JAC Advance Access originally published online on January 29, 2007
Journal of Antimicrobial Chemotherapy 2007 59(3):396-402; doi:10.1093/jac/dkl515
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Studies on the mechanisms of ß-lactam resistance in Bordetella bronchiseptica
1 Institut für Tierzucht, Bundesforschungsanstalt für Landwirtschaft (FAL), Höltystr. 10, 31535 Neustadt-Mariensee, Germany 2 Institut für Medizinische Mikrobiologie, Immunologie und Parasitologie, Pharmazeutische Mikrobiologie, Universität Bonn, Meckenheimer Allee 168, 53115 Bonn, Germany
* Corresponding author. Tel: +49-5034-871-241; Fax: +49-5034-871-246; E-mail: stefan.schwarz{at}fal.de
Received 5 October 2006; returned 17 November 2006; revised 18 November 2006; accepted 20 November 2006
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Abstract |
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Objectives: Little is currently known about ß-lactam resistance in Bordetella bronchiseptica. So far, only a single ß-lactamase gene, blaBOR-1, has been identified. In a previous study, high MICs of ampicillin, cefalotin and ceftiofur were determined among 349 porcine B. bronchiseptica isolates. The aim of this study was to identify genes associated with elevated MICs of ß-lactams and their transferability.
Methods: Selected isolates were investigated by PCR for commonly found bla genes and class 1 integrons; selected amplicons were sequenced. Plasmid location of resistance genes was confirmed by conjugation. ß-Lactamases were characterized by SDS–PAGE and isoelectric focusing. The genomic relatedness of the isolates was investigated by XbaI macrorestriction analysis. Inhibition studies with efflux pump inhibitors were conducted. The permeability of cephalosporins into intact cells was measured exemplarily for one isolate.
Results: Of the 349 B. bronchiseptica isolates, eight isolates carried a class 1 integron with a blaOXA-2 cassette on a conjugative plasmid of ca. 50 kb. In addition, one plasmid-free isolate also carried this class 1 integron. Besides blaBOR-1, no other ß-lactamase gene was detected in the remaining isolates with high MICs of ampicillin of 
32 mg/L. Inhibition experiments suggested that efflux does not play a role in ß-lactam resistance. Instead, membrane permeability for cephalosporins was reduced as shown for B. bronchiseptica isolate B543.
Conclusions: This is to the best of our knowledge the first report of a mobile bla gene in B. bronchiseptica. Reduced membrane permeability of B. bronchiseptica seems to decrease susceptibility against cephalosporins.
Keywords: class D ß-lactamases , blaOXA-2 , class 1 integrons , membrane permeability , efflux , cephalosporins
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Bordetella bronchiseptica is often involved in respiratory tract infections of mammals and plays an important role in farm animals such as pigs and rabbits as well as in pets, e.g. cats and dogs. B. bronchiseptica infections may preferentially develop under conditions where animals are kept at high density, e.g. in intensive animal production systems or animal shelters.1 Infections with B. bronchiseptica may predispose pigs to infections with other respiratory tract pathogens, in particular toxigenic Pasteurella multocida, which then can cause the severe progressive form of atrophic rhinitis.2 B. bronchiseptica is a zoonotic agent and B. bronchiseptica infections causing pneumonia or pertussis-like symptoms in humans are rarely observed. If so, they are most frequently seen in immunocompromised individuals and/or persons with contact to infected animals.3
Little is known about ß-lactam resistance in B. bronchiseptica although high MICs of penicillins and cephalosporins have been described for B. bronchiseptica.4–7 Plasmid-associated resistance to penicillin has also been observed.8,9 An oxacillin hydrolysing protein was described in 197410 and a ß-lactamase with a molecular weight of 46 ± 3 kDa and an isoelectric point (pI) at pH 8.3 was detected in 197511 in porcine B. bronchiseptica isolates. Similar studies on isolates from cats were done more than 20 years later, where a penicillinase with a molecular weight of 49 kDa and a pI at pH 8.45 was detected.12 In none of these studies, however, was the corresponding ß-lactamase gene identified. In 2005, the first ß-lactamase gene sequence from B. bronchiseptica was published for the chromosomally located species-specific blaBOR-1 gene from a human isolate with an MIC of ampicillin of 8 mg/L.13
In the present study, 19 isolates from pigs were investigated for the molecular basis of ampicillin resistance with a focus on the association of resistance genes with mobile genetic elements and the possibility of horizontal transfer of the resistance genes. Moreover, the role of efflux in ß-lactam resistance was investigated and the diffusion of cephalosporins into intact B. bronchiseptica cells was investigated exemplarily in one isolate.
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Isolates and susceptibility testing
From 349 porcine B. bronchiseptica isolates collected in Germany from 2000 to 2003, the results of susceptibility testing against 15 different antimicrobial agents or combinations of agents have been published.7 As reported, 19 isolates showed MICs of ampicillin of 32 mg/L and were included in this study. To better describe the ß-lactam resistance phenotype, these 19 isolates and 7 further B. bronchiseptica isolates that exhibited lower MICs of ampicillin (1–16 mg/L) were tested for susceptibility to additional ß-lactams. The 19 isolates with high MICs of ampicillin, the isolate B543 — used for permeability experiments — and three isolates with lower MICs of ampicillin were chosen to investigate whether efflux mechanisms may play a role. Susceptibility testing was done by broth micro- or macro-dilution or by disc diffusion according to the guideline M31-A2 of the Clinical and Laboratory Standards Institute (CLSI; formerly NCCLS).14
For biochemical ß-lactamase characterization, cells were grown to an OD600 of 1.0 and then harvested by centrifugation at 4°C. Crude protein extracts were either prepared as described previously15 using ultrasound treatment for cell disruption or lysozyme treatment (final concentration 0.2 mg/mL) for 15 min at room temperature with three additional freeze and thaw steps. The protein content in the crude ß-lactamase extracts was determined using BSA as standard.15 In addition, the extract of each isolate was loaded on an SDS–PAGE gel with 13% (w/v) acrylamide and on an isoelectric focusing (IEF) gel with a pH range of 3.0–10.0 (Bio-Rad, Munich, Germany). Gels were stained with 1 mM nitrocefin to detect ß-lactamase activity.15
Genetic basis of ampicillin resistance
Isolation of plasmids by alkaline lysis and whole cell DNA by phenol/chloroform extraction followed previously described standard protocols.16 To detect the most common ampicillin resistance genes by PCR, previously described primer sets were used for the detection of blaTEM,17 blaPSE-1,18 blaSHV,19,20 blaROB-121 and chromosomally and plasmid-encoded AmpC ß-lactamase genes in Enterobacteriaceae.22 PCRs for blaBOR-1,13 the species-specific ß-lactamase gene from B. bronchiseptica, were also done and DMSO was added to a final concentration of 9% (v/v) to the reaction mixture. As ß-lactamase genes are often located on gene cassettes or associated with class 1 integrons, PCRs for conserved regions of class 1 integrons were performed.16
Conjugation experiments were performed by filter mating with the rifampicin-resistant recipient strain E. coli HK225 as described previously.16 In addition, B. bronchiseptica V1645/2 was used as recipient. This isolate has a high MIC of neomycin of 128 mg/L suitable for counter-selection purposes. Transformation into E. coli recipient strains JM109 and JM101 (Stratagene, Amsterdam, The Netherlands) followed a previously described protocol.16 For both experiments, LB or blood agar plates containing ampicillin (30 mg/L) were used.
To sequence selected PCR products, cloning experiments were performed into the vector pCR Blunt and the recombinant plasmids were transformed into chemically competent E. coli TOP10 cells (Invitrogen, Groningen, The Netherlands). Sequence comparisons were carried out using the BLAST® programs blastn and blastp (http://www.ncbi.nlm.nih.gov/BLAST/) and with the ORF finder program (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). The nucleotide sequence has been deposited in the European Molecular Biology Laboratory (EMBL) database under accession number AJ877267 [GenBank] .
Macrorestriction analysis with XbaI and PFGE of the fragment patterns followed a previously described protocol.16
Inhibition of efflux mechanisms
The efflux pump inhibitors Phe-Arg-ß-naphthylamide (PAßN) and carbonyl cyanide m-chlorophenylhydrazone (CCCP) were used for inhibition profiles.23,24 The MICs were determined by macrodilution according to the CLSI guidelines14 and PAßN was added to each tube with a final concentration of 20–80 mg/L or CCCP with 0.5–4 mg/L, representing 
of the strain-specific MICs of these substances.25
Diffusion of cephalosporins into intact cells
We investigated the membrane permeability of one representative B. bronchiseptica isolate (B543) to the cephalosporin cefoxitin by using the Zimmermann and Rosselet technique, which requires that the strain under investigation harbours a suitable ß-lactamase. The test is based on the fact that ß-lactamases in the periplasm of a Gram-negative bacterium act in cooperation with the permeability barrier represented by the outer membrane. At equilibrium, the rate of drug entry equals the rate of hydrolysis within the periplasm.26 In addition to B. bronchiseptica B543, the rifampicin-resistant E. coli strain HK225 was used for comparison. A plasmid carrying the ß-lactamase gene blaCMY-2 was transferred into both strains. The corresponding ß-lactamase CMY-2 is capable of hydrolysing cephalosporins of different generations, including the tested antibiotics cefoxitin and cefalotin, but not ceftiofur.27 The plasmid carrying blaCMY-2 also harbours other resistance genes, including tet(A) for tetracycline resistance, and was isolated from the E. coli clinical isolate no. 56 provided by K. J. Sherwood (Institut für Medizinische Mikrobiologie, Immunologie und Parasitologie, Universität Bonn, Germany). The plasmid was transferred into E. coli HK225 by filter mating as described previously16 with selection on LB agar supplemented with 50 mg/L cefoxitin and 100 mg/L rifampicin. The plasmid carrying blaCMY-2 was also transferred to B. bronchiseptica B543 via electrotransformation as described previously28 for Pasteurella with the Gene Pulser II electroporation system (Bio-Rad, Munich, Germany) and selection was done on blood agar plates containing 20 mg/L tetracycline. In order to confirm the successful transfer of the plasmid into the transformants and transconjugants, PCR for blaCMY-2 was performed using previously described primers.22 Furthermore, the strains were tested for the expression of the ß-lactamase by determination of the specific ß-lactamase activity. ß-Lactamase activity was quantified spectrophotometrically by measuring the change in absorbance at 485 nm using 50 µM nitrocefin (Oxoid, Basingstoke, UK) as substrate and 0.01 M Tris HCl buffer (pH 7.0) as test buffer.
The test for diffusion of cephalosporins into intact B. bronchiseptica B543 and E. coli HK225 cells was performed as described previously29,30 with slight modifications. In brief, overnight cultures were diluted 1:20 for E. coli HK225 and E. coli HK225::blaCMY-2 and 1:10 for B. bronchiseptica B543 and B. bronchiseptica B543::blaCMY-2 and grown in 100 mL of cation-adjusted Mueller-Hinton bouillon (CAMHB; Oxoid, Wesel, Germany) supplemented with 5 mM MgCl2 to an optical density (OD) of 0.8 at 650 nm. Cells were harvested, washed twice with ice-cold phosphate buffer (0.1 M, pH 7) and resuspended in 30 mL of the same buffer supplemented with 5 mM MgCl2 per 1 g cells. Of this bacterial suspension, 1 mL was dried at 105°C to constant weight. At room temperature, 500 µL of 5 mM cefoxitin was added to 4.5 mL of cell suspension. Immediately after addition of the cephalosporin (= time point 0) and after 15, 30 and 60 min aliquots of 1 mL were removed, filtered (0.2 µm pore-size filter units) and the filtrates were frozen at – 20°C. The same approach was performed with 10 mM cefalotin.
The cephalosporin concentration in the filtrates was measured by a bioassay using Klebsiella pneumoniae IV-2-3 as the test organism. For the bioassay 20 mL of CAMH agar (Oxoid, Wesel, Germany) cooled down at 50°C was mixed with 80 µL of the K. pneumoniae suspension (turbidity equivalent to that of a 0.5 McFarland standard) before pouring the plates. After solidification of the bacteria-supplemented agar, cavities were produced with sterile cylinders of 10 mm in diameter. Each cavity was loaded with 100 µL of sample; all samples were measured at least three times. For calibration, five sets of 2-fold dilutions (2 mM to 0.004 mM) of cefoxitin and cefalotin were used. After 16 to 20 h of incubation at 37°C, the inhibition zones were measured and the cephalosporin concentration in the filtrates was determined via the calibration curves. In addition, nitrocefin hydrolysis by the filtrates was measured as described above to check for leaked ß-lactamase activity.
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Molecular and biochemical basis of ampicillin resistance
The species-specific blaBOR-1 gene was detected by PCR analysis in all 19 tested isolates independently of their MICs of ampicillin. The blaBOR-1 genes of four isolates with different PFGE patterns and with different MICs of ampicillin (32–128 mg/L) were cloned and sequenced. These blaBOR-1 genes showed the same nucleotide sequence with 99% identity to the originally described blaBOR-1 sequence.13 Cloning of the complete blaBOR-1 gene into E. coli and subsequent susceptibility testing revealed that all clones were resistant to ampicillin with MICs of 256 mg/L, in comparison with the recipient E. coli TOP10 which had an MIC of 4 mg/L. No blaTEM, blaPSE, blaSHV, blaROB-1 or blaAmpC genes could be detected in any of these 19 isolates by PCR.
Of the 19 isolates, nine carried a class 1 integron with a single blaOXA-2 gene cassette. This blaOXA-2 cassette was indistinguishable in its nucleotide sequence from previously described gene cassettes carrying blaOXA-2.25 The blaOXA-2 gene coded for a protein of 275 amino acids of which the first 21 amino acids represent a leader peptide that is removed during the maturation process. In contrast to most other gene cassettes, the translational termination codon of the blaOXA-2 gene was located downstream of the 1L and 2L integrase binding sites within the 59-base element (Figure 1). The class 1 integron was located on a plasmid of ca. 50 kb in eight isolates. Since this plasmid from each of the eight isolates exhibited the same EcoRI and PstI restriction pattern, a common designation pKBB282 was given. Plasmid pKBB282 proved to be conjugative and conferred resistance to ampicillin in E. coli HK225 recipients with a 4- to 8-fold increase in the MIC to 16–32 mg/L and in B. bronchiseptica V1645/2 with an 8-fold increase in the MIC to 128 mg/L. The same integron was also detected in one of the remaining plasmid-free isolates (Table 1). PCR assays with the primer sets intI1/5'-CS and 3'-CS/sul1 revealed the expected products for all nine isolates and thus confirmed the presence of a complete class 1 integron. The corresponding OXA-2 ß-lactamase had a molecular weight of 29 kDa and a pI at > pH 8. The calculated pI value for the mature protein was 9.07 (Compute pI/Mw tool; ExPASy, Switzerland).
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All isolates carrying blaOXA-2 showed closely related macrorestriction patterns: four of them exhibited the most common pattern A and the other five isolates had pattern A* differing from pattern A in only one band. While five blaOXA-2-negative isolates also showed the most common pattern A, the remaining five blaOXA-2-negative isolates differed from pattern A by at least two XbaI fragments (Table 1).
No other ß-lactamases could be identified in the nine blaOXA-2-positive isolates as well as in the remaining ten blaOXA-2-negative isolates by SDS–PAGE and IEF. Although the carriage of the blaBOR-1 gene was confirmed for all 19 isolates, no band corresponding to the BOR-1 ß-lactamase with the calculated weight of the mature enzyme of 29.6 kDa (Compute pI/Mw tool; ExPASy, Switzerland) could be observed on the SDS–PAGE gel stained with nitrocefin. With a calculated pI value of 9.97 (Compute pI/Mw tool; ExPASy, Switzerland), BOR-1 was not expected to be seen on the IEF gels used.
Additional susceptibility testing
Although MICs of ampicillin varied over more than six dilution steps, all isolates tested showed a similar susceptibility profile for the other ß-lactam antibiotics tested (Table 2). All isolates showed low MICs of piperacillin, piperacillin/tazobactam and meropenem. However, high MICs were observed for cephalosporins and the respective inhibitor combinations as well as for aztreonam.
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Inhibition of efflux pumps
The MICs of ampicillin and cefoxitin either remained unchanged or decreased by not more than two dilution steps in the presence of one of the two different efflux pump inhibitors PAßN or CCCP in any of the isolates.
Diffusion of cephalosporins into intact cells
Both test strains, B. bronchiseptica B543::blaCMY-2 and E. coli HK225::blaCMY-2, produced high levels of the introduced CMY-2 ß-lactamase. Crude protein extracts of the respective parental strains showed only marginal specific ß-lactamase activities towards nitrocefin with 0.02 and 0.01 µmol/min/mg protein, whereas the activities were ca. 1000-fold increased in the protein extracts of both strains transformed with the blaCMY-2-carrying plasmid.
The bioassay with the filtrates of the intact cells revealed that neither the two parental strains, nor B. bronchiseptica B543::blaCMY-2 showed detectable hydrolysis of cefoxitin, whereas for E. coli HK225::blaCMY-2 a cefoxitin hydrolysis rate of 0.96 nmol/min per mg dry cells was measured (Figure 2). Similar results were achieved with cefalotin; the cefalotin hydrolysis rate for E. coli HK225::blaCMY-2 was 42.57 nmol/min per mg dry cells.
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In this study, the gene for a plasmid-located ß-lactamase (OXA-2) was sequenced for the first time for B. bronchiseptica. After the primary description of OXA-231 and the first sequence of blaOXA-2 located on the plasmid R46 from Salmonella Typhimurium,32 the blaOXA-2 gene has been detected in a variety of bacterial species, e.g. Pseudomonas aeruginosa and K. pneumoniae.
As frequently seen in Enterobacteriaceae, the blaOXA-2 gene in this study was also part of a gene cassette in a class 1 integron located on a conjugative plasmid. This plasmid proved to be transferable to E. coli. In contrast to other blaOXA-2-carrying multiresistance plasmids like R4633 or pB10,34 no additional resistance markers except the sulphonamide resistance gene sul1, which is located in the 3'-conserved segment of class 1 integrons, were detected on plasmid pKBB282. In accordance with recently described genes coding for trimethoprim,16 chloramphenicol16 or tetracycline resistance35 in B. bronchiseptica, the blaOXA-2 gene is another example of a resistance gene from the respiratory tract pathogen B. bronchiseptica that is frequently seen in Enterobacteriaceae, but not in other porcine respiratory tract pathogens.
Although this is the first proof of an OXA-2 enzyme in nine porcine B. bronchiseptica isolates, the data from the literature point to a further distribution of class D oxacillinases in B. bronchiseptica. The ß-lactamase described in B. bronchiseptica from pigs in 197410 showed much better hydrolysis of oxacillin than benzylpenicillin or ampicillin — this has been also shown for blaOXA-2.36,37 The two enzymes described later on in B. bronchiseptica, one from a porcine isolate8 and the other from a feline isolate,12 also hydrolysed oxacillin efficiently. The molecular weight for the two enzymes that was given in these studies was approximately 49 kDa. This high molecular weight is unusual for ß-lactamases.38 In both studies column chromatography was used for the determination of the molecular weight and it seems likely that both enzymes were purified as dimers. Dimerization has been confirmed for the OXA-10 ß-lactamase39 and it was also suggested earlier that other class D ß-lactamases, such as OXA-2, form active dimers.40
Only nine out of 19 isolates with high MICs of ampicillin carried blaOXA-2, thus, other mechanisms must contribute to reduced ampicillin susceptibility in the remaining ten isolates. Active efflux from the cells is unlikely to play a relevant role in ampicillin resistance, because the ampicillin MICs did not change distinctly in the presence of efflux pump inhibitors.
A species-specific ß-lactamase from B. bronchiseptica, BOR-1, has been described in a human isolate with an MIC of amoxicillin of 8 mg/L.13 In the present study, the gene blaBOR-1 was sequenced from four porcine isolates with different MICs and different macrorestriction patterns. The sequences were identical and the gene conferred high-level ampicillin resistance to E. coli. The significant reduction of the MIC of amoxicillin by clavulanic acid in all ten blaOXA-2-negative B. bronchiseptica isolates points towards the involvement of a ß-lactamase in the high MICs of ampicillin. Moreover, hydrolysis of nitrocefin by crude protein extracts proved that all isolates produced an active ß-lactamase (data not shown). Since BOR-1 has been shown to be sensitive to inhibition by clavulanic acid13 and as no other ß-lactamases were detected in the isolates, slightly different expression levels of blaBOR-1 might lead to differences in the ampicillin susceptibility.
In accordance with the high MICs previously determined for the two cephalosporins cefalotin and ceftiofur,7 B. bronchiseptica isolates with different ampicillin MICs exhibited the same high MICs of the cephalosporins tested. No significant reduction of resistance was seen in the presence of the ß-lactamase inhibitor clavulanic acid. For E. coli expressing the cloned blaBOR-1 gene, Lartigue et al. observed no differences in MICs compared with the parental strain for cefalotin, cefoxitin, cefotaxime, cefuroxime, ceftazidime, cefepime and cefpirome. In addition, we noticed that the MIC of ceftiofur was also not increased (data not shown). As B. bronchiseptica only produces the narrow-spectrum enzyme BOR-1, other mechanisms have to be proposed to play a role in the low susceptibility to cephalosporins.
One possibility for low susceptibility to cephalosporins is an efflux mechanism. Efflux mechanisms have been shown to contribute to ß-lactam resistance in Gram-negative bacteria,41 but appear to have no impact on the MICs for the tested B. bronchiseptica isolates.
Another option is reduced membrane permeability, which has been described as a possible factor contributing to reduced susceptibility against ß-lactams42 and has also been suggested by Lartigue et al. for B. bronchiseptica.13 In order to investigate the permeability of cephalosporins into intact cells, the broad-spectrum AmpC enzyme CMY-2 was chosen. Cefoxitin hydrolysis by intact B. bronchiseptica cells expressing CMY-2 was not observed during the time period of the experiment, indicating a very low permeability for this antibiotic. Similar results were observed for cefalotin. As low to negligible permeability for two representative cephalosporins was demonstrated, we postulate that reduced outer membrane permeability plays a relevant role in cephalosporin resistance of B. bronchiseptica.
In conclusion, the detected and sequenced ß-lactamase gene blaOXA-2 conferred ampicillin resistance in porcine B. bronchiseptica isolates. In contrast, low susceptibility of porcine B. bronchiseptica isolates to cephalosporins was not based on the production of a ß-lactamase, but seems to be due to low membrane permeability of this pathogen.
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None to declare.
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Acknowledgements |
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We thank Patrice Nordmann and Laurent Poirel for the blaBOR-1 positive control and Kimberley J. Sherwood for providing E. coli isolate no. 56. We thank Inge Luhmer-Becker for excellent technical assistance and Noha Khalaf for helpful discussions. Kristina Kadlec is supported by a scholarship of the H. Wilhelm Schaumann foundation.
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1 Goodnow RA. (1980) Biology of Bordetella bronchiseptica. Microbiol Rev 44:722–38.
2 Magyar T and Lax AJ. (2002) Atrophic rhinitis. In Brogden KA and Guthmiller JM (Eds.). Polymicrobial Diseases(ASM Press, Washington, DC) pp. 169–97.
3
Woolfrey BF and Moody JA. (1991) Human infections associated with Bordetella bronchiseptica. Clin Microbiol Rev 4:243–55.
4
Burton PJ, Thornsberry C, Cheung Yee Y, et al. (1996) Interpretive criteria for antimicrobial susceptibility testing of ceftiofur against bacteria associated with swine respiratory disease. J Vet Diagn Invest 8:464–8.
5
Mortensen JE, Brumbach A, Shryock TR. (1989) Antimicrobial susceptibility of Bordetella avium and Bordetella bronchiseptica isolates. Antimicrob Agents Chemother 33:771–2.
6 Appelbaum PC, Tamim J, Pankuch GA, et al. (1983) Susceptibility of 324 nonfermentative gram-negative rods to 6 cephalosporins and azthreonam. Chemotherapy 29:337–44.[Web of Science][Medline]
7
Kadlec K, Kehrenberg C, Wallmann J, et al. (2004) Antimicrobial susceptibility of Bordetella bronchiseptica isolates from porcine respiratory tract infections. Antimicrob Agents Chemother 48:4903–6.
8
Terakado N, Azechi H, Ninomiya K, et al. (1973) Demonstration of R factors in Bordetella bronchiseptica isolated from pigs. Antimicrob Agents Chemother 3:555–8.
9 Graham AC and Abruzzo GK. (1982) Occurrence and characterization of plasmids in field isolates of Bordetella bronchiseptica. Am J Vet Res 43:1852–5.[Web of Science][Medline]
10
Hedges RW, Jacob AE, Smith JT. (1974) Properties of an R factor from Bordetella bronchiseptica. J Gen Microbiol 84:199–204.
11
Yaginuma S, Terakado N, Mitsuhashi S. (1975) Biochemical properties of a penicillin ß-lactamase mediated by R factor from Bordetella bronchiseptica. Antimicrob Agents Chemother 8:238–42.
12
Speakman AJ, Binns SH, Osborn AM, et al. (1997) Characterization of antibiotic resistance plasmids from Bordetella bronchiseptica. J Antimicrob Chemother 40:811–6.
13
Lartigue MF, Poirel L, Fortineau N, et al. (2005) Chromosome-borne class A BOR-1 ß-lactamase of Bordetella bronchiseptica and Bordetella parapertussis. Antimicrob Agents Chemother 49:2565–7.
14 National Committee for Clinical Laboratory Standards. (2002) Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals—Second Edition: Approved Standard M31-A2(NCCLS, Wayne, PA, USA).
15 Schiefer AM, Wiegand I, Sherwood KJ, et al. (2005) Biochemical and genetic characterization of the ß-lactamases of Y. aldovae, Y. bercovieri, Y. frederiksenii and ‘Y. ruckeri’ strains. Int J Antimicrob Agents 25:496–500.[CrossRef][Web of Science][Medline]
16
Kadlec K, Kehrenberg C, Schwarz S. (2005) Molecular basis of resistance to trimethoprim, chloramphenicol and sulphonamides in Bordetella bronchiseptica. J Antimicrob Chemother 56:485–90.
17
Pasquali F, Kehrenberg C, Manfreda G, et al. (2005) Physical linkage of Tn3 and part of Tn1721 in a tetracycline and ampicillin resistance plasmid from Salmonella Typhimurium. J Antimicrob Chemother 55:562–5.
18
Ng LK, Mulvey MR, Martin I, et al. (1999) Genetic characterization of antimicrobial resistance in Canadian isolates of Salmonella serovar Typhimurium DT104. Antimicrob Agents Chemother 43:3018–21.
19
Brinas L, Moreno MA, Zarazaga M, et al. (2003) Detection of CMY-2, CTX-M-14, and SHV-12 ß-lactamases in Escherichia coli fecal-sample isolates from healthy chickens. Antimicrob Agents Chemother 47:2056–8.
20
Pitout JD, Thomson KS, Hanson ND, et al. (1998) ß-Lactamases responsible for resistance to expanded-spectrum cephalosporins in Klebsiella pneumoniae, Escherichia coli, and Proteus mirabilis isolates recovered in South Africa. Antimicrob Agents Chemother 42:1350–4.
21
Juteau JM, Sirois M, Medeiros AA, et al. (1991) Molecular distribution of ROB-1 ß-lactamase in Actinobacillus pleuropneumoniae. Antimicrob Agents Chemother 35:1397–402.
22
Stock I, Burak S, Sherwood KJ, et al. (2003) Natural antimicrobial susceptibilities of strains of ‘unusual’ Serratia species: S. ficaria, S. fonticola, S. odorifera, S. plymuthica and S. rubidaea. J Antimicrob Chemother 51:865–85.
23 Li XZ and Nikaido H. (2004) Efflux-mediated drug resistance in bacteria. Drugs 64:159–204.[CrossRef][Web of Science][Medline]
24 Miro E, Verges C, Garcia I, et al. (2004) Resistance to quinolones and ß-lactams in Salmonella enterica due to mutations in topoisomerase-encoding genes, altered cell permeability and expression of an active efflux system. Enferm Infecc Microbiol Clin 22:204–11.[CrossRef][Web of Science][Medline]
25
Preisler A and Mraheil MA Heisig P. (2006) Role of novel gyrA mutations in the suppression of the fluoroquinolone resistance genotype of vaccine strain Salmonella Typhimurium vacT (gyrA D87G). J Antimicrob Chemother 57:430–6.
26
Zimmermann W and Rosselet A. (1977) Function of the outer membrane of Escherichia coli as a permeability barrier to ß-lactam antibiotics. Antimicrob Agents Chemother 12:368–72.
27
Bauvois C, Ibuka AS, Celso A, et al. (2005) Kinetic properties of four plasmid-mediated AmpC ß-lactamases. Antimicrob Agents Chemother 49:4240–6.
28 Kehrenberg C and Schwarz S. (2005) Molecular analysis of tetracycline resistance in Pasteurella aerogenes. Antimicrob Agents Chemother 45:2885–90.
29
Bellido F, Pechere JC, Hancock RE. (1991) Novel method for measurement of outer membrane permeability to new ß-lactams in intact Enterobacter cloacae cells. Antimicrob Agents Chemother 35:68–72.
30
Bellido F, Pechere JC, Hancock RE. (1991) Reevaluation of the factors involved in the efficacy of new ß-lactams against Enterobacter cloacae. Antimicrob Agents Chemother 35:73–8.
31 Anderson ES and Datta N. (1965) Resistance to penicillins and its transfer in Enterobacteriaceae. Lancet 191:407–9.[CrossRef][Medline]
32 Dale JW, Godwin D, Mossakowska D, et al. (1985) Sequence of the OXA2 ß-lactamase: comparison with other penicillin-reactive enzymes. FEBS Lett 191:39–44.[CrossRef][Web of Science][Medline]
33
Hall RM and Vockler C. (1987) The region of the IncN plasmid R46 coding for resistance to ß-lactam antibiotics, streptomycin/spectinomycin and sulphonamides is closely related to antibiotic resistance segments found in IncW plasmids and in Tn21-like transposons. Nucleic Acids Res 15:7491–501.
34 Szcepanowski R, Krahn I, Puhler A, et al. (2004) Different molecular rearrangements in the integron of the IncP-1ß resistance plasmid pB10 isolated from a wastewater treatment plant result in elevated ß-lactam resistance levels. Arch Microbiol 182:429–35.[CrossRef][Web of Science][Medline]
35
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 58:225–6.
36 Ledent P and Frere JM. (1993) Substrate-induced inactivation of the OXA2 ß-lactamase. Biochem J 295:871–8.
37 Naas T and Nordmann P. (1999) OXA-type ß-lactamases. Curr Pharm Des 5:865–79.[Web of Science][Medline]
38 Bush K, Jacoby GA, Medeiros AA. (1995) A functional classification scheme for ß-lactamases and its correlation with molecular structure. Antimicrob Agents Chemother 39:1211–33.[Web of Science][Medline]
39 Paetzel M, Danel F, de Castro L, et al. (2000) Crystal structure of the class D ß-lactamase OXA-10. Nat Struct Biol 7:918–25.[CrossRef][Web of Science][Medline]
40 Danel F, Frere JM, Livermore DM. (2001) Evidence of dimerisation among class D ß-lactamases: kinetics of OXA-14 ß-lactamase. Biochim Biophys Acta 1546:132–42.[CrossRef][Medline]
41 Poole K. (2004) Resistance to ß-lactam antibiotics. Cell Mol Life Sci 61:2200–23.[Web of Science][Medline]
42 Nikaido H. (1985) Role of permeability barriers in resistance to ß-lactam antibiotics. Pharmacol Ther 27:197–231.[CrossRef][Web of Science][Medline]
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