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Antimicrobial Agents and Chemotherapy, December 2004, p. 4903-4906, Vol. 48, No. 12
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.12.4903-4906.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Antimicrobial Susceptibility of Bordetella bronchiseptica Isolates from Porcine Respiratory Tract Infections

Kristina Kadlec,1 Corinna Kehrenberg,1 Jürgen Wallmann,2 and Stefan Schwarz1*

Institut für Tierzucht, Bundesforschungsanstalt für Landwirtschaft (FAL), Neustadt-Mariensee,1 Bundesamt für Verbraucherschutz und Lebensmittelsicherheit (BVL), Berlin, Germany2

Received 7 July 2004/ Returned for modification 6 August 2004/ Accepted 9 August 2004


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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).


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TEXT
 
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).


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  		TABLE 1. Comparison of MIC data from porcine B. bronchiseptica isolates from 2000 to 2003

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 ß-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.


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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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FOOTNOTES
 
* Corresponding author. Mailing address: Institut für Tierzucht der Bundesforschungsanstalt für Landwirtschaft (FAL), Höltystr. 10, 31535 Neustadt-Mariensee, Germany. Phone: 49-5034-871-241. Fax: 49-5034-871-246. E-mail: stefan.schwarz{at}fal.de. Back


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Antimicrobial Agents and Chemotherapy, December 2004, p. 4903-4906, Vol. 48, No. 12
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.12.4903-4906.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Kadlec, K., Wiegand, I., Kehrenberg, C., Schwarz, S. (2007). Studies on the mechanisms of {beta}-lactam resistance in Bordetella bronchiseptica. J Antimicrob Chemother 59: 396-402 [Abstract] [Full Text]  
  • Kadlec, K., Kehrenberg, C., Schwarz, S. (2007). Efflux-mediated resistance to florfenicol and/or chloramphenicol in Bordetella bronchiseptica: identification of a novel chloramphenicol exporter. J Antimicrob Chemother 59: 191-196 [Abstract] [Full Text]  
  • Kadlec, K., Kehrenberg, C., Schwarz, S. (2006). tet(A)-mediated tetracycline resistance in porcine Bordetella bronchiseptica isolates is based on plasmid-borne Tn1721 relics. J Antimicrob Chemother 58: 225-227 [Full Text]  
  • Kadlec, K., Kehrenberg, C., Schwarz, S. (2005). Molecular basis of resistance to trimethoprim, chloramphenicol and sulphonamides in Bordetella bronchiseptica. J Antimicrob Chemother 56: 485-490 [Abstract] [Full Text]  

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