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. 2004 Dec;48(12):4903–4906. doi: 10.1128/AAC.48.12.4903-4906.2004

Antimicrobial Susceptibility of Bordetella bronchiseptica Isolates from Porcine Respiratory Tract Infections

Kristina Kadlec 1, Corinna Kehrenberg 1, Jürgen Wallmann 2, Stefan Schwarz 1,*
PMCID: PMC529238  PMID: 15561878

Abstract

MICs for 349 Bordetella bronchiseptica isolates from respiratory tract infections of swine were determined by broth microdilution. The lowest MIC at which 90% of isolates tested are inhibited (MIC90) was that of tetracycline and enrofloxacin (0.5 μg/ml), whereas the highest MIC90s were those of tilmicosin and cephalothin (32 μg/ml) as well as streptomycin (256 μg/ml).

Porcine respiratory diseases represent the leading cause of mortality in nursery and finishing units (12). Bordetella bronchiseptica is often involved in porcine respiratory tract infections, along with viruses and other bacteria (1). It has been shown that infections with B. bronchiseptica predispose pigs to secondary infections with toxigenic strains of Pasteurella multocida and thus play an important role in the pathogenesis of severe atrophic rhinitis (1, 8). Various antimicrobial agents are licensed and used for the control of bacteria involved in porcine respiratory diseases and atrophic rhinitis, including aminopenicillins, cephalosporins, aminoglycosides, tetracyclines, macrolides, lincosamides alone or in combination with spectinomycin, potentiated sulfonamides, fluoroquinolones, pleuromutilins, and florfenicol. In contrast to well-studied pathogens such as P. multocida (for a review see reference 5), comparatively little is known about the antimicrobial susceptibility of porcine B. bronchiseptica isolates (3, 5, 6, 9-11, 14, 16, 20).

Between 2000 and 2003, 349 B. bronchiseptica isolates were collected from cases of bronchopneumonia and/or atrophic rhinitis of swine in Germany. This study includes 78 isolates from 2000, 98 isolates from 2001, 91 isolates from 2002, and 82 isolates from 2003. All isolates were collected from diseased animals on the basis of one isolate per herd. The animals had not been treated with antimicrobial agents in the 3 weeks prior to sample collection. Samples included nasal swabs sent to the diagnostic laboratories by veterinarians and lung tissue obtained during postmortem inspections at diagnostic laboratories. Microbiological sample processing and biochemical confirmation of species assignment followed standard procedures (7). All bacterial isolates were investigated for their in vitro susceptibility to antimicrobial agents by the microdilution broth method with microtiter plates (Sensititre, Westlake, Ohio) that contained the antimicrobial agents in serial twofold dilutions. The layouts of the microtiter plates corresponded to those used in the German resistance monitoring program for veterinary pathogens (GERM-VET). The antimicrobial agents and concentrations tested are shown in Table 1. Performance and evaluation of the susceptibility tests followed the recommendations given in document M31-A2 of the National Committee for Clinical Laboratory Standards (13). Specifically, an inoculum that corresponded to a 0.5 McFarland standard was prepared in cation-supplemented Mueller-Hinton broth and then further diluted to yield a final concentration of 105 CFU/ml. After incubation for 16 to 20 h at 35°C, the wells of the microtiter plates were inspected macroscopically for growth. The reference strain Escherichia coli ATCC 25922 served for quality control purposes (13).

TABLE 1.

Comparison of MIC data from porcine B. bronchiseptica isolates from 2000 to 2003

Antimicrobial agent and yra No. of isolates for which MIC (μg/ml) isb:
MIC (μg/ml)c
0.015 0.03 0.06 0.12 0.25 0.5 1 2 4 8 16 32 64 128 256 50% 90%
Ampicillin
    2000 0 0 0 8 2 7 56 2 0 2 1 0 8 8
    2001 0 0 0 2 0 5 78 7 1 3 2 0 8 16
    2002 0 0 0 1 3 6 36 41 1 0 3 0 16 16
    2003 0 0 0 1 1 13 30 31 5 1 0 0 8 16
        Total 0 0 0 12 6 31 200 81 7 6 6 0 8 16
Amoxicillin clavulanic acid (2:1)d
    2000 0 0 0 8 3 47 20 0 0 0 2 4
    2001 0 0 0 2 49 45 2 0 0 2 4
    2002 0 0 0 3 5 42 40 1 0 0 2 4
    2003 0 0 0 1 9 31 38 2 1 0 2 4
        Total 0 0 0 14 17 169 143 5 1 0 2 4
Chloramphenicol
    2000 0 0 0 0 4 31 34 1 2 2 0 3 1 4 8
    2001 0 0 0 0 0 23 67 5 1 1 1 0 0 4 4
    2002 0 0 0 0 1 35 43 10 0 0 0 1 1 4 8
    2003 0 0 0 0 11 30 33 3 0 0 2 2 1 2 4
        Total 0 0 0 0 16 119 177 19 3 3 3 6 3 4 8
Florfenicol
    2000 67 7 2 2 0 2 4
    2001 71 26 1 0 0 2 4
    2002 73 17 0 1 0 2 4
    2003 67 11 0 0 4 2 4
        Total 278 61 3 3 4 2 4
Tetracycline
    2000 29 39 7 3 0 0 0 0 0 0 0 0 0.25 0.5
    2001 5 65 27 1 0 0 0 0 0 0 0 0 0.25 0.5
    2002 6 63 17 1 1 0 0 0 0 3 0 0 0.25 0.5
    2003 8 60 11 2 1 0 0 0 0 0 0 0 0.25 0.5
        Total 48 227 62 7 2 0 0 0 0 3 0 0 0.25 0.5
Gentamicin
    2000 0 0 0 0 4 74 0 0 0 0 0 0 0 2 2
    2001 0 0 0 0 2 95 1 0 0 0 0 0 0 2 2
    2002 0 0 0 1 21 69 0 0 0 0 0 0 0 2 2
    2003 0 0 1 1 33 45 2 0 0 0 0 0 0 2 2
        Total 0 0 1 2 60 283 3 0 0 0 0 0 0 2 2
Neomycin
    2000 0 0 0 0 0 0 32 46 0 0 0 0 0 8 8
    2001 0 0 0 0 0 0 28 70 0 0 0 0 0 8 8
    2002 0 0 0 0 0 30 47 13 0 0 1 0 0 4 8
    2003 0 0 1 0 1 43 21 15 0 0 0 1 0 8 8
        Total 0 0 1 0 1 73 128 144 0 0 1 1 0 8 8
Streptomycin
    2000 0 0 0 0 0 0 0 0 7 52 2 17 64 256
    2001 0 0 0 0 0 0 0 0 2 78 3 15 64 256
    2002 0 0 0 0 0 0 0 0 1 49 28 13 64 256
    2003 0 0 0 0 1 0 0 1 1 57 16 6 64 128
        Total 0 0 0 0 1 0 0 1 11 236 49 51 64 256
Nalidixic acid
    2000 1 1 14 59 1 2 8 8
    2001 1 0 11 80 5 1 8 8
    2002 0 3 7 69 11 1 8 16
    2003 3 7 4 31 36 1 8 16
        Total 5 11 36 239 53 5 8 16
Enrofloxacin
    2000 0 1 1 8 59 7 0 2 0 0.25 0.5
    2001 0 0 0 9 51 37 1 0 0 0.25 0.5
    2002 0 0 5 13 18 53 2 0 0 0.5 0.5
    2003 1 1 4 5 32 36 2 1 0 0.25 0.5
        Total 1 2 10 35 160 133 5 3 0 0.25 0.5
Trimethoprim
    2000 32 13 22 4 3 4 4 16
    2001 25 9 24 38 1 1 8 16
    2002 40 24 12 11 3 1 4 16
    2003 63 5 10 4 0 0 2 8
        Total 160 51 68 57 7 6 4 16
Trimethoprim/sulfamethoxazole (1:19)e
    2000 31 6 4 3 1 4 14 10 1 1 2 1 0.12 4
    2001 36 20 8 0 0 3 11 15 4 0 1 0 0.12 4
    2002 47 8 1 0 1 4 14 10 3 2 1 0 0.06 4
    2003 52 7 1 1 6 7 2 6 0 0 0 0 0.06 1
        Total 166 41 14 4 8 18 41 41 8 3 4 1 0.12 4
Tilmicosin
    2000 0 0 0 0 2 12 32 22 10 8 32
    2001 0 0 0 0 1 1 8 54 34 16 32
    2002 0 0 0 0 0 10 5 51 25 16 32
    2003 0 0 0 1 0 10 13 35 23 16 32
        Total 0 0 0 1 3 33 58 162 92 16 32
Ceftiofur
    2000 0 0 0 0 0 0 0 0 0 5 73 16 16
    2001 0 0 0 0 0 0 0 0 0 1 97 16 16
    2002 0 0 0 0 0 0 0 0 1 78 12 8 16
    2003 0 0 0 0 0 0 0 1 2 57 22 8 16
        Total 0 0 0 0 0 0 0 1 3 141 204 16 16
Cephalothin
    2000 0 0 0 0 0 0 2 68 7 0 1 0 0 8 16
    2001 0 0 0 0 0 0 0 57 36 3 2 0 0 8 16
    2002 0 0 0 0 0 0 0 30 38 22 0 1 0 16 32
    2003 0 0 0 0 0 0 0 18 11 50 1 2 0 32 32
        Total 0 0 0 0 0 0 2 173 92 75 4 3 0 8 32
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a

n = 78, 98, 91, and 82 for 2000, 2001, 2002, and 2003, respectively.

b

MICs equal to or lower than the lowest concentration tested are given as the lowest concentration, whereas MICs equal to or higher than the highest concentration tested are given as the highest concentration.

c

50% and 90%, MIC50 and MIC90, respectively.

d

The MICs of amoxicillin/clavulanic acid (2:1) are expressed as MICs of amoxicillin.

e

The MICs of trimethoprim/sulfamethoxazole (1:19) are expressed as MICs of trimethoprim.

The distribution of the MICs of the B. bronchiseptica isolates tested in this study is shown in Table 1. A year-by-year comparison of the data obtained for each antimicrobial agent revealed virtually no variations in the MICs at which 50 and 90% of isolates tested are inhibited (MIC50s and MIC90s, respectively) over the 4-year period. The maximum difference seen was two dilution steps in the MIC50s of cephalothin and trimethoprim and in the MIC90s of trimethoprim/sulfamethoxazole.

Using National Committee for Clinical Laboratory Standards-approved B. bronchiseptica-specific breakpoints for florfenicol (susceptible, ≤2 μg/ml; intermediate, 4 μg/ml; resistant, ≥8 μg/ml), 10 (2.9%) isolates were classified as resistant and another 61 (17.5%) as intermediate. This confirms the results of two florfenicol-specific monitoring studies conducted in Germany in 2000 to 2001 (16) and 2002 to 2003 (4). The MICs of chloramphenicol for all florfenicol-resistant strains were also high (≥128 μg/ml). A comparison of the MICs of ampicillin and amoxicillin/clavulanic acid suggested that the presumable β-lactamases which may account for the high MICs of ampicillin are susceptible to inhibition by clavulanic acid. Different distributions of MICs were recorded for the three aminoglycoside antibiotics gentamicin, neomycin, and streptomycin. While the MICs of streptomycin for 336 (96.3%) of the isolates were ≥64 μg/ml, those of gentamicin ranged between 0.25 and 4 μg/ml, with the MICs for 343 (98.3%) isolates 1 or 2 μg/ml. In the case of neomycin, the MICs for 345 (98.9%) isolates were 2 to 8 μg/ml, while distinctly higher MICs of 64 and 128 μg/ml were seen for single isolates. With tetracycline, the MICs for 346 isolates were ≤2 μg/ml and that for the remaining 3 isolates was 64 μg/ml. Although sulfonamides were not included in the test panels, a comparison of the MICs of trimethoprim and trimethoprim/sulfamethoxazole suggested that sulfonamides had some effect against isolates for which MICs of trimethoprim were elevated. The overall MICs of both cephalosporins tested for the B. bronchiseptica isolates in this study were high: ceftiofur, MIC90 of ≥16 μg/ml; and cephalothin, MIC90 of 32 μg/ml. A similar situation was seen with tilmicosin, with MIC50 and MIC90 of 16 and 32 μg/ml, respectively. In contrast, a low MIC50 and MIC90 of 0.25 and 0.5 μg/ml, respectively, were recorded for enrofloxacin.

Comparison of the results of this study with those of other studies is often problematic for several reasons: (i) different methodologies were used for susceptibility testing, including agar dilution (9, 10, 14, 17, 18), E-test (17, 18), and disk diffusion (6, 15, 19, 20); (ii) different antimicrobial agents were tested (6, 9, 10, 14); (iii) the evaluation of the results followed different guidelines (6, 9, 10, 14, 19); and/or (iv) isolates from animals other than pigs were tested (15, 17, 18). However, three studies from the United States (2, 3, 11) were suitable for comparisons with our data. In the first study (11), the range of MICs as well as the MIC90s of various antimicrobial agents were determined in 1988 for 48 porcine B. bronchiseptica isolates collected in the United States. The results for ampicillin, gentamicin, chloramphenicol, cephalothin, and trimethoprim/sulfamethoxazole corresponded closely to those of the present study, whereas the values for tetracycline were lower in the current study of German isolates (11). The second study described the in vitro susceptibility to tilmicosin of porcine respiratory tract pathogens collected between 1994 and 1998 in the United States (3). There was a close similarity between their observed range and MIC90 of tilmicosin and those found in the present study. The third study dealt with the in vitro susceptibility of porcine respiratory tract pathogens to ceftiofur and revealed that B. bronchiseptica isolates are rather insensitive to ceftiofur; the MICs for these isolates were ≥8 μg/ml (2). This was in good accordance with our observation that the ceftiofur MIC for 345 (98.9%) of the 349 B. bronchiseptica isolates was ≥8 μg/ml.

The classification of B. bronchiseptica isolates as susceptible, intermediate, or resistant based on the MIC data presents some problems. Interpretive criteria that can be used explicitly for B. bronchiseptica are currently only available for florfenicol, but not for the other antimicrobial agents tested in this study. Nevertheless, the data presented in this study allow a reliable estimate of the resistance status of German B. bronchiseptica isolates from porcine respiratory diseases based on testing a large number of isolates and using internationally accepted methods. In addition to other data such as pharmacokinetic and pharmacodynamic parameters or clinical efficacy, the MIC data of this study may help to establish breakpoints for antimicrobial agents for which no breakpoints approved for B. bronchiseptica are currently available.

Acknowledgments

Kristina Kadlec is supported by a scholarship of the H. Wilhelm Schaumann foundation.

We thank Thomas R. Shryock and the NCCLS Subcommittee on Veterinary Antimicrobial Susceptibility Testing as well as Joseph W. Carnwath for helpful discussions.

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