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Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 1998 Jul;36(7):1907–1911. doi: 10.1128/jcm.36.7.1907-1911.1998

Effects of Different Test Conditions on MICs of Food Animal Growth-Promoting Antibacterial Agents for Enterococci

Patrick Butaye 1,*, Luc A Devriese 1, Freddy Haesebrouck 1
PMCID: PMC104950  PMID: 9650934

Abstract

The influence of the addition of sheep blood to Mueller-Hinton II agar and the effects of aerobic incubation with or without CO2 and of anaerobic incubation were tested with bacitracin, tylosin, avoparcin, virginiamycin, avilamycin, narasin, and flavomycin on enterococci. The antibacterial activity of bambermycin (Flavomycin) was strongly inhibited by the addition of blood, except with the species Enterococcus faecium, Enterococcus mundtii, Enterococcus hirae, Enterococcus casseliflavus, and Enterococcus gallinarum, which were not susceptible to this antibiotic on blood-free medium. With all other antimicrobials except avoparcin and tylosin, the presence of blood resulted in MIC increases of 1 to 3 log2 differences. Incubation in aerobic or anaerobic atmospheres enriched with CO2 lowered the susceptibility of enterococci to tylosin and increased their susceptibility to avilamycin, narasin, and avoparcin. This effect was most pronounced in tests on blood-free media. Results of susceptibility tests incubated under anaerobiosis and in a CO2-enriched atmosphere did not differ. For all enterococcal species, the preferred conditions for testing the susceptibility are Mueller-Hinton II medium supplemented with blood and incubation in a CO2-enriched atmosphere. However, when only E. faecium and Enterococcus faecalis are being tested, Mueller-Hinton II medium without blood incubated aerobically gives satisfactory results.


Several enterococcal species belong to the normal intestinal flora of humans and animals. Enterococcus faecalis, Enterococcus faecium, and more rarely other species may be involved in human infections. Antibiotic therapy is problematic because of resistances. In particular, penicillin, aminoglycoside, and glycopeptide resistances cause problems (12, 15).

To date, in animals, enterococcal infections are not known to play an important role, possibly because geriatric medicine and intensive care are less well developed. Recently the susceptibility of these intestinal bacteria to food animal growth-promoting antimicrobial agents not used in therapy has become a subject of concern (1, 11, 18). However, few data on the in vitro susceptibility of intestinal bacteria to these agents are available (7). Enterococci from animals have been tested for susceptibility and resistance to growth promoters and therapeutic agents by the disc diffusion method (13), agar dilution tests in Mueller-Hinton agar (6), and the broth dilution method (18). These differences in test methods make test results difficult to compare.

Enterococci need rich media for rapid growth (3). Most probably because the usual standardized Mueller-Hinton II medium was unsatisfactory in this respect, a modification of culture conditions, consisting of the use of brain heart infusion, was suggested by the National Committee for Clinical Laboratory Standards (NCCLS) (standard M100-S7 for use with M7-A4) (14) for testing resistance of E. faecalis and E. faecium to gentamicin, streptomycin, and vancomycin. This complex medium has not been standardized for antibiotic susceptibility testing, and differences may occur between different manufacturers. Furthermore, several species of enterococci belonging to the normal flora of animals require special growth conditions. A CO2-enriched environment is necessary for the growth of Enterococcus columbae and Enterococcus cecorum (4). Other species show enhanced growth when incubated in a CO2-enriched environment. The natural habitat of the intestinal enterococci is, however, largely anaerobic, and these facultative anaerobic bacteria have a preference for anaerobic conditions (3). These special requirements imply a need for specialized culture conditions.

The activity of several antibiotics is known to be influenced by the composition of the medium and/or by the incubation conditions. The possible effects of the composition of the medium and/or incubation conditions are much less well-known for the growth-promoting antibacterials. The only well-known phenomenon involving these substances is the influence of CO2 on the pH of the medium, which affects the activity of tylosin, a macrolide antibiotic (9, 10, 16).

Therefore, we wished to investigate the effects of different modifications of the standardized Mueller-Hinton II agar dilution procedure on inhibitory activity of growth promoters. Such modifications have been proposed by the NCCLS for use with fastidious bacteria (standard M100-S7 for use with M7-A4) (14). The effects of sheep blood (5%), CO2 incubation, and anaerobic incubation on the MICs of seven growth-promoting agents used in Europe for collection strains of animal-associated species of the genus Enterococcus were determined.

MATERIALS AND METHODS

Strains.

The tested collection strains of intestinal enterococci belonging to different species groups are shown in Table 1 (5, 21). The E. faecalis ATCC 29212 and Staphylococcus aureus ATCC 29213 control strains used for susceptibility tests were included in all tests.

TABLE 1.

Enterococcal collection strains tested

Species group Species Straina
E. faecalis group E. faecalis LMG 7937T
ATCC 29213
CCM 5613
E. faecium group E. faecium LMG 11423T
LMG 1620
LMG 10741
Enterococcus mundtii LMG 10748T
LMG 13044
LMG 12308
Enterococcus hirae LMG 6399T
NCFB 1632
LMG 11492
Enterococcus durans LMG 12691T
NCFB 498
LMG 122283
E. avium group Enterococcus avium LMG 10744T
LMG 12303
LMG 12304
Enterococcus raffinosus LMG 12300
Enterococcus malodoratus LMG 10747T
LMG 13602
E. gallinarum group Enterococcus casseliflavus LMG 10745T
LMG 16281
LMG 16285
Enterococcus gallinarum LMG 13129T
LMG 16204
LMG 16201
E. cecorum group E. columbae LMG 11740T
LMG 11747
LMG 12295
E. cecorum LMG 12902T
CCUG 27881
LMG 11743
a

ATCC, American Type Culture Collection, Rockville, Md.; LMG, Collectie Laboratorium voor Microbiologie, Ghent, Belgium; CCM, Collection of Microorganisms, Brno, Czechia; NCFB, National Collection of Food-Borne Bacteria, now merged with National Collection of Industrial and Marine Bacteria, Aberdeen, United Kingdom; CCUG, Culture Collection of the University of Göteborg, Göteborg, Sweden. 

Antibiotics.

The following antibiotic preparations, manufactured for analytical purposes, were tested: avoparcin (American Cyanamid, Princeton, N.J.), bambermycin (Flavomycin; Hoechst, Frankfurt, Germany), virginiamycin (Pfizer, Rixensart, Belgium), bacitracin (67,000 U/mg) (Sigma, St. Louis, Mo.), tylosin (Sigma), avilamycin (Eli Lilly, Indianapolis, Ind.), and narasin (Eli Lilly). Antibiotics were dissolved in appropriate solvents to make stock solutions containing 1,000 μg/ml and then further diluted in sterile distilled water according to the methods recommended by the NCCLS (standard M100-S7 for use with M7-A4) (14).

Antimicrobial susceptibility tests.

MIC tests were carried out on Mueller-Hinton II agar (Becton Dickinson, Cockeysville, Md.) with or without 5% sheep blood containing doubling dilutions of the antibiotics and used on the day of preparation. Appropriate antibiotic-free agar plates were included as controls. Inocula were prepared by diluting brain heart infusion (Oxoid, Basingstoke, United Kingdom) cultures in buffered saline overnight to a density of 0.5 on the McFarland turbidity scale and diluted 40-fold before inoculation. Plates were seeded with approximately 105 CFU.

Plates with or without sheep blood added were incubated aerobically, anaerobically (GasPak Plus; Becton Dickinson) with more than 4% but less than 10% CO2, or in a CO2 (5%)-enriched aerobic environment for 24 h.

RESULTS

Effect of blood supplementation.

When plates were incubated aerobically, major differences between Mueller-Hinton media with and without blood were noted in bambermycin MICs for several strains (44% of the strains) (Table 2). This increase in MIC on blood-containing media was not evident in the range of concentrations tested with strains for which MICs obtained on blood-free media were high. These all belonged to the E. faecium and E. gallinarum species groups. Narasin MICs were almost systematically (72% of the strains) 2 or 3 doubling dilutions higher on blood-supplemented agar. With bacitracin, differences of 1 or 2 doubling dilutions were seen with most strains (72%), and for some strains, a much higher MIC was seen when blood was added. With virginiamycin and avilamycin, the MIC was 1 or 2 doubling dilutions higher for 48 and 44% of the strains, respectively. With avoparcin and tylosin, no important differences were found. It should be noted that none of the strains of the E. cecorum group grew in these aerobically incubated tests. The same was seen with certain of the E. avium group strains but only on blood-free media.

TABLE 2.

Log2 differences in the comparison of test results on Mueller-Hinton II medium either supplemented with 5% sheep blood or not supplemented

Incubation condition and antibiotic na No. of strains with log2 differenceb:
−1 0 1 2 3 4 5 6 7 8 9 11
Aerobic
 Bambermycin 25 12 2 2 2 3 1 2 1
 Virginiamycin 25 13 11 1
 Avilamycin 25 14 7 4
 Narasin 25 5 2 8 10
 Avoparcin 25 1 17 7
 Bacitracin 25 4 12 6 1 1 1
 Tylosin 25 1 16 6 2
Anaerobic
 Bambermycin 33 13 1 1 9 6 3
 Virginiamycin 33 1 12 20
 Avilamycin 33 6 22 5
 Narasin 33 2 4 15 10 2
 Avoparcin 33 23 10
 Bacitracin 33 1 7 14 8 2 1
 Tylosin 33 1 24 6 1 1
CO2-enriched atmosphere
 Bambermycin 33 11 1 1 4 3 5 3 3 2
 Virginiamycin 33 14 17 1 1
 Avilamycin 33 8 16 9
 Narasin 33 1 13 15 1 3
 Avoparcin 33 2 16 15
 Bacitracin 33 7 15 7 4
 Tylosin 33 3 26 3 1
a

Number of strains tested. 

b

A positive difference is a higher MIC of the antibiotic incubated with 5% sheep blood; a negative difference is a higher MIC of the antibiotic incubated without 5% sheep blood. Strains were tested for log2 differences of between −7 and 11; however, no differences of −7 to −2 or 10 were found. 

All strains grew when incubations were performed anaerobically (Table 2), and differences regarding blood supplementation effects were similar to those seen in the aerobically incubated tests. For bambermycin, major differences were seen in 61% of the strains tested. Virginiamycin, avilamycin, and bacitracin tended to have MICs 1 doubling dilution higher on blood-supplemented media for most of the strains (60, 80, and 76%, respectively). With narasin, the effect of blood was the same as that observed when plates were incubated aerobically. Tylosin MICs were only slightly different for 24% of the strains.

Upon incubation in a CO2-enriched atmosphere (Table 2), the same tendencies in MIC differences were seen as when incubation was performed anaerobically.

Anaerobic versus aerobic incubation.

When blood-supplemented plates were used (Table 3), the antibiotics bambermycin, avilamycin, narasin, and avoparcin showed slightly higher MICs upon anaerobic incubation than upon aerobic incubation for 27, 30, 65, and 34% of the strains, respectively. With tylosin, the opposite was seen in 46% of the strains tested. For virginiamycin and bacitracin, there was an almost equal distribution between higher and lower MICs when the aerobic test results were compared with the anaerobic ones.

TABLE 3.

Log2 differences in the comparison of test results from incubation occurring aerobically and anaerobically

Supplement and antibiotic na No. of strains with log2 differenceb
−7 −6 −4 −3 −2 −1 0 1 2 3 6 8
Blood
 Bambermycin 26 7 17 2
 Virginiamycin 26 3 17 6
 Avilamycin 26 5 3 15 3
 Narasin 26 1 16 6 3
 Avoparcin 26 1 8 14 3
 Bacitracin 26 3 21 1 1
 Tylosin 26 1 13 9 2 1
None
 Bambermycin 25 1 1 1 4 14 2 1 1
 Virginiamycin 25 3 19 1 1 1
 Avilamycin 25 3 13 8 1
 Narasin 25 1 1 5 14 4
 Avoparcin 25 1 12 12
 Bacitracin 25 1 7 11 6
 Tylosin 25 1 14 6 2 2
a

Number of strains tested. 

b

A positive difference is a higher MIC of the antibiotic incubated anaerobically; a negative difference is a higher MIC of the antibiotic incubated aerobically. Strains were tested for log2 differences of between −7 and 11; however, no differences of −5, 4, 5, 7, or 9 to 11 were found. 

No uniform effect of anaerobic incubation was seen on Mueller-Hinton plates without blood supplement (Table 3). Avilamicin, narasin, and avoparcin had slightly lower MICs when incubated anaerobically for 64, 84, and 52% of the strains, respectively. Virginiamycin and bacitracin anaerobic and aerobic test results did not differ, while tylosin showed a tendency that was again opposite to that of the other antibiotics tested: MICs for 40% of the strains were higher when the strains were incubated anaerobically.

Aerobic incubation versus incubation in a CO2-enriched atmosphere.

On Mueller-Hinton plates supplemented with blood, no major differences were noted (Table 4). For avilamycin, narasin, avoparcin, and to a lesser extent bacitracin, there was a tendency towards slightly lower MICs on plates incubated in CO2 with 46, 77, 58, and 31% of the strains, respectively. In tests with tylosin, the opposite was seen with 50% of the strains. Bambermycin and virginiamycin results did not differ when strains were incubated aerobically or in a CO2-enriched atmosphere.

TABLE 4.

Log2 differences in the comparison of test results from incubation occurring aerobically and in a CO2-enriched atmosphere

Supplement and antibiotic na No. of strains with log2 differenceb
−7 −5 −4 −3 −2 −1 0 1 2 4 6
Blood
 Bambermycin 26 23 2 1
 Virginiamycin 26 4 19 3
 Avilamycin 26 5 7 8 3 3
 Narasin 26 7 13 6
 Avoparcin 26 1 14 11
 Bacitracin 26 8 17 1
 Tylosin 26 13 10 3
None
 Bambermycin 25 1 1 2 13 3 3 1 1
 Virginiamycin 25 5 18 2
 Avilamycin 25 1 14 10
 Narasin 25 2 1 4 7 10 1
 Avoparcin 25 20 5
 Bacitracin 25 16 8 1
 Tylosin 25 8 11 6
a

Number of strains tested. 

b

A positive difference is a higher MIC of the antibiotic incubated in an CO2-enriched atmosphere; a negative difference is a higher MIC of the antibiotic incubated aerobically. Strains were tested for log2 differences of between −7 and 11; however, no differences of −6, 3, 5, or 7 to 11 were found. 

When Mueller-Hinton plates without blood were used (Table 4), differences between results obtained with aerobic incubation and in a CO2-enriched atmosphere were relatively unimportant and similar to the ones noted in the comparative tests with anaerobic incubation.

Anaerobic incubation versus incubation in a CO2-enriched atmosphere.

Between anaerobic incubation or incubation in a CO2-enriched atmosphere, there were no substantial differences in MIC results on plates with and without blood (Table 5).

TABLE 5.

Log2 differences in the comparison of test results from incubation occurring anaerobically and in a CO2-enriched atmosphere

Supplement and antibiotic na No. of strains with log2 differenceb
−3 −2 −1 0 1 2 3 4 5
Blood
 Bambermycin 33 2 1 20 10
 Virginiamycin 33 1 25 7
 Avilamycin 33 1 22 5 5
 Narasin 33 9 20 4
 Avoparcin 33 2 28 3
 Bacitracin 33 3 27 2 1
 Tylosin 33 1 29 2 1
None
 Bambermycin 33 1 4 20 6 1 1
 Virginiamycin 33 3 22 6 2
 Avilamycin 33 1 26 4 2
 Narasin 33 1 2 19 7 3 1
 Avoparcin 33 3 17 13
 Bacitracin 33 6 18 6 2 1
 Tylosin 33 1 1 6 22 3
a

Number of strains tested. 

b

A positive difference is a higher MIC of the antibiotic incubated in a CO2- enriched atmosphere; a negative difference is a higher MIC of the antibiotic incubated aerobically. Strains were tested for log2 differences of between −7 and 11; however, no differences of −7 to −4 or 6 to 11 were found. 

MICs obtained in tests on Mueller-Hinton II with blood and incubated in 5% CO2 are tabulated in Table 6.

TABLE 6.

MICs of different growth-promoting antibiotics for enterococci tested on blood-supplemented Mueller-Hinton II agar and incubated in CO2

Antibiotic and strain na MIC (μg/ml)
Median Range
Bambermycin
E. faecalis ATCC 29212 4 12 8–16
 S. aureus ATCC 29213 4 2 1–4
 E. faecalis 2 32 32   
 E. faecium species group 12 >64 64–>64
 E. gallinarum species group 6 32 32–64
 E. avium species group 6 >64 >64   
 E. cecorum species group 6 1.5 0.5–4
Virginiamycin
 E. faecalis ATCC 29212 3 2 2–4
 S. aureus ATCC 29213 3 0.25 0.25
 Enterococcus (all species)b 32 1 0.12–4
Avilamycin
 E. faecalis ATCC 29212 3 0.5 0.5–2
 S. aureus ATCC 29213 3 2 1–2
 Enterococcus (all species) 32 0.5 0.06–2
Narasin
 E. faecalis ATCC 29212 3 0.5 0.25–0.5
 S. aureus ATCC 29213 3 0.5 0.25–0.5
 Enterococcus (all species) 32 0.5 0.06–1
Avoparcin
 E. faecalis ATCC 29212 3 2 2   
 S. aureus ATCC 29213 3 4 4   
 Enterococcus (all species)c 26 0.5 0.25–2
Bacitracind,e
 E. faecalis ATCC 29212 3 4 4–8
 S. aureus ATCC 29213 3 2 2   
 E. faecalis 2 5 2–8
 E. faecium species group 11 8 0.25–8
 E. gallinarum species group 6 8 8–16
 E. avium species group 6 2 0.5–16
 E. cecorum species group 6 ≤0.03 ≤0.03–0.12
Tylosind
 E. faecalis ATCC 29212 3 2 1   
 S. aureus ATCC 29213 3 1 1–2
 Enterococcus (all species) 24 2 0.5–2
a

Number of repeats for E. faecalis ATCC 29212 and S. aureus ATCC 29213, and number of strains tested for the other enterococci. 

b

All Enterococcus species except E. faecalis ATCC 29212. 

c

All species except E. faecalis ATCC 29212 and the species from the E. gallinarum species group, which possess the vanC gene (8). 

d

Excluding strains with acquired resistance. 

e

The MIC of bacitracin is expressed in units/milliliter. 

DISCUSSION

Blood supplementation of Mueller-Hinton plates has been shown to have an effect on the MICs of sulfonamide, trimethoprim, cephalosporins, novobiocin, and nafcillin for enterococci (22). The addition of blood reduces MICs of cephalosporins, but not all cephalosporins react in the same way: some remain unaffected, and for others the effect does not exceed 1 or 2 doubling dilutions. This is dependent on the chemical structure of the cephalosporins (2, 17). In the present study, major detrimental effects of blood on antibacterial activity were seen with bambermycin. In the original description of this antibiotic (20), which at that time was called moenomycin, it was indicated that serum inactivates its in vitro activity by 10 to 0.1% compared to protein-free medium. The effects of blood seen in the present tests were much more dramatic, at least on the inhibitory activity on species and strains which were susceptible to relatively low concentrations of this antibiotic. A similar effect of blood may possibly be found in higher concentration ranges for the other strains.

Few reports deal with the effects of anaerobic incubation on MIC results. The activity of aminoglycosides is significantly reduced with Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis incubated anaerobically without CO2 (19). The reason for the negative effect on tylosin activity is found in the presence of CO2, which has an effect on the pH of the medium (9, 10, 16). The causes of the slightly positive effects of CO2 and anaerobic incubation on the inhibitory activity of avilamycin, narasin, and avoparcin observed in the present investigation are unknown, though they are most probably found in the effect of CO2 on the activity of the antibiotics. To our knowledge, nothing is known about a pH effect on the MICs of these growth promoters. The activity of bambermycin has been reported to be affected by pH increases (20). This was not evident in our tests on blood-containing media, not even with strains such as the S. aureus control strain and the few other strains that were inhibited by relatively low concentrations of bambermycin in this medium (Table 6). The effects of this antibiotic on blood-free medium were strongly variable and strain dependent. It should be noted that the comparisons between aerobic incubation results and the results obtained in aerobic or aerobic CO2-supplemented atmospheres are somewhat hindered by the presence of E. cecorum and of E. avium group strains which did not grow or which grew poorly aerobically. These strains were not included in the comparative tables.

Many changes in MICs corresponded to the species groups, or to several species of a species group, indicating that there may be species-specific growth requirements and that the species-specific metabolism may play a role in the differences noted. For the capnophilic bacteria, CO2 dependency is clearly species group related; none of the strains in the E. cecorum group grow without CO2, and some of the strains in the E. avium group have such poor growth without CO2 that MICs could not be evaluated. However, for the other species-related changes, no obvious reason can be found for the differences.

It can be concluded that Mueller-Hinton II agar can be used for testing of the inhibitory activity of growth-promoting agents on enterococci. The addition of blood decreases the activity of these products to various degrees. The addition of CO2 under both aerobic and anaerobic conditions enhances the inhibitory effects of narasin, avoparcin, and avilamycin and is detrimental to the activity of tylosin. When CO2 supplementation is necessary because capnophilic strains are to be tested, the choice between anaerobic incubation with 4 to 10% CO2 in the jars or incubation in a CO2 incubator can be left open. The preferred conditions for testing the susceptibility when all enterococcal species are being compared is Mueller-Hinton II medium supplemented with blood and incubated in a CO2-enriched atmosphere. When only E. faecium and E. faecalis are being tested, Mueller-Hinton II medium incubated aerobically gives satisfactory results. It should be recognized, however, that the omission of blood strongly influences results obtained with certain antibiotics, especially bambermycin and narasin.

ACKNOWLEDGMENTS

This work has been supported by the Research Fund of the University of Ghent, Codenr. BOZF97/N2/022.

We thank A. Van de Kerckhove for her skilled technical assistance.

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