British isolates of B. hyodysenteriae with decreased tiamulin susceptibility and one susceptible isolate digested with MluI. Lane 1, Lambda Ladder PFG Marker (New England BioLabs), lanes 25, isolates E3, E6, E7 and E9.

Three of the British tiamulin-susceptible isolates were analysed by PFGE (Table 2). These isolates are from the same region in England and from the same years as the clone with a high tiamulin MIC (1632 µg ml^-1). The two isolates with the lowest MICs (0.030.06 µg ml^-1) had a different pattern (pattern H, Fig. 2) compared with the clone with a high MIC. Interestingly, isolate E7, which had a slightly elevated tiamulin MIC (0.5 µg ml^-1) had the same PFGE pattern as the high-MIC clone. Tiamulin resistance in B. hyodysenteriae develops gradually both in vitro and in vivo (Karlsson et al., 2001, 2002). The stepwise increase of the MICs indicates several mutations causing different levels of resistance. The results described above could be explained by E7 being a representative of the first step towards the high MICs against E1E6.

All the isolates tested had low MICs for salinomycin, doxycycline and chloramphenicol. No cross-resistance with tiamulin was recorded, but the British clone had an aivlosin MIC of >32 µg ml^-1 for all tested isolates. Chloramphenicol binds to the ribosome and the site is very close to the presumed binding site of tiamulin (Poulsen et al., 2001). Despite this, chloramphenicol did not show any cross-resistance to tiamulin in the isolates studied. However, this does not rule out that modification of the binding site is responsible for the decreased tiamulin susceptibility. The binding sites for the two drugs are probably not identical but instead overlap, and the size and interacting groups of the molecules may differ.

The Swedish tiamulin-susceptible isolates (Table 3) had a similar antimicrobial-susceptibility pattern as the other isolates tested. One exception was that the doxycycline MICs showed a biphasic distribution, with a highly susceptible group. The tylosin MICs obtained for the Swedish isolates followed the aivlosin MICs, indicating that the 23S rRNA mutation causing macrolide resistance in B. hyodysenteriae (Karlsson et al., 1999) also affects the binding of aivlosin. This was expected because aivlosin is a substance obtained by refermentation of tylosin.

Table 3. Antimicrobial susceptibility for 20 tiamulin-susceptible Swedish field isolates of B. hyodysenteriae and the B. hyodysenteriae type strain, B78T (ATCC 27164T) Antimicrobials: Tiam., tiamulin; Tylo., tylosin; Aivl., aivlosin; Doxy., doxycyline; Salin., salinomycin; Chlor., chloramphenicol; Avil., avilamycin.

No mechanism for the decreased susceptibility to tiamulin in Brachyspira spp. has been described. There are possibly several different mutations or even different mechanisms causing the increased tiamulin MICs. A fragment including the complete domain V of 23S rRNA, which is folded in the active centre of the ribosome where tiamulin binds, was sequenced for ten isolates with decreased susceptibility to tiamulin and eight susceptible isolates. The few sequence differences that were found were either too far from the tiamulin binding site to be of any importance and/or had no connection to the tiamulin MIC (unpublished data).

In conclusion, the German isolates studied represented several different PFGE patterns, which shows that the selective pressure, most likely from tiamulin usage, has been sufficient to promote clones with decreased susceptibility at different locations. The British isolates have been spread clonally through animal movement. The tiamulin MICs for the German isolates varied, whereas those for the British isolates were uniform; German isolates with PFGE pattern C had MICs of 232 µg ml^-1, compared with the British clone that consistently had an MIC of 1632 µg ml^-1. This indicates different mechanisms of resistance for the German and the British isolates. No consistent cross-resistance between tiamulin and the other antimicrobials studied was found.

We thank K. Perry for supplying field isolates from the UK and J. Rohde for supplying field isolates from Germany. This work was supported by Formas, the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning and SLF, the Swedish Farmers Foundation for Agricultural Research.