1 Institut für Tierzucht der Bundesforschungsanstalt für Landwirtschaft (FAL), Höltystrasse 10, 31535 Neustadt-Mariensee, Germany; 2 US Biologicals Clinical Affairs, Pfizer Animal Health, Kalamazoo, MI 49001; 3 US Biologicals Development, Pfizer Animal Health, Kalamazoo, MI 49001, USA
Received 15 July 2003; returned 1 October 2003; revised 3 November 2003; accepted 6 November 2003
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Abstract |
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Methods: MIC determination for all 302 strains was performed by the broth dilution method and evaluated according to NCCLS standards. MIC50 and MIC90 values were calculated. Strains resistant to spectinomycin were subjected to PCR assays for genes known to mediate spectinomycin resistance in Gram-negative and Gram-positive bacteria.
Results: With the exception of resistance to sulfamethoxazole in P. multocida and M. haemolytica, and resistance to ampicillin in M. haemolytica, an overall low level of resistance was detected. A total of 93.5% of the P. multocida and 98.6% of the M. haemolytica strains were susceptible to spectinomycin, with MIC90s of 32 mg/L. PCR analysis showed that none of the spectinomycin-resistant strains carried any of the aadA gene subtypes, nor the genes spc or aad(9).
Conclusions: Prior to the renewal of spectinomycin, only a small number of spectinomycin-resistant strains was detected among bovine P. multocida and M. haemolytica. The genes responsible for spectinomycin resistance in these strains seemed to be different from those so far known to occur in other Gram-negative and Gram-positive bacteria.
Keywords: antibiotic resistance, aminocyclitol antibiotics, MIC determination, PCR analysis
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Introduction |
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Therefore, the aim of the present study was to generate pre-renewal data for spectinomycin from strains collected all over Germany, using the NCCLS guideline M31-A2,2 and to compare the MICs of spectinomycin with those of other antimicrobials. Moreover, spectinomycin-resistant strains were investigated by PCR for the presence of genes commonly associated with spectinomycin resistance in Gram-negative and Gram-positive bacteria.
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Materials and methods |
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During September to December 1999, 154 P. multocida and 148 M. haemolytica strains were collected on the basis of one strain per herd from various diagnostic laboratories all over Germany. All strains were from nasopharyngeal swabs or lung tissues of animals suffering or dead from BRD. Biochemical confirmation of the species assignment was performed as described previously.4 The strains were cultivated on sheep blood agar plates (blood agar base; Oxoid, Wesel, Germany; supplemented with 5% sheep blood). Resistance patterns were determined by the broth microdilution method using microtitre plates (Sensititre, West Lake OH, USA) that contained the following antimicrobials, in serial two-fold dilutions: ampicillin (0.0332 mg/L), cefquinome (0.03 32 mg/L), ceftiofur (0.0332 mg/L), gentamicin (0.0332 mg/L), neomycin (0.12128 mg/L), spectinomycin (0.5512 mg/L), sulfamethoxazole (0.5512 mg/L) and trimethoprim/sulfamethoxazole (0.016/0.316/304 mg/L). In addition, all strains were investigated for florfenicol susceptibility (0.124 mg/L), and the spectinomycin-resistant strains were checked for streptomycin resistance (32128 mg/L) by the broth macrodilution method. Susceptibility testing was performed and evaluated according to NCCLS criteria.2,3 MIC50 and MIC90 data were calculated as the lowest concentrations of the antimicrobials that inhibited growth of 50% or 90% of the strains, respectively.
DNA preparation and PCR analysis
Plasmid DNA and whole-cell DNA of the P. multocida and M. haemolytica strains was prepared as described previously.4 For PCR analysis the following primers were used: forward, 5'-GTGGATGGCGGCCTG AAGCC-3' and reverse, 5'-ATTGCCCAGTCGGCAGCG-3', which perfectly matched the sequences of aadA1a (GenBank accession no. X02340), aadA1b (M95287), aadA2 (X68227), aadA2b (D43625) and aadA3 (AF047479). Three to four mismatches in the forward primer and two to five mismatches in the reverse primer were detected when comparing these primer sequences with the sequences of aadA5 (AF137361), aadA6 (AF140629) and aadA7 (AF224733). Therefore, PCR assays with different annealing temperatures were conducted: annealing at 56°C for specific binding and also at 45°C to allow binding to the closely related aadA5aadA7 sequences. The amplification followed standard procedures, with an initial denaturation step for 3 min at 94°C, followed by 35 cycles of 1 min at 94°C, 1 min at the respective annealing temperature and 30 s at 72°C for primer extension. After a final step for 7 min at 72°C, the reaction was cooled at 4°C, and 10 µL aliquots were subjected to gel electrophoresis in 1.5% (w/v) agarose gels.
PCRs were also conducted to check for the presence of the spectinomycin resistance genes spc from Staphylococcus aureus (X02588) (annealing temperature 58°C; forward, 5'-ACGCATTAACAGCGATGTAGATG-3' and reverse, 5'-AGTCCTTCCCACTTATCATCA CAC-3'), and aad(9) from Enterococcus faecalis (M69221) (annealing temperature 56°C; forward, 5'-TGGATCAGGAGTTGAGAGTGG-3' and reverse, 5'-GAGAAGATTCAGCCACTGCATT-3'). Plasmids carrying the genes spc and aad(9), as well as various aadA subtypes, served as positive controls. The 12 spectinomycin-resistant strains were also investigated by PCR for the presence of the streptomycin resistance gene strA, previously described to be widespread among Pasteurella and Mannheimia strains.4
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Results and discussion |
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Receiving and maintaining approval for a specific antimicrobial agent in a country such as Germany requires that strains of the target bacteria collected in the respective country are studied for their susceptibility patterns. For this, the bacteria should be obtained from various locations to exclude bias arising from multiple inclusion of certain clones prevalent in a specific geographical area, or from higher resistance rates due to local preferences in the use of certain antimicrobial agents. To date, only few studies dealing with susceptibility of bovine P. multocida and M. haemolytica strains to various antimicrobial agents have been conducted in Germany.1,810 Unfortunately, spectinomycin was included in only one of these studies.1 Striking differences in the percentage of spectinomycin-resistant strains were observed between the present study and that of Klarmann in 1997.1 The use of different breakpoints for resistance (32 mg/L in the Klarmann study and the NCCLS approved breakpoint of
128 mg/L in this study) might account for the different percentages of resistant strains in the two studies.
In summary, only the use of a standardized methodology in combination with veterinary-specific breakpoints as recommended in the NCCLS document M31-A2 allow a reliable assessment of the number of spectinomycin-resistant strains among P. multocida and M. haemolytica strains from BRD cases in Germany. The small number of resistant strains indicates that there is a low basal level of resistance to spectinomycin among bovine P. multocida and M. haemolytica strains collected in the pre-renewal period.
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Acknowledgements |
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Footnotes |
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References |
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2 . National Committee for Clinical Laboratory Standards. (2002). Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from AnimalsSecond Edition: Approved Standard M31-A2. NCCLS, Wayne, PA, USA.
3 . National Committee for Clinical Laboratory Standards. (1999). Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals: Approved Standard M31-A. NCCLS, Wayne, PA, USA.
4 . Kehrenberg, C. & Schwarz S. (2001). Occurrence and linkage of genes coding for resistance to sulfonamides, streptomycin and chloramphenicol in bacteria of the genera Pasteurella and Mannheimia. FEMS Microbiology Letters 205, 28390.[CrossRef][ISI][Medline]
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7 . Sandvang, D. (2001). Aminoglycoside resistance genes and their mobility in gramnegative bacteria from production animals. PhD thesis. The Royal Veterinary and Agricultural University, Copenhagen, Denmark.
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