This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Vicca, J.
Right arrow Articles by Haesebrouck, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Vicca, J.
Right arrow Articles by Haesebrouck, F.

 Previous Article  |  Next Article 

Antimicrobial Agents and Chemotherapy, November 2004, p. 4470-4472, Vol. 48, No. 11
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.11.4470-4472.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

In Vitro Susceptibilities of Mycoplasma hyopneumoniae Field Isolates

J. Vicca,1,2* T. Stakenborg,3 D. Maes,1 P. Butaye,3 J. Peeters,3 A. de Kruif,1 and F. Haesebrouck2

Department of Reproduction, Obstetrics and Herd Health,1 Department of Bacteriology, Pathology and Poultry Diseases, Faculty of Veterinary Medicine, Ghent University, Merelbeke,2 CODA-CERVA, Veterinary and Agrochemical Research Centre, Brussels, Belgium3

Received 31 March 2004/ Returned for modification 27 May 2004/ Accepted 27 July 2004


arrow
ABSTRACT
 
The in vitro susceptibilities of 21 Mycoplasma hyopneumoniae field isolates were determined using a broth microdilution technique. One isolate showed acquired resistance to lincomycin, tilmicosin, and tylosin, while five isolates were resistant to flumequine and enrofloxacin. Acquired resistance against these antimicrobials in M. hyopneumoniae field isolates was not reported previously.


arrow
TEXT
 
Mycoplasma hyopneumoniae causes enzootic pneumonia, a chronic respiratory disease in pigs resulting in considerable economic losses. Although appropriate vaccines are available to reduce the consequences of infection, medication with antimicrobials in feed or water is still a common practice. The use of antimicrobials, however, results in the selection of resistant bacteria. The antimicrobial susceptibility of M. hyopneumoniae field isolates has rarely been determined, and limited numbers of strains have been considered in these studies (5, 7, 8, 14, 16), mainly due to the fact that M. hyopneumoniae is a fastidious and slowly growing microorganism, making it difficult to obtain large numbers of field strains. In this study, the antimicrobial susceptibility of recently isolated M. hyopneumoniae field strains was determined, and the antibiotic use on the originating herds was monitored.

For MIC determination, 21 M. hyopneumoniae field strains, isolated between 2000 and 2002 from 21 different farrow-to-finish pig herds in Belgium, were used (18, 19). The isolation and cultivation of M. hyopneumoniae were optimized by using earlier reports (12). The antibiotics used on the herds at the three main production stages during the year before M. hyopneumoniae isolation are mentioned in Table 1.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Antibiotics used on the herd during 1 year before isolation of M. hyopneumoniae

The MIC determination was performed according to guidelines written by Hannan (6). Briefly, 96-well, round-bottom microtiter plates (Sensititre Ltd., East Grinstead, England) containing stabilized, freeze-dried antimicrobials (Table 2) were used. Three wells on each plate were left antimicrobial free as a positive growth control. Freshly thawed M. hyopneumoniae isolates with known titers were diluted in nonselective Friis medium until the number of organisms reached 104 color-changing units/ml. Fifty microliters of the diluted culture was transferred into each well of the Sensititre plates. The M. hyopneumoniae type strain, ATCC 25634 (J strain), was used as the control strain and tested three times in order to estimate the reproducibility of the procedure. The plates were sealed using an adhesive foil and incubated at 36 ± 1°C for 14 days and observed daily. Growth of M. hyopneumoniae organisms was observed when the color of the medium changed from red to yellow (phenol red indicator). The initial and final MICs were recorded. The initial MIC was defined as the lowest antibiotic concentration to show no change in color when the growth control turned yellow, and the final MIC was defined as the lowest antibiotic concentration to show no change in color at 14 days after inoculation (13).


View this table:
[in this window]
[in a new window]
  		TABLE 2. Initial and final MIC50s, MIC90s, and MIC ranges of antimicrobials against Belgian M. hyopneumoniae field isolates

In Table 2, the initial and final MICs at which 50 and 90% of the isolates tested were inhibited (MIC50 and MIC90, respectively) and the MIC ranges are presented for the 21 M. hyopneumoniae field strains and the three replicates of the J strain. The values for these replicates were equal or differed from each other by only one doubling dilution, indicating good reproducibility of the test. The initial MICs for the J strain were in agreement with values reported previously (7, 8, 14, 16).

A bimodal frequency distribution of MICs of the macrolides tylosin and tilmicosin as well as for the lincosamide antibiotic lincomycin was seen. The MICs of these antibiotics were clearly higher for one isolate, indicating acquired resistance. The MIC of tylosin for this isolate was also higher than the suggested breakpoint (7). Macrolides and lincosamides are chemically distinct but have similar modes of action and overlapping binding sites on the 23S rRNA of the 50S subunit of the bacterial ribosome. They act by blocking protein synthesis on assembled and functioning 50S ribosomal subunits (20). Acquired resistance against these antibiotics has not been described before for M. hyopneumoniae and has not been reported often for other mycoplasmas, most probably due to a limited number of strains having been tested. Only two M. pneumoniae strains, one Ureaplasma urealyticum strain, and two resistant Mycoplasma hominis isolates were isolated from humans (2). In animal mycoplasmas, acquired resistance to tylosin has been described for Mycoplasma gallisepticum (11), Mycoplasma hyosynoviae (1, 10), Mycoplasma hyorhinis (9, 10), and Mycoplasma bovis (17). No coresistance to lincomycin was observed for the tylosin-resistant M. hyosynoviae isolates. Lincomycin was not evaluated by Levisohn (11), and although tylosin- and lincomycin-resistant M. bovis strains were found by Thomas et al. (17), the existence of coresistance was not reported. ter Laak et al. (15) found lincomycin-resistant M. bovis isolates, but those were susceptible to tylosin. The use of lincomycin in growth-finishing pigs from the herd where our macrolide-lincosamide-resistant M. hyopneumoniae strain was isolated may have contributed to the selection of antibiotic resistance.

For five isolates, the MIC of flumequine was >16 µg/ml, which is higher than the previously proposed breakpoint of ≥16 µg/ml (7). For these isolates, the MIC of enrofloxacin was ≥0.5 µg/ml, while the MIC50 was 0.06 µg/ml. This rather high frequency of acquired resistance against fluoroquinolones is unusual. Although fluoroquinolone-resistant M. hominis isolates have been reported (3), resistance against these antibiotics in human-associated mycoplasmas has rarely been reported. Hannan et al. (7) described acquired resistance against flumequine in avian, porcine (but not M. hyopneumoniae), bovine, ovine, and caprine mycoplasmas, and Thomas et al. (17) isolated enrofloxacin-resistant strains from bovines. A possible explanation for the high prevalence of fluoroquinolone resistance in the present study might be the frequent use of enrofloxacin to treat Escherichia coli diarrhea in suckling and nursery piglets. The MIC of enrofloxacin for M. hyopneumoniae isolate 19 was >1 µg/ml. In the originating herd, fluoroquinolones were used in suckling, nursery, and growth-finishing pigs.

The MICs of oxytetracycline and doxycycline did not show a clear bimodal frequency distribution range (4), and the MICs of oxytetracycline were lower than the suggested breakpoint (7), indicating no acquired resistance against these antibiotics; nevertheless, in 62% of the herds selected for this study tetracycline antibiotics were used to treat nursery and growth-finishing pigs.

In conclusion, this study is the first description of acquired resistance in M. hyopneumoniae field isolates to macrolides, lincosamides, and fluoroquinolones. Resistance against other antimicrobials was not detected, confirming that antimicrobial resistance does not yet pose a major problem for the treatment of M. hyopneumoniae infections (7, 8, 16). However, the rather high frequency of fluoroquinolone resistance is worrying and warrants prudent use of these antibiotics.


arrow
ACKNOWLEDGMENTS
 
This work was supported by the Federal Public Service of Public Health, Food Chain Security and Environment, Brussels, Belgium, grant no. S-6039.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium. Phone: 32 (0)9 264 75 49. Fax: 32 (0)9 264 77 98. E-mail: Jo.Vicca{at}UGent.be. Back


arrow
REFERENCES
 
    1
  1. Aarestrup, F. M., and N. F. Friis. 1998. Antimicrobial susceptibility testing of Mycoplasma hyosynoviae isolated from pigs during 1968-1971 and during 1995 and 1996. Vet. Microbiol. 61:33-39.[CrossRef][Medline]
    2. 2
  2. Bébéar, C., and C. Bébéar. 2002. Antimycoplasmal agents, p. 545-566. In S. Razin and R. Herrmann (ed.), Molecular biology and pathogenicity of mycoplasmas. Kluwer Academic/Plenum Publishers, New York, N.Y.
    3. 3
  3. Bebear, C. M., J. Renaudin, A. Charron, H. Renaudin, B. De Barbeyrac, T. Schaeverbeke, and C. Bebear. 1999. Mutations in the gyrA, parC, and parE genes associated with fluoroquinolone resistance in clinical isolates of Mycoplasma hominis. Antimicrob. Agents Chemother. 43:954-956.[Abstract/Free Full Text]
    4. 4
  4. Butaye, P., L. A. Devriese, and F. Haesebrouck. 2003. Antimicrobial growth promoters used in animal feed: effects of less well known antibiotics on gram-positive bacteria. Clin. Microbiol. Rev. 16:175-188.[Abstract/Free Full Text]
    5. 5
  5. Friis, N. F., and J. Szancer. 1994. Sensitivity of certain porcine and bovine mycoplasmas to antimicrobial agents in a liquid medium test compared to a disc assay. Acta Vet. Scand. 35:389-394.[Medline]
    6. 6
  6. Hannan, P. C. T. 2000. Guidelines and recommendations for antimicrobial minimum inhibitory concentration (MIC) testing against veterinary mycoplasma species. Vet. Res. 31:373-395.[CrossRef][Medline]
    7. 7
  7. Hannan, P. C. T., H. M. Windsor, A. de Jong, N. Schmeer, and M. Stegemann. 1997. Comparative susceptibilities of various animal-pathogenic mycoplasmas to fluoroquinolones. Antmicrob. Agents Chemother. 41:2037-2040.[Abstract]
    8. 8
  8. Hannan, P. C. T., H. M. Windsor, and P. H. Ripley. 1997. In vitro susceptibilities of recent field isolates of Mycoplasma hyopneumoniae and Mycoplasma hyosynoviae to valnemulin (Econor), tiamulin and enrofloxacin and the in vitro development of resistance to certain antimicrobial agents in Mycoplasma hyopneumoniae. Res. Vet. Sci. 36:157-160.
    9. 9
  9. Kobayashi, H., T. Morozumi, G. Munthali, K. Mitani, N. Ito, and D. Yamamoto. 1996. Macrolide susceptibility of Mycoplasma hyorhinis isolated from pigs. Antimicrob. Agents Chemother. 40:1030-1032.[Abstract]
    10. 10
  10. Kobayashi, H., N. Sonmez, T. Morozumi, K. Mitani, N. Ito, H. Shiono, and K. Yamamoto. 1996. In vitro susceptibility of Mycoplasma hyosynoviae and M. hyorhinis to antimicrobial agents. J. Vet. Med. Sci. 58:1107-1111.[Medline]
    11. 11
  11. Levisohn, S. 1981. Antibiotic sensitivity patterns in field isolates of Mycoplasma gallisepticum as a guide to chemotherapy. Isr. J. Med. Sci. 17:661-666.[Medline]
    12. 12
  12. Stakenborg, T., J. Vicca, P. Butaye, H. Imberechts, J. Peeters, H. de Kruif, F. Haesebrouck, and D. Maes. A multiplex PCR to identify poraine mycoplasmas present in broth cultures. Vet. Res. Commun., in press.
    13. 13
  13. Tanner, A. C., and C. C. Wu. 1992. Adaptation of the Sensititre broth microdilution technique to antimicrobial susceptibility testing of Mycoplasma gallisepticum. Avian Dis. 36:714-717.[CrossRef][Medline]
    14. 14
  14. Tanner, A. C., B. Z. Erickson, and R. F. Ross. 1993. Adaptation of the Sensititre broth microdilution technique to antimicrobial susceptibility testing of Mycoplasma hyopneumoniae. Vet. Microbiol. 36:301-306.[CrossRef][Medline]
    15. 15
  15. ter Laak, E. A., J. H. Noordergraaf, and M. H. Verschure. 1993. Susceptibilities of Mycoplasma bovis, Mycoplasma dispar, and Ureaplasma diversum strains to antimicrobial agents in vitro. Antimicrob. Agents Chemother. 37:317-321.[Abstract/Free Full Text]
    16. 16
  16. ter Laak, E. A., A. Pijpers, J. H. Noordergraaf, E. C. Schroevers, and J. H. M. Verheijden. 1991. Comparison of methods for in vitro testing of susceptibility of porcine Mycoplasma species to antimicrobial agents. Antimicrob. Agents Chemother. 35:228-233.[Abstract/Free Full Text]
    17. 17
  17. Thomas, A., C. Nicolas, I. Dizier, J. Mainil, and A. Linden. 2003. Antibiotic susceptibilities of recent Belgian Mycoplasma bovis isolates. Vet. Rec. 153:428-431.[Abstract/Free Full Text]
    18. 18
  18. Vicca, J., D. Maes, L. Thermote, J. Peeters, F. Haesebrouck, and A. de Kruif. 2002. Patterns of Mycoplasma hyopneumoniae infections in Belgian farrow-to-finish pig herds with diverging disease-course. J. Vet. Med. B 49:349-353.[CrossRef]
    19. 19
  19. Vicca, J., T. Stakenborg, D. Maes, P. Butaye, J. Peeters, A. de Kruif, and F. Haesebrouck. 2003. Evaluation of virulence of Mycoplasma hyopneumoniae field isolates. Vet. Microbiol. 97:177-190.[CrossRef][Medline]
    20. 20
  20. Weisblum, B. 1995. Insights into erythromycin action from studies of its activity as inducer of resistance. Antmicrob. Agents Chemother. 39:797-805.[Medline]

Antimicrobial Agents and Chemotherapy, November 2004, p. 4470-4472, Vol. 48, No. 11
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.11.4470-4472.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Antunes, N. T., Tavio, M. M., Mercier, P., Ayling, R. D., Al-Momani, W., Assuncao, P., Rosales, R. S., Poveda, J. B. (2007). In Vitro Susceptibilities of Mycoplasma putrefaciens Field Isolates. Antimicrob. Agents Chemother. 51: 3452-3454 [Abstract] [Full Text]  
  • Assuncao, P., Antunes, N. T., Rosales, R. S., Poveda, C., Poveda, J. B., Davey, H. M. (2006). Flow Cytometric Determination of the Effects of Antibacterial Agents on Mycoplasma agalactiae, Mycoplasma putrefaciens, Mycoplasma capricolum subsp. capricolum, and Mycoplasma mycoides subsp. mycoides Large Colony Type.. Antimicrob. Agents Chemother. 50: 2845-2849 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Vicca, J.
Right arrow Articles by Haesebrouck, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Vicca, J.
Right arrow Articles by Haesebrouck, F.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»