In vitro activities of spectinomycin and comparator agents against Pasteurella multocida and Mannheimia haemolytica from respiratory tract infections of cattle

Stefan Schwarz1,*, Corinna Kehrenberg1, Sarah A. Salmon2 and Jeffrey L. Watts3

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


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Objectives: Prior to the renewal of spectinomycin licensing for veterinary uses in Germany, 154 Pasteurella multocida and 148 Mannheimia haemolytica strains from respiratory tract infections in cattle were investigated for their MICs of spectinomycin and other antimicrobial agents. The data obtained should serve as a baseline from which to judge the future development of resistance. Moreover, the in vitro activity of spectinomycin in comparison with other antimicrobials should be assessed.

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


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The aminocyclitol antimicrobial agent spectinomycin has been available in Germany for the control of bacterial infections in a wide variety of animals (including cattle), either alone or in combination with lincomycin. Spectinomycin was recently renewed in Germany, according to the Federal Drug Law, and approved in the USA for the treatment of bovine respiratory disease (BRD) caused by Pasteurella multocida, Mannheimia haemolytica and Haemophilus somnus. In contrast to P. multocida and M. haemolytica, H. somnus has been detected rarely in bovine respiratory tract infections in Germany. Continuous monitoring of the main target bacteria for resistance to spectinomycin represents a prerequisite for a reliable detection of emergence of resistance. However, to validate resistance data obtained after approval or renewal of a specific antimicrobial agent, a sufficiently high number of unrelated strains of the target bacteria sampled prior to approval/renewal has to be tested. Such data represent a baseline for the evaluation of the development of resistance in the period following approval/renewal. The only pre-renewal data on spectinomycin resistance of bovine P. multocida and M. haemolytica strains from Germany published so far showed high percentages of 77.7% resistant P. multocida and 70.2% resistant M. haemolytica strains.1 However, it was questionable whether these data could be regarded as representative for the situation among bovine P. multocida and M. haemolytica strains, since all strains were collected in a specific geographical area in Northern Germany, and the methodology of susceptibility testing and breakpoints used differed distinctly from those described in the NCCLS guidelines.2,3

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.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Bacterial strains and antimicrobial susceptibility testing

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 (0.03–32 mg/L), cefquinome (0.03– 32 mg/L), ceftiofur (0.03–32 mg/L), gentamicin (0.03–32 mg/L), neomycin (0.12–128 mg/L), spectinomycin (0.5–512 mg/L), sulfamethoxazole (0.5–512 mg/L) and trimethoprim/sulfamethoxazole (0.016/0.3–16/304 mg/L). In addition, all strains were investigated for florfenicol susceptibility (0.12–4 mg/L), and the spectinomycin-resistant strains were checked for streptomycin resistance (32–128 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


    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The MICs of the antimicrobials tested are listed in Table 1. All 154 P. multocida and 148 M. haemolytica strains were highly susceptible to cefquinome and ceftiofur, with MIC90s of <=0.03 mg/L. The vast majority of the P. multocida strains were also susceptible to ampicillin. In contrast, a bimodal distribution was seen among the MICs of ampicillin for the M. haemolytica strains, with 99 (66.9%) of the strains exhibiting MICs <= 1 mg/L and 48 (32.4%) showing MICs >= 4 mg/L. All strains were susceptible to florfenicol, with MIC50s and MIC90s similar to those described for bovine P. multocida and M. haemolytica collected in 2000/2001.5 Resistance to sulfamethoxazole was common, with 66.2% of P. multocida and 73.6% of M. haemolytica strains showing MICs >= 512 mg/L. In contrast, resistance to the combination trimethoprim/sulfamethoxazole was only seen in three P. multocida (1.9%) and 11 M. haemolytica (7.4%) strains. None of the P. multocida strains and only a single M. haemolytica strain exhibited gentamicin resistance. The MICs of neomycin for both species varied over a wide range, with most of the P. multocida and M. haemolytica strains showing MICs of 2–16 and 2–8 mg/L, respectively. Determination of the number of neomycin-resistant strains was not possible, because no NCCLS-approved breakpoints are currently available for neomycin. A bimodal distribution of the MICs for spectinomycin was recorded (Figure 1). Most P. multocida and M. haemolytica strains showed MICs of 8–32 mg/L, while 10 P. multocida (6.5%) and two M. haemolytica (1.4%) strains exhibited MICs > 128 mg/L. For the majority of the antimicrobials tested, the MIC50s and MIC90s were either identical or differed by not more than two serial two-fold dilution steps. Exceptions were the values for ampicillin and trimethoprim/sulfamethoxazole among M. haemolytica strains (Table 1).


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Table 1. MIC data for the bovine P. multocida and M. haemolytica strains studied
 


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Figure 1. Distribution of the MICs of spectinomycin for the P. multocida (grey bars) and M. haemolytica (open bars) strains investigated in this study.

 
Various genes involved in resistance to aminoglycosides and aminocyclitols have been described, with some inactivating enzymes mediating cross-resistance between selected aminoglycosides and aminocyclitols.6 One such enzyme, 3-N-aminoglycoside acetyltransferase type IV, was found to mediate resistance to the aminoglycoside gentamicin and the aminocyclitol apramycin,7 while several aminoglycoside adenyltransferases, encoded by aad genes, have been reported to mediate resistance to streptomycin and spectinomycin.6 Cross-resistance between spectinomycin and aminoglycosides other than streptomycin has yet to be described. Therefore, we tested the 12 spectinomycin-resistant strains for streptomycin resistance and the presence of aad genes, as well as the streptomycin resistance gene strA. Only five of the 12 strains were resistant to streptomycin, with MICs >= 128 mg/L, while the remaining seven strains exhibited MICs <= 32 mg/L. PCR analysis did not reveal the presence of any of the aadA gene subtypes among the spectinomycin-resistant strains. Either no amplicon or only amplicons that differed distinctly from the expected size were detected. However, four of the five streptomycin- and spectinomycin-resistant strains carried the gene strA. Further attempts to identify the gene responsible for spectinomycin resistance included PCR assays for the genes spc from the staphylococcal transposon Tn554 and aad(9) from E. faecalis. Again, no amplicons specific for these two genes were obtained. These observations suggest that the highly resistant P. multocida and M. haemolytica strains may carry spectinomycin resistance genes that have yet to be described.

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.


    Acknowledgements
 
The authors thank Vera Nöding, Vivian Hensel and Roswitha Becker for their expert technical assistance. Microtitre plates were kindly provided by Pfizer Animal Health, Kalamazoo, MI, USA.


    Footnotes
 
* Corresponding author. Tel: +49-5034-871-241; Fax: +49-5034-871-246; E-mail: stefan.schwarz{at}fal.de Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Klarmann, D. (1997). Antibiotic resistance of important bacteria of infectious disease in 1996 in the district of Weser-Ems in Lower Saxony. Deutsche Tierärztliche Wochenschrift 104, 325–35.[ISI][Medline]

2 . 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.

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, 283–90.[CrossRef][ISI][Medline]

5 . Priebe, S. & Schwarz, S. (2003). In vitro activities of florfenicol against bovine and porcine respiratory tract pathogens. Antimicrobial Agents and Chemotherapy 47, 2703–5.[Abstract/Free Full Text]

6 . Johnson, A. P., Burns, L., Woodford, N. et al. (1994). Gentamicin resistance in clinical isolates of Escherichia coli encoded by genes of veterinary origin. Journal of Medical Microbiology 40, 221–6.[Abstract]

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.

8 . Hörmansdorfer, S. & Bauer, J. (1996). Resistance pattern of bovine pasteurellae. Berliner und Münchener Tierärztliche Wochenschrift 109, 168–71.[ISI][Medline]

9 . Hörmansdorfer, S. & Bauer, J. (1998). Resistance of bovine and porcine pasteurellae against florfenicol and other antibiotics. Berliner und Münchener Tierärztliche Wochenschrift 111, 422–6.[ISI][Medline]

10 . Böttner, A., Schmid, P. & Humke, R. (1995). In vitro efficacy of cefquinome (INN) and other anti-infective drugs against bacterial isolates from Belgium, France, Germany, The Netherlands, and the United Kingdom. Journal of Veterinary Medicine Series B 42, 377–83.[ISI][Medline]

11 . National Committee for Clinical Laboratory Standards. (2000). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Fifth Edition: approved Standard M7-A5. NCCLS, Wayne, PA, USA.