Abstract
Outbreaks of swine dysentery, caused by Brachyspira hyodysenteriae and the recently discovered “Brachyspira hampsonii,” have reoccurred in North American swine herds since the late 2000s. Additionally, multiple Brachyspira species have been increasingly isolated by North American diagnostic laboratories. In Europe, the reliance on antimicrobial therapy for control of swine dysentery has been followed by reports of antimicrobial resistance over time. The objectives of our study were to determine the antimicrobial susceptibility trends of four Brachyspira species originating from U.S. swine herds and to investigate their associations with the bacterial species, genotypes, and epidemiological origins of the isolates. We evaluated the susceptibility of B. hyodysenteriae, B. hampsonii, Brachyspira pilosicoli, and Brachyspira murdochii to tiamulin, valnemulin, doxycycline, lincomycin, and tylosin by broth microdilution and that to carbadox by agar dilution. In general, Brachyspira species showed high susceptibility to tiamulin, valnemulin, and carbadox, heterogeneous susceptibility to doxycycline, and low susceptibility to lincomycin and tylosin. A trend of decreasing antimicrobial susceptibility by species was observed (B. hampsonii > B. hyodysenteriae > B. murdochii > B. pilosicoli). In general, Brachyspira isolates from the United States were more susceptible to these antimicrobials than were isolates from other countries. Decreased antimicrobial susceptibility was associated with the genotype, stage of production, and production system from which the isolate originated, which highlights the roles of biosecurity and husbandry in disease prevention and control. Finally, this study also highlights the urgent need for Clinical and Laboratory Standards Institute-approved clinical breakpoints for Brachyspira species, to facilitate informed therapeutic and control strategies.
INTRODUCTION
Swine dysentery is a mucohemorrhagic enteric disease that affects the health and welfare of pigs and limits production globally (1). The spirochete Brachyspira hyodysenteriae is considered to be the primary etiological agent of swine dysentery (1), but the advent of more-discriminatory microbiological methods has revealed considerable genetic diversity among Brachyspira species (2–4). A decade ago, Brachyspira suanatina was isolated from pigs with dysentery-like disease in Sweden and Denmark (5). More recently, a novel pathogenic species designated “Brachyspira hampsonii” was isolated in North America from cases of mucohemorrhagic diarrhea that were clinically indistinguishable from swine dysentery (3). A milder enteric syndrome of chronic mucodiarrheal disease of grower-finisher pigs, termed porcine intestinal spirochetosis, is caused by Brachyspira pilosicoli (1). Brachyspira murdochii is commonly isolated from healthy pigs; however, some studies have found it to be associated with mild diarrhea and/or colitis (6, 7).
Although historically a prevalent disease worldwide, changes in industry structure and operations in the United States made swine dysentery a relatively uncommon and sporadic problem in the early 1990s. Outbreaks of bloody mucoid diarrhea in pigs in the late 2000s, however, suggested the reemergence of swine dysentery in North America (2, 8). Since then, increasing numbers of Brachyspira isolations have been reported by veterinary diagnostic laboratories in the United States (9). It was hypothesized that the reemergence of swine dysentery may be due to emerging antimicrobial resistance, increased virulence/pathogenicity, or changes in pig nutrition (8).
Several antimicrobials, including tiamulin, tylosin, lincomycin, carbadox, virginiamycin, and bacitracin, are licensed in the United States to treat, to control, and/or to prevent swine dysentery (10). Pleuromutilins (tiamulin and valnemulin) have been used to treat and to control swine dysentery in many countries, due to their relatively short withdrawal periods and the sensitivity of Brachyspira species to them. In the United States, carbadox has also been used to control dysentery, due to the high susceptibility of Brachyspira species, although its use in older pigs can be limited by its withdrawal period of 42 days. Tylosin, lincomycin, virginiamycin, and bacitracin are available in many countries but are less often used for the treatment and control of swine dysentery (11). These four agents have also been used for production benefits (increased rate of weight gain and improved feed efficiency) in pigs in some countries (10, 12).
Two methods that have been used to determine the antimicrobial susceptibilities of Brachyspira species include an agar dilution (AD) method and a commercial broth microdilution (BMD) method (13). A recent study confirmed the utility of the BMD method for testing of the susceptibility of various Brachyspira species to multiple antimicrobials (14). Clinical interpretation of in vitro antimicrobial susceptibility results have been hindered by the absence of internationally accepted and Clinical and Laboratory Standards Institute (CLSI)-approved clinical breakpoints. Some researchers have made attempts to bridge this gap in knowledge by proposing epidemiological cutoff values (15) and interpretive criteria (16–19).
B. hyodysenteriae and/or B. pilosicoli strains from numerous countries, including Australia (13), Belgium (20), the Czech Republic (21), Germany (22), Italy (23), Japan (24), Netherlands (25), Poland (26), Spain (27), Sweden (15), and the United Kingdom (28), were reported to have low susceptibilities to one or more antimicrobial agents. Some studies have also identified resistant strains in several farms or regions as a result of clonal dissemination (23, 28). Such trends of low susceptibilities of B. hyodysenteriae and B. pilosicoli to multiple antimicrobials complicate disease control efforts. Although substantial information on the antimicrobial susceptibilities of Brachyspira isolates is available from Europe (15, 21–23, 26–28), Australia (13), and Asia (24), limited information is available from North America (9). Recent studies have characterized the genotypes of B. hyodysenteriae and B. hampsonii circulating in commercial swine herds in the United States (2, 8). However, it is not known whether antimicrobial resistance has played a role in the apparent reemergence of swine dysentery in North America. This study was undertaken to evaluate the in vitro antimicrobial susceptibilities of U.S. Brachyspira isolates in the context of their epidemiological origin, bacterial species, and genotype and to compare them to susceptibility patterns reported for other countries.
MATERIALS AND METHODS
Brachyspira isolates.
A total of 124 Brachyspira field isolates (see Table S1 in the supplemental material) obtained from the University of Minnesota Veterinary Diagnostic Laboratory were evaluated for susceptibility to six antimicrobial compounds. The isolates included B. hyodysenteriae (n = 40), B. hampsonii (n = 40), B. pilosicoli (n = 24), and B. murdochii (n = 20) isolates. They were purposefully selected to represent diverse epidemiological sources, including 97 swine farms within 41 swine production systems in 14 U.S. states (i.e., Arkansas, Iowa, Illinois, Kansas, Minnesota, Missouri, North Carolina, Nebraska, New York, Oklahoma, South Carolina, South Dakota, Virginia, and Wisconsin), different stages of production (breeding versus finisher farms), and different years of isolation (2009 to 2014). A swine production system refers to a production company that owns multiple swine farms. All frozen pure cultures were passaged at least twice on tryptic soy agar (BD, Franklin Lakes, NJ) plates containing commercial 5% defibrinated sheep blood (I-Tek Medical Technologies, White Bear Lake, MN) and were checked for purity by phase-contrast microscopy before being evaluated for antimicrobial susceptibility.
Antimicrobial agents.
The antimicrobials evaluated in this study were selected because they are commonly used to treat and/or to control Brachyspira-induced diarrhea or dysentery and/or were included in the commercially available VetMIC Brachy panel (Swedish National Veterinary Institute, Uppsala, Sweden). The six antimicrobials evaluated included two pleuromutilins (tiamulin and valnemulin), a lincosamide (lincomycin), a macrolide (tylosin), a tetracycline (doxycycline), and a quinoxaline (carbadox). Antimicrobial concentrations in 2-fold dilutions were evaluated within the following ranges: tiamulin, 0.063 to 8 μg/ml; valnemulin, 0.031 to 4 μg/ml; lincomycin, 0.5 to 64 μg/ml; tylosin, 2 to 128 μg/ml; doxycycline, 0.125 to 16 μg/ml; carbadox, 0.001 to 1 μg/ml.
In vitro antimicrobial susceptibility testing.
All antimicrobials were evaluated by the BMD method with the exception of carbadox, which was evaluated by the AD method, as described previously (14). Each batch of isolates evaluated included the quality control strain B. hyodysenteriae B-78 (ATCC 27164). Briefly, 3-day-old Brachyspira cultures were used to make inocula of either 2 × 106 organisms or 2 × 107 organisms per ml of brain heart infusion (BHI) broth (BD, Franklin Lakes, NJ) containing 10% fetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, MO) (10% FBS-BHI), for the BMD or AD method, respectively. For the BMD method, each well of a commercial VetMIC Brachy panel (Swedish National Veterinary Institute) containing a defined concentration of antimicrobial was inoculated with about 1 × 106 Brachyspira organisms per ml of 10% FBS-BHI and incubated anaerobically at 37°C for 4 days on a shaker. A single well with no antimicrobial served as a growth control for each panel. For the AD method, test and control plates were prepared by pouring a molten mixture of tryptic soy agar, commercial sheep blood, and defined concentrations of carbadox (Sigma-Aldrich, St. Louis, MO) or distilled water (for test plates or control plates, respectively) into sterile petri dishes. After cooling, an inoculum of about 1 × 105 organisms was spotted onto the agar surface, with a cut for visualization of hemolysis, and the plates were incubated anaerobically at 37°C for 4 days. The MIC, defined as the lowest concentration of antimicrobial that inhibited visible growth (observed as turbidity for the BMD method or hemolysis for the AD method), was used to record the in vitro antimicrobial susceptibility. Finally, each isolate was retested for purity by phase-contrast microscopy and subculturing of growth from the control well (for the BMD method) or by phase-contrast microscopy of growth from the control plate (for the AD method).
Analysis. (i) U.S. Brachyspira isolates.
The in vitro antimicrobial susceptibility results were summarized on both the Brachyspira genus and Brachyspira species levels using the following measures: MIC range, MIC mode, MIC50, and MIC90. Prior to the application of any interpretive guidelines, antimicrobial susceptibility results were characterized as indicating high susceptibility or low susceptibility, relative to the range of antimicrobials evaluated. In addition, various published interpretation guidelines (Table 1) were used to categorize the antimicrobial susceptibility results. Isolates with MICs above the epidemiological cutoff values (15) or above the values from other interpretive criteria (as reported by Burch [16], the Swedish National Veterinary Institute [19], Ronne and Szancer [18], and Duhamel et al. [17]) for each antimicrobial-Brachyspira isolate combination were characterized as having decreased susceptibility or resistance, respectively. The percentage of isolates characterized as having decreased susceptibility or resistance for each antimicrobial agent was determined. Genotypic information represented by sequence types (STs) of U.S. B. hyodysenteriae and B. hampsonii isolates was obtained from previous studies (2, 8). Analyses to detect associations between the antimicrobial susceptibility results for isolates and their genotypes or epidemiological origins were carried out qualitatively using categorical variables (high susceptibility versus decreased susceptibility) and quantitatively using nonparametric analysis of actual MICs. For categorical variables, Fisher's exact test and odds ratios were used to evaluate qualitatively the following questions: (i) whether decreased susceptibilities of B. hyodysenteriae and B. hampsonii isolates to pleuromutilin were statistically associated with their genotypes; (ii) whether decreased susceptibilities of B. hyodysenteriae, B. hampsonii, and B. pilosicoli to pleuromutilin were statistically associated with the swine production systems from which the isolates originated; and (iii) whether the susceptibilities of B. hyodysenteriae and B. hampsonii to lincomycin and tylosin were statistically associated with the stage of production from which the isolates originated. These associations were also evaluated quantitatively using Wilcoxon's rank sum test. In addition, similar associations between the carbadox susceptibility of Brachyspira isolates and their epidemiological sources were evaluated quantitatively with Wilcoxon's rank sum test. All statistical analyses were performed using R software (R Foundation for Statistical Computing, Vienna, Austria), and all analyses were supported by P values or 95% confidence intervals (CIs) for interpretation of statistical significance.
TABLE 1.
Guidelines for interpretation of antimicrobial susceptibility for Brachyspira species
| Drug | MIC (μg/ml)a |
||||
|---|---|---|---|---|---|
| Epidemiological cutoff (15) (BMD) | Criteria of Burch (16) (BMD) | SVARM criteria (19) (BMD) | Criteria of Ronne and Szancer (18) (AD) | Criteria of Duhamel et al. (17) (AD) | |
| Tiamulin | >0.25 (DS) | >0.5 (R) | >2 (R) | >4 (R) or >1 and ≤4 (I) | >2 (R) or ≥1 and ≤2 (I) |
| Valnemulin | >0.125 (DS) | >0.125 (R) | NA | NA | NA |
| Lincomycin | >1 (DS) | >50 (R) | NA | >36 (R) or >4 and ≤36 (I) | >75 (R) or ≥25 and ≤75 (I) |
| Tylosin | >16 (DS) | >16 (R) | >16 (R) | >4 (R) or >1 and ≤4 (I) | NA |
| Doxycycline | >0.5 (DS) | NA | NA | NA | NA |
| Carbadox | NA | NA | NA | NA | >1 (R) or ≥0.125 and ≤1 (I) |
BMD, broth microdilution; AD, agar dilution; DS, decreased susceptibility; R, resistance; I, intermediate susceptibility; NA, not applicable.
(ii) International B. hyodysenteriae genotypes with decreased tiamulin susceptibility.
Although isolates characterized as having decreased tiamulin susceptibility (using epidemiological cutoff values) were identified previously in many countries (9, 11, 13, 15, 20–26, 28, 29), only a few have been genotyped (2, 8, 23, 30, 31). Information on international B. hyodysenteriae genotypes and corresponding information on tiamulin susceptibility was obtained from published studies (23, 28–31). Using various interpretive criteria (15–19), these genotypes were characterized as having tiamulin resistance and/or decreased tiamulin susceptibility.
RESULTS
General trends in antimicrobial susceptibility.
The antimicrobial MICs and respective epidemiological information for the 124 Brachyspira isolates evaluated are listed in Table S1 in the supplemental material. The MIC50, MIC90, MIC mode, and range of MICs at the Brachyspira genus and Brachyspira species level are listed in Table 2. At the genus level, the observed ranges of MICs for each antimicrobial were as follows: tiamulin, ≤0.063 to >8 μg/ml; valnemulin, ≤0.031 to >4 μg/ml; lincomycin, ≤0.5 to >64 μg/ml; tylosin, ≤2 to >128 μg/ml; doxycycline, ≤0.125 to 4 μg/ml; carbadox, 0.002 to 0.5 μg/ml. The antimicrobial susceptibility results extended across the evaluated ranges (about 8 log2 dilutions) and, in general, the MIC90 values varied between zero and six dilutions higher than the MIC50 values. The patterns and trends of susceptibility of Brachyspira to multiple antimicrobials are illustrated in Fig. 1 to 3. In general, most Brachyspira isolates were highly susceptible to tiamulin, valnemulin, and carbadox (Fig. 1). In contrast, most Brachyspira isolates showed low susceptibilities to lincomycin and tylosin, except for B. hampsonii isolates, which showed high susceptibilities to lincomycin and tylosin (Fig. 1). Brachyspira isolates displayed heterogeneous susceptibility to doxycycline. At the species level, in general, B. hampsonii had the highest susceptibilities, followed by B. hyodysenteriae, while B. murdochii and B. pilosicoli showed lower susceptibilities (Fig. 2). The distribution of MICs for the six antimicrobials at the genus level is shown in Fig. 3. The percentages of isolates deemed to be antimicrobial resistant varied according to the criteria used (16–19) but, in general, were similar to or lower than the percentages characterized as having decreased antimicrobial susceptibility using the epidemiological cutoff values described by Pringle et al. (15) (Table 3).
TABLE 2.
Summarized MIC measures of six antimicrobials to Brachyspira species and genus
| Species and drug | MIC50 (μg/ml) | MIC90 (μg/ml) | MIC mode (μg/ml) | MIC range (μg/ml) |
|---|---|---|---|---|
| B. hampsonii | ||||
| Tiamulin | ≤0.063 | 0.25 | ≤0.063 | ≤0.063 to 0.5 |
| Valnemulin | ≤0.031 | ≤0.031 | ≤0.031 | ≤0.031 to 0.125 |
| Lincomycin | ≤0.5 | 16 | ≤0.5 | ≤0.5 to 32 |
| Tylosin | 4 | >128 | >128 | ≤2 to >128 |
| Doxycycline | 0.25 | 1 | ≤0.125 | ≤0.125 to 2 |
| Carbadox | 0.004 | 0.016 | 0.004 | 0.004–0.25 |
| B. hyodysenteriae | ||||
| Tiamulin | ≤0.063 | 2 | ≤0.063 | ≤0.063 to >8 |
| Valnemulin | ≤0.031 | 1 | ≤0.031 | ≤0.031 to 2 |
| Lincomycin | 16 | 32 | 32 | ≤0.5 to >64 |
| Tylosin | >128 | >128 | >128 | ≤2 to >128 |
| Doxycycline | 0.5 | 2 | 2 | ≤0.125 to 4 |
| Carbadox | 0.008 | 0.25 | 0.004 | 0.002–0.25 |
| B. murdochii | ||||
| Tiamulin | 0.25 | 2 | ≤0.063 | ≤0.063 to >8 |
| Valnemulin | 0.125 | 0.5 | ≤0.031 | ≤0.031 to >4 |
| Lincomycin | 16 | 32 | 16 | ≤0.5 to >64 |
| Tylosin | >128 | >128 | >128 | ≤2 to >128 |
| Doxycycline | 0.5 | 2 | 1 | ≤0.125 to 4 |
| Carbadox | 0.004 | 0.008 | 0.004 | 0.002–0.016 |
| B. pilosicoli | ||||
| Tiamulin | 0.5 | >8 | >8 | ≤0.063 to >8 |
| Valnemulin | 0.25 | 2 | 1 | ≤0.031 to 4 |
| Lincomycin | 32 | 64 | 32 | ≤0.5 to >64 |
| Tylosin | >128 | >128 | >128 | 8 to >128 |
| Doxycycline | 2 | 4 | 4 | ≤0.125 to 4 |
| Carbadox | 0.008 | 0.016 | 0.008 | 0.004–0.016 |
| Brachyspira | ||||
| Tiamulin | ≤0.063 | 2 | ≤0.063 | ≤0.063 to >8 |
| Valnemulin | ≤0.031 | 1 | ≤0.031 | ≤0.031 to >4 |
| Lincomycin | 8 | 32 | ≤0.5 | ≤0.5 to >64 |
| Tylosin | >128 | >128 | >128 | ≤2 to >128 |
| Doxycycline | 0.5 | 2 | 0.25 | ≤0.125 to 4 |
| Carbadox | 0.004 | 0.125 | 0.004 | 0.002–0.5 |
FIG 1.
Distributions of antimicrobial MICs for 124 U.S. Brachyspira isolates, stratified by species (B. hyodysenteriae, B. hampsonii, B. pilosicoli, and B. murdochii) and genus. (A) Percentages of Brachyspira isolates exhibiting various tiamulin MICs. (B) Percentages of Brachyspira isolates exhibiting various valnemulin MICs. (C) Percentages of Brachyspira isolates exhibiting various lincomycin MICs. (D) Percentages of Brachyspira isolates exhibiting various tylosin MICs. (E) Percentages of Brachyspira isolates exhibiting various doxycycline MICs. (F) Percentages of Brachyspira isolates exhibiting various carbadox MICs.
FIG 3.
Distribution of antimicrobial MICs for 124 U.S. Brachyspira isolates, stratified by antimicrobial (carbadox, valnemulin, tiamulin, doxycycline, lincomycin, and tylosin).
FIG 2.
Distributions of antimicrobial MICs for 124 U.S. Brachyspira isolates of various species, stratified by antimicrobial (carbadox, valnemulin, tiamulin, doxycycline, lincomycin, and tylosin). (A) Percentages of B. hyodysenteriae isolates exhibiting various MICs for six antimicrobials. (B) Percentages of B. hampsonii isolates exhibiting various MICs for six antimicrobials. (C) Percentages of B. pilosicoli isolates exhibiting various MICs for six antimicrobials. (D) Percentages of B. murdochii isolates exhibiting various MICs for six antimicrobials.
TABLE 3.
Percentages of isolates of each Brachyspira species with decreased antimicrobial susceptibility or resistance, according to different guidelines
| Drug and species | % of isolates with decreased susceptibility or resistancea |
||||
|---|---|---|---|---|---|
| Epidemiological cutoff (15)b | Criteria of Burch (16)c | SVARM criteria (19)c | Criteria of Ronne and Szancer (18)c | Criteria of Duhamel et al. (17)c | |
| Tiamulin | |||||
| B. hampsonii | 10 | 0 | 0 | 0 | 0 |
| B. hyodysenteriae | 25 | 17.5 | 10 | 10 | 10 |
| B. murdochii | 45 | 25 | 5 | 5 | 5 |
| B. pilosicoli | 54.2 | 41.7 | 29.2 | 25 | 29.2 |
| Valnemulin | |||||
| B. hampsonii | 0 | 0 | NA | NA | NA |
| B. hyodysenteriae | 27.5 | 27.5 | NA | NA | NA |
| B. murdochii | 50 | 50 | NA | NA | NA |
| B. pilosicoli | 58.3 | 58.3 | NA | NA | NA |
| Lincomycin | |||||
| B. hampsonii | 37.5 | 0 | NA | 0 | 0 |
| B. hyodysenteriae | 75 | 5 | NA | 5 | 5 |
| B. murdochii | 85 | 10 | NA | 10 | 10 |
| B. pilosicoli | 91.7 | 16.7 | NA | 16.7 | 4.2 |
| Tylosin | |||||
| B. hampsonii | 25 | 25 | 25 | 30 | NA |
| B. hyodysenteriae | 75 | 75 | 75 | 92.5 | NA |
| B. murdochii | 80 | 80 | 80 | 80 | NA |
| B. pilosicoli | 87.5 | 87.5 | 87.5 | 100 | NA |
| Doxycycline | |||||
| B. hampsonii | 25 | NA | NA | NA | NA |
| B. hyodysenteriae | 47.5 | NA | NA | NA | NA |
| B. murdochii | 50 | NA | NA | NA | NA |
| B. pilosicoli | 54.2 | NA | NA | NA | NA |
| Carbadox | |||||
| B. hampsonii | NA | NA | NA | NA | 0 |
| B. hyodysenteriae | NA | NA | NA | NA | 0 |
| B. murdochii | NA | NA | NA | NA | 0 |
| B. pilosicoli | NA | NA | NA | NA | 0 |
NA, not applicable.
Values indicate percentages with decreased susceptibility.
Values indicate percentages with resistance.
Epidemiological associations with Brachyspira antibiograms.
Some novel associations between the antimicrobial susceptibility results and the epidemiological characteristics were identified (Table 4). Two genotypes of B. hyodysenteriae (ST94 and ST107) had significantly lower susceptibilities to tiamulin and valnemulin in both qualitative and quantitative analyses. Similarly, one genotype of B. hampsonii (ST7) had significantly lower susceptibilities to tiamulin, valnemulin, and carbadox in quantitative analyses. Isolates from one swine production system from which B. hyodysenteriae was isolated (system M) and one swine production system from which B. hampsonii was isolated (system J) showed statistically significant associations with decreased tiamulin susceptibilities in qualitative and quantitative analyses. Similarly, isolates from one swine production system from which B. hyodysenteriae was isolated (system M), another swine production system from which B. hampsonii was isolated (system J), and a third swine production system from which B. pilosicoli was isolated (system R) showed statistically significant associations with decreased valnemulin susceptibilities in quantitative analyses. This was also true when isolates from one swine production system (system M) from which B. hyodysenteriae was isolated were analyzed qualitatively. Although the association of isolates from system K was not statistically significant (Table 4), five of six B. pilosicoli isolates evaluated from that system showed decreased pleuromutilin susceptibility (see Table S1 in the supplemental material). Furthermore, 11 of 12 Brachyspira isolates identified as tiamulin resistant using Swedish veterinary antimicrobial resistance monitoring (SVARM) criteria (19) originated from different farms belonging to system M (four B. hyodysenteriae isolates), system R (three B. pilosicoli isolates and one B. murdochii isolate), or system K (three B. pilosicoli isolates) (see Table S1). Interestingly, these isolates also demonstrated similar profiles of susceptibility to the other five antimicrobials evaluated, suggesting that they likely represent three strains (one B. hyodysenteriae strain and two B. pilosicoli strains) (see Table S1). Finally, compared to isolates from breeding farms, both B. hyodysenteriae and B. hampsonii isolates from finisher farms had significantly lower susceptibilities to lincomycin and tylosin in both qualitative and quantitative analyses.
TABLE 4.
Associations between antimicrobial susceptibility patterns and Brachyspira species, genotype, stage of production, and production systema
| Variable and species | Unit | Antimicrobial | P from Fisher's exact test | Odds ratio (95% CI) | P from Wilcoxon's test |
|---|---|---|---|---|---|
| Genotype | |||||
| B. hampsonii | ST1 | Tiamulin | 0.55 | 0 (0–5.44) | 0.07 |
| Valnemulin | ND | ND | 0.36 | ||
| ST3 | Tiamulin | 0.56 | 0 (0–6.81) | 1.00 | |
| Valnemulin | ND | ND | 0.41 | ||
| ST7 | Carbadox | NA | NA | 0.02b | |
| Tiamulin | 0.07 | 10.75 (0.53–241.07) | 0.002b | ||
| Valnemulin | ND | ND | 0.006b | ||
| B. hyodysenteriae | ST93 | Carbadox | NA | NA | 0.47 |
| Tiamulin | 0.07 | 0 (0–1.40) | 0.13 | ||
| Valnemulin | 0.07 | 1 (0–1.40) | 0.04 | ||
| ST94 | Tiamulin | 0.003b | ∞ (2.41 to ∞)c | 0.007b | |
| Valnemulin | 0.003b | ∞ (2.41 to ∞)c | 0.002b | ||
| ST107 | Tiamulin | 0.01b | 18.01 (1.37–1071.96)c | 0.01b | |
| Valnemulin | 0.01b | 18.01 (1.37–1071.96)c | 0.005b | ||
| Stage of production | |||||
| B. hampsonii | Finisher | Lincomycin | 0.009b | 6.25 (1.35–34.16)c | 0.007b |
| Tylosin | 0.009b | 8.76 (1.38–100.75)c | 0.002b | ||
| B. hyodysenteriae | Lincomycin | <0.001b | 65.18 (6.19–3563.17)c | <0.001b | |
| Tylosin | <0.001b | 65.18 (6.19–3563.17)c | <0.001b | ||
| Production system | |||||
| B. hampsonii | U | Tiamulin | 1.00 | 0 (0–16.77) | 0.25 |
| Valnemulin | ND | ND | 0.59 | ||
| V | Tiamulin | 1.00 | 0 (0–12.23) | 0.19 | |
| Valnemulin | ND | ND | 0.53 | ||
| J | Carbadox | NA | NA | 0.08 | |
| Tiamulin | <0.001b | ∞ (3.54 to ∞)c | <0.001b | ||
| Valnemulin | ND | ND | <0.001b | ||
| M | Tiamulin | 1.00 | 0 (0–9.51) | 0.14 | |
| Valnemulin | ND | ND | 0.48 | ||
| X | Tiamulin | 1.00 | 0 (0–16.77) | 0.25 | |
| Valnemulin | ND | ND | 0.59 | ||
| B. hyodysenteriae | M | Carbadox | NA | NA | 0.19 |
| Tiamulin | <0.001b | 33.98 (3.70–1712.45)c | <0.001b | ||
| Valnemulin | <0.001b | 33.98 (3.70–1712.45)c | <0.001b | ||
| B. pilosicoli | R | Tiamulin | 0.60 | 2.88 (0.19–172.77) | 0.13 |
| Valnemulin | 0.62 | 2.37 (0.16–142.81) | 0.03b | ||
| K | Tiamulin | 0.17 | 5.81 (0.5–323.85) | 0.07 | |
| Valnemulin | 0.34 | 4.70 (0.40–261.82) | 0.21 |
Fisher's exact test and odds ratios were used to determine the statistical associations using categorical values. Wilcoxon's test was used to determine the statistical association using numerical values. P values were unadjusted. NA, not applicable; ND, not determined.
Statistically significant result (P < 0.05).
The odds ratio is statistically significant, as the 95% confidence interval does not contain the value 1.
International B. hyodysenteriae genotypes with decreased tiamulin susceptibility.
Using the SVARM criteria (19) and the criteria described by Ronne and Szancer (18), 43 B. hyodysenteriae isolates (representing 22 STs) from various countries that were reported to have decreased tiamulin susceptibilities were characterized with different interpretations of sensitivity (Table 5). Of the 22 genotypes (from Germany, Italy, Spain, the United Kingdom, and the United States) showing decreased tiamulin susceptibilities (15), 17 and 16 genotypes were characterized as tiamulin resistant using the SVARM criteria (19) and the criteria described by Ronne and Szancer (18), respectively (Table 5).
TABLE 5.
Categorization of tiamulin susceptibility of B. hyodysenteriae genotypes according to three interpretive criteria
| Genotype | No. of isolates | Country of origin | Tiamulin MIC (μg/ml)a | Susceptibility interpretation |
Reference(s) | ||
|---|---|---|---|---|---|---|---|
| Epidemiological cutoff (15) | SVARM criterion (19)b | Criterion of Ronne and Szancer (18)c | |||||
| ST8 | 2 | UK | >8 | Decreased susceptibility | Resistant | Resistant | 28, 30 |
| Italy | >8 | Decreased susceptibility | Resistant | Resistant | 23 | ||
| ST51 | 1 | Germany | >8 | Decreased susceptibility | Resistant | Resistant | 28, 30 |
| ST52 | 2 | Spain | 2 | Decreased susceptibility | Intermediate | Intermediate | 29, 31 |
| Italy | 4 | Decreased susceptibility | Resistant | Intermediate | 23 | ||
| ST73 | 1 | Spain | >8 | Decreased susceptibility | Resistant | Resistant | 29, 31 |
| ST74 | 2 | Italy | 0.5, >8d | Decreased susceptibility | Resistantd | Resistantd | 23 |
| ST75 | 3 | Italy | 0.5, 8, >8 | Decreased susceptibility | Resistant | Resistant | 23 |
| ST76 | 3 | Italy | 1, 8, >8 | Decreased susceptibility | Resistant | Resistant | 23 |
| ST77 | 4 | Italy | 0.5, 1, 8, >8 | Decreased susceptibility | Resistant | Resistant | 23 |
| ST78 | 5 | Italy | 0.5, 2, 4, 8, >8 | Decreased susceptibility | Resistant | Resistant | 23 |
| ST79 | 3 | Italy | 0.5, 2, >8 | Decreased susceptibility | Resistant | Resistant | 23 |
| ST80 | 1 | Italy | 0.5 | Decreased susceptibility | Intermediate | Sensitive | 23 |
| ST83 | 1 | Italy | >8 | Decreased susceptibility | Resistant | Resistant | 23 |
| ST84 | 1 | Italy | 0.5 | Decreased susceptibility | Sensitive | Sensitive | 23 |
| ST86 | 1 | Italy | 8 | Decreased susceptibility | Resistant | Resistant | 23 |
| ST94 | 4 | USA | 05, 1, 2, >8 | Decreased susceptibility | Resistant | Resistant | 8, this study |
| ST97 | 1 | Italy | 0.5 | Decreased susceptibility | Intermediate | Sensitive | 23 |
| ST98 | 1 | Italy | >8 | Decreased susceptibility | Resistant | Resistant | 23 |
| ST100 | 1 | Italy | 1 | Decreased susceptibility | Intermediate | Sensitive | 23 |
| ST101 | 1 | Italy | 0.5 | Decreased susceptibility | Intermediate | Sensitive | 23 |
| ST102 | 1 | Italy | 0.5 | Decreased susceptibility | Intermediate | Sensitive | 23 |
| ST103 | 2 | Italy | 2, >8 | Decreased susceptibility | Resistant | Resistant | 23 |
| ST107 | 2 | USA | 1, >8 | Decreased susceptibility | Resistant | Resistant | 8, this study |
Includes available susceptibility results (by the broth microdilution method) for all isolates characterized as a specific genotype from a specific country. Results for individual isolates are presented.
SVARM criteria are based on the broth microdilution susceptibility testing method. MICs that were below the SVARM cutoff value for resistance but above the epidemiological cutoff value were considered to indicate intermediate susceptibility.
The criteria described by Ronne and Szancer (18) are based on the agar dilution susceptibility testing method.
In the case of multiple isolates of a genotype, the STs were characterized as resistant by the SVARM criteria and the criteria described by Ronne and Szancer (18) if at least one isolate was above the cutoff value for resistance.
DISCUSSION
Veterinary and/or medical practitioners require interpretations of susceptibility test results (namely, sensitive, intermediate, or resistant) in order to select an antimicrobial appropriately and to determine its dosage for treatment. Internationally accepted and CLSI-approved clinical breakpoints, which are used to determine whether an antimicrobial agent is potentially useful for treating a microbial infection, are set by integrating information on the epidemiological distributions of antimicrobial MICs (i.e., epidemiological cutoff values), pharmacokinetic (PK) and pharmacodynamic (PD) characteristics (i.e., PK/PD cutoff values), and clinical outcomes (i.e., clinical cutoff values) (32). Swine dysentery has been recognized as a major disease of pigs for almost 100 years, and the use of antimicrobials has played a central role in the treatment, control, prevention, and eradication of the disease on farms throughout the world. However, no internationally accepted set of CLSI-approved susceptibility testing methods or clinical breakpoints exists for antimicrobials used for Brachyspira species. Several research groups have proposed guidelines (Table 1) for interpreting the antimicrobial susceptibility results for Brachyspira species, including those for detection of decreased susceptibility (the epidemiological cutoff values described by Pringle et al. [15]) and those for detection of resistance (criteria described by Burch [16], Ronne and Szancer [18], Duhamel et al. [17], and the Swedish National Veterinary Institute [19]). These guidelines (Table 1) are based on the results of either AD or BMD methods, which are known to differ by approximately 1 doubling dilution (11, 33). Unfortunately, since there are no CLSI-approved clinical breakpoints for Brachyspira, diagnostic laboratories cannot interpret and report to clients the antimicrobial susceptibility results for Brachyspira species as clinically sensitive or resistant. For interpretation of results and comparisons between studies, the proposed epidemiological cutoff values can serve as criteria to distinguish wild-type populations of bacteria from those with acquired or selected resistance mechanisms; although such findings do not indicate clinical resistance, they can be used to indicate decreased susceptibility to an antimicrobial (15).
Our observations suggest that B. hyodysenteriae isolates from the United States (Table 2) are more susceptible to tiamulin and valnemulin than are isolates from Germany, Italy, Japan, Poland, Spain, the Czech Republic, and the United Kingdom (21–24, 26–28), which have reported strains resistant to tiamulin and/or valnemulin. Interestingly, we found a similar MIC50 value but higher MIC90 value for B. hyodysenteriae (Table 2), compared with those reported for Australia and Sweden (13, 15). This indicates the presence of occasional isolates with low pleuromutilin susceptibility in the United States. The observation of tiamulin and/or valnemulin resistance reported from Europe has been associated with the intensive use of pleuromutilins for the treatment of swine dysentery (20, 21, 28). Swine dysentery continues to be a major clinical problem in most of Europe, and the lack of commercial vaccines has led to a reliance on antimicrobial therapy to control the disease (22). Several antimicrobials, including carbadox, have been banned for use in swine production in Europe since 1999. Of the antimicrobials licensed and available for swine dysentery treatment and/or control in Europe, tiamulin and valnemulin were most effective in controlling swine dysentery, but reports of resistant strains have emerged over the past decade (22). In contrast, after clinical swine dysentery largely disappeared from the United States in the early 1990s, the disease was rarely reported until the recent reemergence of the disease in the late 2000s (8). This may explain the relatively greater susceptibility to pleuromutilins of B. hyodysenteriae isolates from the United States, compared to isolates from Europe. Our data (Table 2 and Fig. 1 and 2) generally concur with previous observations of a relatively high prevalence of pleuromutilin susceptibility among U.S. isolates of B. hyodysenteriae, B. pilosicoli, and B. murdochii (9). However, the detection of occasional Brachyspira isolates of U.S. origin (Fig. 3), especially B. pilosicoli (Fig. 1 and 2), with low susceptibility to tiamulin is unique to our study. Interestingly, a previous study also found that a significant proportion of Swedish B. pilosicoli isolates showed decreased susceptibility (and possibly resistance) to tiamulin (15). The relatively greater susceptibilities of B. hampsonii to both tiamulin and valnemulin (Fig. 1 and 2), compared to other Brachyspira species, could possibly reflect a shorter history of exposure of this species to antimicrobials.
The differences in the interpretive criteria (15–19) were responsible for the different percentages of Brachyspira isolates categorized as having decreased susceptibility to or being resistant to tiamulin (Table 3). In general, Brachyspira isolates showed high pleuromutilin susceptibility in the United States; however, a few strains, especially those of B. pilosicoli, appeared to have developed resistance to tiamulin (Table 3). Several point mutations in domain V of the 23S rRNA gene and/or the ribosomal protein L3 gene are associated with decreased pleuromutilin susceptibility of clinical and laboratory-selected B. hyodysenteriae isolates (27, 34). Although cross-resistance has not been specifically studied in Brachyspira, valnemulin resistance has been observed in B. pilosicoli during selection for tiamulin resistance in vitro (35). The stepwise development of the mutations observed in vitro and in vivo suggests that multiple mutations are likely required for the development of increasing degrees of resistance, gradually altering the conformation of the drug binding site (27, 35). This gradual development of pleuromutilin-resistant strains may be facilitated by the frequent and long-term use of these antimicrobials in a herd (20, 21, 28) and could possibly explain the presence of a few tiamulin-resistant strains of Brachyspira species among the U.S. isolates.
The lincomycin and tylosin susceptibility trends of B. hyodysenteriae isolates from Australia, Germany, Poland, Spain, and Sweden (13, 15, 22, 26, 27) generally concurred with our findings (Table 2), although isolates from the Czech Republic showed lower lincomycin susceptibilities than did isolates from most other countries (21). Previous studies (9, 15) found trends similar to those in our study (Fig. 1 and 2) for B. pilosicoli isolates from the United States for lincomycin susceptibility and from Sweden for lincomycin and tylosin susceptibility. Interestingly, although B. murdochii and B. hampsonii isolates showed similar patterns of low lincomycin and tylosin susceptibility, compared with other Brachyspira species (Fig. 3), some B. hampsonii isolates with surprisingly high susceptibility were also detected (Fig. 1 and 2).
The differences in the interpretive criteria (15–19) were also responsible for the different percentages of Brachyspira isolates categorized as having decreased susceptibility to or being resistant to lincomycin (Table 3). Nevertheless, our findings suggest that, in general, resistance or decreased susceptibility to tylosin and lincomycin is prevalent among Brachyspira species in the United States (Table 3 and Fig. 1, 2, and 3). Cross-resistance between macrolides and lincosamides can be observed because they share a target site for drug binding (36). A single mutation in one position of the 23S rRNA gene of B. hyodysenteriae and B. pilosicoli is sufficient to cause resistance to both tylosin and lincomycin, and a previous study demonstrated rapid in vitro development of the causative mutation in the 23S rRNA gene of B. hyodysenteriae (36, 37). Rapid development of resistance followed by multiplication and transmission of such strains can lead to widespread tylosin and lincomycin resistance within a population. The decreased susceptibility of Brachyspira species to lincomycin and especially tylosin may have been augmented by the availability of these antimicrobials for both therapeutic and growth promotion purposes in U.S. swine production.
Our findings suggest that B. hyodysenteriae isolates from the United States (Table 2 and Fig. 1 and 2) are relatively more susceptible to doxycycline than are those from Germany, Poland, and the United Kingdom (26, 28). However, both B. hyodysenteriae and B. pilosicoli isolates from the United States are less susceptible to doxycycline than are isolates from Sweden (28). The heterogeneous distribution of Brachyspira susceptibilities to doxycycline (Fig. 1) differs from the trends observed for other evaluated antimicrobials. A single mutation in one position of the 16S rRNA gene of B. hyodysenteriae is associated with in vitro doxycycline resistance, and the spread of resistant strains likely depends on the multiplication and transmission of such mutant clones (38).
Our findings of high carbadox susceptibility of B. hyodysenteriae and/or B. pilosicoli isolates from the United States concurred with the trends observed for Sweden and Japan (24, 33). The genetic mechanism of carbadox resistance has not been studied for Brachyspira species. Although a study reported the presence of an R plasmid conferring carbadox resistance to Escherichia coli that was found to be conjugally transmissible to Salmonella species and Shigella flexneri, it was not found to be transmissible to B. hyodysenteriae (39). Currently, the only available criteria (17) for the interpretation of carbadox susceptibility results are based on the AD method, which prompted us to use the AD method for carbadox susceptibility testing of Brachyspira species in this study. However, carbadox MIC results for Brachyspira species obtained by the BMD method can be one to five dilutions higher than those obtained by the AD method (14). Therefore, the susceptibility results determined by the BMD method could be significantly higher than those reported here from the AD method. The evaluation of carbadox susceptibilities by the AD method in this study identified a bimodal distribution for each Brachyspira species, with concentrations of ≥0.125 μg/ml forming a second peak (Fig. 1, 2, and 3). Although an epidemiological cutoff value for carbadox is not currently available for Brachyspira species, our results, similar to those reported by Duhamel et al. (17), suggest that a MIC of ≥0.125 μg/ml obtained by the AD method might represent such a cutoff value for decreased carbadox susceptibility. Future studies investigating the genetic mechanisms of resistance and surveying a larger number of Brachyspira isolates may provide further support for such a bimodal distribution and a potential epidemiological cutoff value. In our study, one B. hampsonii isolate and seven B. hyodysenteriae isolates with carbadox MICs of ≥0.125 μg/ml by the AD method were identified. Based on the criteria described by Duhamel et al. (17), no carbadox-resistant isolates were detected among the Brachyspira isolates evaluated.
Interestingly, the more important pathogens B. hampsonii and B. hyodysenteriae showed greater susceptibilities to the evaluated antimicrobials than did the less clinically important pathogen B. pilosicoli and the commensal B. murdochii (Fig. 1 and 2). Furthermore, B. pilosicoli, which is known to be a highly recombinant species that demonstrates a substantial amount of genomic variation (40), could be prone to developing mutations associated with antimicrobial resistance. The tendency of B. pilosicoli to develop resistance more rapidly than other Brachyspira species is supported by the “skipped well” phenomenon, i.e., spontaneous mutations associated with in vitro resistance to an antimicrobial that develops in the inoculated medium during susceptibility testing (36). Two previous studies observed skipped wells while testing the B. pilosicoli type strain P43/6/78 (ATCC 51139) and several field isolates for tylosin and lincomycin susceptibility (14, 36). In our study, we identified six B. pilosicoli field isolates that exhibited this skipped well phenomenon with respect to tylosin and/or valnemulin (data not shown).
Since tiamulin is commonly used for the treatment of swine dysentery in many countries, it is important to identify and to monitor strains with decreased tiamulin susceptibility. Eight B. hyodysenteriae isolates (representing genotypes ST94 and ST107) characterized as being tiamulin resistant and/or having decreased tiamulin susceptibility (Table 5) originated from different finisher farms of a common production system (system M) in the United States (see Table S1 in the supplemental material). A previous study (28) evaluated tiamulin-resistant B. hyodysenteriae isolates (Table 5) from different farms in the United Kingdom that were known to be connected epidemiologically by pig movement. Those isolates were found to have the same MICs for six antimicrobials (including tiamulin) and the same pulsed-field gel electrophoresis patterns, which indicates that the tiamulin-resistant strain spread clonally between farms (28). Similarly, tiamulin-resistant genotypes ST8 (from Italy and the United Kingdom) and ST52 (from Italy and Spain) (Table 5) could represent scenarios of transmission of resistant strains between geographical regions (23). None of the tiamulin-resistant B. hyodysenteriae genotypes from the United States was identified in other countries. Resistant strains can disseminate in the population through the movement of infected pigs, fomites, or vectors. Therefore, early detection of isolates with decreased susceptibility, followed by strain monitoring, can help control the rapid spread of resistant strains.
The statistically significant finding of Brachyspira isolates originating from specific production systems having decreased pleuromutilin susceptibility (Table 4) highlighted the role of husbandry in swine production. Production systems that own and/or manage multiple farms or sites often have epidemiological connections between the farms, including the sources of the animals and the movement of animals, trucks, workers, and/or fomites. It is possible that the use of tiamulin in a herd with a history of swine dysentery could have selected for a resistant strain that clonally disseminated to other farms in the same production system. Interestingly, both B. hyodysenteriae genotypes (ST94 and ST107) associated with tiamulin resistance originated from system M (see Table S1 in the supplemental material). B. hyodysenteriae genotypes are known to be associated with production systems (8), and the antimicrobial usage therein may determine the resultant antimicrobial susceptibility of the genotypes. Although not all B. hyodysenteriae isolates of ST94 and ST107 were tiamulin resistant (see Table S1), the association of those genotypes with the susceptibility phenotype (and production system M) can facilitate monitoring of the spread of resistant strains.
Preliminary observations indicated that most isolates with decreased susceptibility originated from finisher farms, while most isolates with high susceptibility originated from breeding farms (see Table S1 in the supplemental material). Our analysis confirmed that there were significantly greater odds of having decreased tylosin or lincomycin susceptibility for B. hampsonii isolates and particularly B. hyodysenteriae isolates originating from finisher farms than for B. hyodysenteriae isolates originating from breeding farms (Table 4). It is known that B. hyodysenteriae resistance to tylosin and lincomycin is commonly observed. The surprising finding of tylosin- and lincomycin-susceptible Brachyspira isolates in breeding farms may be due to less antimicrobial exposure. Specialized breeding farms generally have stricter biosecurity practices and house more-mature animals than growing pig farms, and these factors may reduce the need for antimicrobial use. A previous study identified a link between biosecurity and both production-related and treatment-related measures in swine herds, suggesting that improved biosecurity practices may decrease antimicrobial use (41). Unfortunately, little information exists regarding the association of antibiograms and biosecurity and/or husbandry practices for different stages of swine production; our findings suggest that this topic warrants further investigation.
Over the past 4 decades, some countries in the European Union have banned the use of certain antimicrobials for growth promotion purposes in pigs (12). Specifically, tylosin was banned as a growth promoter in the European Union in 1999, and all antimicrobials were banned for growth promotion purposes across the European Union in 2006 (12). Additionally, the use of carbadox as a therapeutic and/or growth-promoting additive in food was banned in Australia, Japan, the European Union (12), and Canada. As of 1 January 2017, the U.S. Food and Drug Administration (FDA) guidance (42) will prohibit the use of medically important antimicrobials (which excludes carbadox and tiamulin) for growth promotion purposes and will require veterinary oversight of their therapeutic use in feed or water. Over the past 2 decades, the unavailability of an effective commercial vaccine, the ban on the use of several antimicrobials (including carbadox), and the high prevalence of swine dysentery in the European Union have led to a reliance on pleuromutilins for control of the disease (22). This reliance has been followed by the emergence of pleuromutilin-resistant strains in many countries (20–23, 28, 29). Since swine dysentery had a low prevalence in North American pigs during that time, likely less antimicrobial usage was directed to swine dysentery control, thus favoring the susceptibility trends in the United States. However, the FDA recently announced its recommendation to rescind approval for carbadox use in pigs in the United States (43). If successful, this action will further limit the number of effective antimicrobials available for control of swine dysentery in the United States. Additionally, the lack of CLSI-approved susceptibility testing methods and clinical breakpoints for Brachyspira species and the variability in interpretations of susceptibility test results according to different criteria or cutoff values continue to complicate elimination and treatment programs and therapeutic decisions globally. These findings emphasize the urgent need for internationally accepted and CLSI-approved clinical breakpoints for Brachyspira species, in order to facilitate judicious use of antimicrobial agents in the treatment of dysentery. In conclusion, the recent reemergence of dysentery in North America, the emergence of a novel pathogen (B. hampsonii), and the reports of Brachyspira strains with reduced susceptibility to multiple antimicrobials all highlight the need to monitor pathogens and their susceptibility to commonly used antimicrobials, in order to control and to treat Brachyspira-related diseases.
Supplementary Material
ACKNOWLEDGMENTS
We thank the University of Minnesota Veterinary Diagnostic Laboratory for providing us with B. hampsonii, B. hyodysenteriae, B. pilosicoli, and B. murdochii field isolates originating from the United States. We also thank Aaron Rendahl for his assistance with statistical analyses.
Funding Statement
The funders had no role in study design, data collection, data analysis, interpretation of findings, preparation of the manuscript, or the decision to publish.
Footnotes
Supplemental material for this article may be found at http://dx.doi.org/10.1128/JCM.00834-16.
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