Influence of different medium components on the in vitro activity of the growth-promoting antibiotic flavomycin against enterococci

Patrick Butaye*, Luc A. Devriese and Freddy Haesebrouck

Laboratory of Veterinary Bacteriology and Mycology, Department of Pathology, Bacteriology and Poultry Diseases, Faculty of Veterinary Medicine, University of Ghent, Salisburylaan 133, B-9820 Merelbeke, Belgium


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The growth-promoting antibiotic flavomycin (also called bambermycin, flavophospholipol and moenomycin) has a complex spectrum of activity against enterococci, with some species being naturally resistant and others susceptible. In this study, proteins added to Mueller–Hinton II medium had a strong deleterious effect on the activity of flavomycin, glucose had no effect and starch decreased the activity of flavomycin. The fatty substances Tween 80 and tributyrin increased the activity of flavomycin for several enterococcal species. Slight differences in the composition of the susceptibility test medium affected the MIC results obtained, indicating that strict standardization of the test medium is necessary.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Flavomycin is an antibiotic used solely as a growth-promoting antibiotic in food animals. It inhibits cell wall synthesis by impairing the transglycolase activities of penicillinbinding proteins as a competitive enzyme inhibitor1 and is nearly exclusively active against Gram-positive bacteria.2

Previous studies in our laboratory have shown an inhibitory effect of blood on the in vitro activity of flavomycin.3 Dutta & Devriese4 and Butaye et al.3,5 reported that Enterococcus faecium was naturally resistant to this antibiotic whereas Aarestrup et al.6 reported that 72–93% of Danish E. faecium strains showed resistance. The latter finding of strains with different susceptibilities in the same species implies that acquired resistance to this antibiotic exists. However, such a phenomenon has never been demonstrated convincingly with flavomycin.

It is not known which substances affect the action of flavomycin and how much, so we investigated the influence of different substances on the in vitro action of flavomycin.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
We studied well-characterized collection strains of E. faecium (LMG11423T, LMG16709), Enterococcus durans (LMG12691T, LMG12285, LMG12903, LMG10741), Enterococcus hirae (LMG6399T, LMG11492, LMG11424), Enterococcus faecalis (LMG7937T, CCM5613, ATCC29213), Enterococcus gallinarum (LMG13129T, LMG16204), Enterococcus casseliflavus (LMG10745T, CCM2478) and Enterococcus avium (LMG10744T, LMG11394); Staphylococcus aureus ATCC 29213 was used as a control strain.

Flavomycin (Hoechst, Frankfurt, Germany) was dissolved in sterile distilled water to make stock solutions containing 1000 mg/L and then further diluted as recommended by the NCCLS.7

In a first series of tests, Mueller–Hinton II agar (Becton Dickinson, Cockeysville, MD, USA) supplemented with Tween 80 (Merck, Darmstadt, Germany), glucose (Merck), starch (Merck), fresh egg yolk, sheep blood (E&O Laboratories, Bonnybridge, UK), washed sheep blood cells, horse blood or liver concentrate (Sigma, St Louis, MO, USA) was compared with unsupplemented Mueller–Hinton II agar (Table IGo). In a second series of tests, different concentrations of bovine albumin, fraction V, bovine haemoglobin and casein from bovine milk (all from Sigma) were added to Mueller–Hinton II agar (Table IIGo) and compared with unsupplemented Mueller–Hinton II agar. In a third series of tests, the casein hydrolysates HY-case M, N-Z amine B and N-Z amine E (all from Sigma) were added to Mueller–Hinton II agar and compared with the unsupplemented agar (Table IIGo). Finally, tributyrin medium (Oxoid, Basingstoke, UK) was compared with its basic medium, yeast extract agar (Oxoid) and the latter was compared with unsupplemented Mueller–Hinton II agar. Doubling concentrations between 0.06 and 256 mg/L were tested. Antibiotic plates were inoculated, incubated aerobically and read as described before.3


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Table I. Two-fold differences between test results using Mueller–Hinton II medium or yeast extract medium (only compared with tributyrin medium) and various supplements; the table shows results of tests with 18 strains, nine of which were susceptible to flavomycin and nine resistant
 

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Table II. Two-fold differences between test results using varying concentrations and molecular weights of proteins added to Mueller–Hinton II medium; the table shows results of tests with susceptible strains
 

    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
In unsupplemented Mueller–Hinton II medium, all strains of E. faecium, E. hirae, E. gallinarum and E. casseliflavus showed high flavomycin MICs (ranging from 128 to >256 mg/L), thus showing natural resistance to flavomycin. Much lower MICs, ranging from 0.12 to 1 mg/L, were found with strains of E. faecalis, E. durans and E. avium. This might explain some remarkable differences seen in the reported prevalence of resistance,3–6 since phenotypic identification errors may occur, especially with strains other than E. faecalis.8 The natural resistance of some enterococcal species towards this antibiotic may result from differences in penicillin-binding proteins (PBPs) between enterococcal species.9 However, the PBPs on which flavomycin acts in enterococci are still to be determined. MICs on yeast extract medium were generally slightly (one or two doubling dilutions) higher. The MIC of flavomycin for the ATCC 29213 S. aureus control strain was from <=0.06 to 0.12 mg/L on both media.

The different medium supplements had very diverse effects (Table IGo). Sheep blood increased MICs by up to 11 doubling dilutions (an MIC of 256 mg/L instead of 0.12 mg/L), whereas washed sheep blood cells or whole horse blood had much smaller effects. Only the MICs for the intrinsically resistant strains did not change. Addition of egg yolk gave a similar result to addition of sheep blood. Liver homogenate had a much smaller effect, with some strains (one E. gallinarum strain and one E. casseliflavus strain) even becoming more susceptible. Complex compositions of proteins, lipids and sugars had different effects on the MICs as shown by the addition of blood, egg yolk or liver homogenate. The presence of glucose in the medium did not influence the MICs, while susceptible strains became more resistant to flavomycin when 1% starch was added to the medium. The effects of the glycolipid Tween 80 differed according to the species tested. The MICs for the susceptible strains and the naturally resistant E. gallinarum and E. casseliflavus decreased, whereas those for the similarly resistant E. faecium and E. hirae remained unchanged. Tributyrin, a lipid, had no effect on susceptible E. durans and E. faecalis strains. E. avium strains showed an increased susceptibility and all the naturally resistant E. faecium, E. gallinarum and the E. casseliflavus strains, but only one E. hirae strain, became more susceptible. The diverse effects on flavomycin of the fatty substances tested are difficult to explain. It has previously been observed that growth conditions might affect the expression of PBPs. This has been shown to affect the penicillin susceptibility of enterococci.10 It is not clear whether this is also the case for flavomycin.

The influence of proteins added to the medium was investigated by comparing different concentrations and different molecular weights (Table IIGo). The effect on the MICs for the flavomycin-susceptible strains of adding a protein increased with increasing concentrations. Concentrations as low as 0.0007% adversely influenced the activity of flavomycin. The molecular weight of the protein molecules also had an effect: the higher the molecular weight of the protein, the higher the effect of a constant concentration of a protein. Among the high molecular weight proteins, haemoglobin had the greatest effect on MICs and casein the least. A similar increase in MIC associated with increasing molecular weight was seen with the low molecular weight proteins, the casein hydrolysates.

The antimicrobial action of flavomycin in the intestines, where it is supposed to act on the Gram-positive flora, is difficult to explain as it is inhibited by so many compounds. Some enterococcal species seem to be influenced differently from others. The complex composition of the intestine does not allow us to predict any effect of flavomycin. In vivo studies are necessary to determine the activity of flavomycin in the intestines. Clostridium perfringens has been found to be resistant to flavomycin in vitro,11 but had a suppressive effect on this bacterium in chicken gut in in vivo trials.12

The existence of acquired resistance to flavomycin has not been documented convincingly to date. Species of the E. faecium group (E. faecium, E. hirae and E. durans) and E. faecalis do not require special growth conditions, and so can be tested on unsupplemented Mueller–Hinton II medium. Variation resulting from small differences in composition of this medium should be avoided as far as possible. However, no satisfactory medium is available at present to test the flavomycin susceptibility of the more fastidious species.


    Acknowledgments
 
We wish to thank Mrs A. Van de Kerckhove for her skilled technical assistance. This work was supported by the Research Fund of the University of Ghent, Belgium, code BOZF97/N2/022.


    Notes
 
* Corresponding author. Tel: +32-9-264-74-35; Fax: +32-9-264-74-94; E-mail: pbutaye{at}allserv.rug.ac.be Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . van Heijenoort, Y., Leduc, M., Singer, H. & van Heijenoort, J. (1987). Effects of moenomycin on Escherichia coli. Journal of General Microbiology 133, 667–74.[ISI][Medline]

2 . von Wasielewski, E., Muschaweck, R. & Schütze E. (1965). Moenomycin, a new antibiotic. III. Biological properties. Antimicrobial Agents and Chemotherapy 5, 743–8.

3 . Butaye, P., Devriese, L. A. & Haesebrouck, F. (1998). Effects of different test conditions on MICs of food animal growth-promoting antibacterial agents for enterococci. Journal of Clinical Microbiology 36, 1907–11.[Abstract/Free Full Text]

4 . Dutta, G. N. & Devriese, L. A. (1982). Susceptibility of fecal streptococci of poultry origin to nine growth-promoting agents. Applied and Environmental Microbiology 44, 832–7.[ISI][Medline]

5 . Butaye, P., Van Damme, K., Devriese, L. A., Van Damme, L., Baele, M., Lauwers, S. et al. (2000). In vitro susceptibility of E. faecium isolated from food to growth-promoting and therapeutic antibiotics. International Journal of Food Microbiology 54, 181–7.[ISI][Medline]

6 . Aarestrup, F. M., Bager, F., Jensen, N. E., Madsen, M., Meyling, A. & Wegener, H. C. (1998). Surveillance of antimicrobial resistance in bacteria isolated from food animals to antimicrobial growth promoters and related therapeutic agents in Denmark. Acta Pathologica Microbiologica et Immunologica Scandinavica 106, 606–22.

7 . National Committee on Clinical Laboratory Standards. (1997). Scheme for preparing dilutions of antimicrobial agents to be used in agar dilution susceptibility tests. Approved Standard M100-S7. NCCLS, Wayne, PA.

8 . Bascomb, S. & Manafi, M. (1998). Use of enzyme tests in characterization and identification of aerobic and facultatively aerobic Gram-positive cocci. Clinical Microbiology Reviews 11, 318–40.[Abstract/Free Full Text]

9 . Williamson, R., Gutmann, L., Horaud, T., Delbos, F. & Acar, J. F. (1986). Use of penicillin-binding proteins for the identification of enterococci. Journal of General Microbiology 132, 1929–37.[ISI][Medline]

10 . Fontana, R., Amalfitano, G., Rossi, L. & Satta, G. (1992). Mechanisms of resistance to growth inhibition and killing by ß-lactam antibiotics in enterococci. Clinical Infectious Diseases 15, 486–9.[ISI][Medline]

11 . Devriese, L. A., Daube, G., Hommez, J. & Haesebrouck, F. (1993). In vitro susceptibility of Clostridium perfringens isolated from farm animals to growth-enhancing antibiotics. Journal of Applied Bacteriology 75, 55–7.[ISI][Medline]

12 . Bolder, N. M., Wagenaar, J. A., Putirulan, F. F., Veldman K. T. & Sommer, M. (1999). The effect of flavophospholipol (Flavomycin) and salinomycin sodium (Sacox) on the excretion of Clostridium perfringens, Salmonella enteritidis, and Campylobacter jejuni in broilers after experimental infection. Poultry Science 78, 1681–9.[ISI][Medline]

Received 31 January 2000; returned 17 April 2000; revised 15 May 2000; accepted 12 June 2000