impact of the agar medium and disc type on disc diffusion susceptibility testing against teicoplanin...

APMIS 102: 94-10-7. 1994 Copyright Q A P M I S 1994 Prinred in Denmark . All rights reserved

APhlJU8 ISSN 0903-4641

~

Impact of the agar medium and disc type on disc ~ ~ ~ ~

diffusion susceptibility testing against teicoplanin and vancomycin

KJELD TRUBERG JENSEN,' HENRIK SCH0NHEYDER,' ADAM GOTTSCHAU,' and VILLY FR0LUND THOMSEN3

'Department of Clinical Microbiology, 'Department of Biostatistics, and 'Department of Hospital Infections and Antibiotics, Statens Seruminstitut, Copenhagen, Denmark

Jensen, K. T., Sch~lnheyder, H., Gottschau, A. & Thomsen, V. F. Impact of the agar medium and disc type on disc diffusion susceptibility testing against teicoplanin and vancomycin. APMIS 102: 94-102, 1994.

Susceptibility to teicoplanin and vancomycin was assessed by three disc types: two commercially available discs (NeoSensitabs and PDM disc (30 pg)) and one locally prepared 30 pg disc (SS disc) on four different medium types: Mueller-Hinton agar (MH medium), MH medium and PDM agar I1 supplemented with 5% horse blood (HMB medium and PDM medium, respectively), and Danish blood agar (DBA medium). Two previously studied groups of Gram-positive bacteria were tested: group B (N = 75) comprised miscellaneous cocci, and group C (N = 59) mostly rods. With NeoSensitabs, mean zone diameters were larger than with PDM and SS discs on all medium types, and mean zone diameters were larger on DBA medium than on MHB and PDM medium with all disc types. The impact of the medium type on the zone diameter was evaluated for 121 strains growing on MHB medium, PDM medium, and DBA medium. Bacterial groups B and C each divided into three MIC groups were analysed separately. We compared mean zone diameters for each specific group with the average zone diameter, i.e. the mean value for all zone diameters obtained. The smallest deviations from the average zone diameters were observed on PDM medium for both teicoplanin and vancomycin. Thirty-seven percent of strains failed to grow on MH medium, but supplementation of MH medium with horse blood significantly reduced the zone diameter for group B strains both for teicoplanin and vancomycin. Poor predictability of MIC from the zone diameter was found especially for strains with MICs s 1 pg/ml. The medium type hardly affected the results of regression analysis. In contrast, the medium type markedly affected the results of error-rate bounded analysis. No errors were recorded with the SS disc on MHB medium for either teicoplanin or vancomycin, but no strains with MICs of vancomycin within the intermediate group could be correctly classified on DBA medium.

Key words: Disc susceptibility test; agar medium; antibiotic disc; teicoplanin; vancomycin.

Kjeld Truberg Jensen, Department of Clinical Microbiology, Esbjerg Central Hospital, Ostergade 80, DK-6700 Esbjerg, Denmark.

The glycopeptide antibiotics teicoplanin and serious infections due to p-lactam-resistant vancomycin are the drugs of choice as alterna- Gram-positive bacteria or of patients with p- tive agents for the treatment of patients with lactam allergy (2, 12). Reliable and practical

methods for the determination of susceptibility to glycopeptide antibiotics should be available to the clinical microbiologist, and it is worrying that disc diffusion methods may produce er-

Received March 3, 1993. Accepted November 11, 1993.

94

TEICOPLANIN AND VANCOMYCIN

roneous results (4,9, 11). Thus, a previous study demonstrated a poor correlation between zone diameter as determined by disc diffusion on Danish blood agar and the MIC of teicoplanin (4). A risk of reporting false susceptibility as well as false resistance with commercially available discs was pointed out. The results for streptococci and a group of miscellaneous Gram-positive bacteria (mostly rods) were especially poor, and therefore these bacteria were chosen for further evaluation of susceptibility tests with emphasis on the impact of different medium types and disc types.

MATERIALS AND METHODS

Bacterial strains Two bacterial groups (N = 134), referred to as B

and C in a previous study (4), included Gram-positive isolates from human clinical and veterinary sources. Group B (N = 75) comprised Streptococcus pneumoniae 11; P-haemolytic streptococci 12 (3 from each of the Lancefield groups A, B, C and G); Streptococcus sanguis 4; Streptococcus bovis 4; Streptococcus mitis 4; Streptococcus mutans 4; Enterococcus durans 5 ; Enterococcus faecalis 10; Enterococcus faecium 10; motile Enterococcus spp. 4: Pediococcus spp. 2, and Aerococcus-like organisms ( A m ) 5. Group C (N= 59) comprised mostly Gram-positive rods (Listeria monocytogenes 6; Erysipelothrix rhusiopathiae 8; Co- rynebacterium spp. 23 (C. diphtheriae 6, C. jeikeium 9, and CDC group D2 8); Arcanobacterium haemolyticum 7; Lactobacillus spp. 3; Rothia dentocariosa 3; Nocardia spp. 5; Rhodococcus equi 1; Leuconostoc

Staphylococcus aureus ATCC 25923 was used as a SPP. 3).

control strain. All strains were kept at -80°C.

Antibiotic discs NeoSensitabsB with teicoplanin (60 pg) and vanco-

mycin (70 pg) were purchased from Rosco Diagnos- tics (Denmark) and PDM@; AB BIODISK (PDM disc) with vancomycin (30 pg) and teicoplanin (30 pg) was from Pharmavit ApS (Denmark). Discs con- taining vancomycin (30 pg) and teicoplanin (30 pg) (SS disc) were prepared locally as described previously (4).

Media Agar plates, 14 cm in diameter and 6 mm in depth,

were prepared with four different medium types: Mueller-Hinton agar (MH medium), Mueller-Hinton agar supplemented with 5% defibrinated horse blood (MHB medium), PDM agar 11, PDM@, AB BIO- DISK, supplemented with 5% defibrinated horse

blood (PDM medium), and Danish blood agar (DBA medium) (4).

Disc diffusion assays Each strain was tested for susceptibility to teico-

planin and vancomycin on the four medium types with NeoSensitabs, PDM disc, and S S disc as described previously (direct method) (4). Inoculations were adjusted to obtain semiconfluent growth after overnight incubation at 35°C. Leuconostoc spp. were, however, incubated at 25°C. Zone diameters were measured in mm.

Minimal inhibitory concentration MIC as determined by the agar dilution method

has previously been reported for bacteria of groups B and C (4).

Statistical methods For bacteria with no zone of inhibition 8, 8, and

10 mm were assigned the PDM disc, SS disc, and NeoSensitabs, respectively.

The influence of disc type and medium type on the zone diameter was investigated for strains that were able to grow on MHB medium, PDM medium, and DBA medium, making 121 bacteria available for analysis (72 strains from group B and 49 from group C). The effect of blood in the test agar was investigated for a total of 83 strains that grew on both MH medium and MHB medium.

The impact of medium type, disc type, bacteria group, and MIC group (1) 0.06-0.5 pg/ml, 2) I .0-4.0 pg/ml, and 3) 8-> 128.0 pg/ml) on the zone diameter was evaluated by analysis of variance. Interactions between medium types and disc types were included in order to test whether the differences between the disc types were the same on all medium types. Also interactions between bacteria groups and MIC groups were tested.

The association between MIC and zone diameter was estimated by linear regression analysis. The error of prediction of MIC based on the zone diameter was evaluated as twice the mean square error.

Moreover, data were analysed using the error-rate bounded method of Metzler & DeHaun (1974) (6) as previously described (4). The National Committee for Clinical Laboratory Standards (NCCLS) MIC breakpoints were applied (5 8 pg/ml for susceptibility and 2 32 pg/ml for resistance to teicoplanin, and 5 4 pg/ml for susceptibility and 232 pg/ml for resistance to vancomycin (8).

RESULTS

A total of 49 (37%) of the 134 bacteria failed to grow on MH medium: 21 (28%) from group B and 28 (47%) from group C. Twelve

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JENSEN ei al.

strains (9%) failed to grow on MHB medium (group B 3 strains, group C 9 strains), whilst all strains but 2 and 3 grew on PDM and DBA medium, respectively. MICs for the 121 strains that grew on MHB and PDM as well as DBA medium are shown in Table 1.

Influence of disc and medium type on the zone diameter

For both teicoplanin and vancomycin the mean zone diameters with NeoSensitabs were larger than with PDM and SS discs on all medium types, and the mean zone diameters on DBA medium were larger than on MHB and PDM medium with all disc types (Table 2).

Comparisons between disc types as related to MIC and bacteria groups

For both teicoplanin and vancomycin larger mean zone diameters were observed with Neo- Sensitabs than with PDM and SS discs, irrespective of MIC and bacteria group (data not shown). The differences between the me-

dium types were calculated for each disc type and were found not to depend on the disc type used (data not shown). The impact of the disc type on the zone diameter is shown in Fig. 1. The PDM disc and SS disc were comparable through all MIC and bacteria groups.

Comparisons between medium types as related to MIC and bacteria groups

Larger mean zone diameters were observed on DBA medium than on PDM and MHB medium in all MIC and bacteria groups for both teicoplanin and vancomycin (data not shown). The differences between the disc types were calculated for each medium type and were found not to depend on the medium type used (data not shown).

The impact of the medium type on the zone diameter is shown in Fig. 2. Overall there was a clear medium-dependent deviation from the mean zone diameter. On MHB and DBA medium, deviations for teicoplanin were larger for group C than for group B strains in MIC

TABLE 1. Minimal inhibitory concentration (pg/ml) of teicoplanin and vancomycin against I21 Gram-positive bacteria

Strains Teicoplanin Vancom ycin Range Median Range Median

E. faecalis (10) 0.254.5 0.5 1-4 1 E. faecium (1 0) 0.25-2 1 1-4 1 E. durans (5) 0.12-0.5 0.25 1 1 Motile enterococci (4) 2 2 16 16 S. pneumoniae (1 1) 0.06-0.12 0.12 0.5 0.5 P-haemolytic streptococci

Lancefield gr. A (3) 0.06-0.12 0.06 0.5 -1 0.5

Lancefield gr. C (3) 0.12-0.25 0.12 0.5 -1 0.5 Lancefield gr. G (3) 0.12-0.25 0.25 0.5 -1 0.5

S. bovis (4) 0.25-0.5 0.5 0.5 -1 0.5

Lancefield gr. B (3) 0.12 0.12 1 1

S. sanguis (4) 0.06-0.25 0.19 0.5 -1 1

S. mitis (4) 0.12 0.12 1 1 S. mutans (2) 0.5 -1 1

Rhodococcus equi (1) 0.5 1 L. monocytogenes (6) 0.5 0.5 1-2 2

C. jeikeium (9) 1 -2 2 1 1 C. D2 (8) 1 1 1 1 C. diphtheriae (6) 0.254.5 0.25 1 1 A. haemolyticum (7) 0.124.25 0.12 0.5 -1 1

Rothia sp. (1) 1 2

Pediococcus spp. (2) 0.25-0.5 0.5 Aerococcus spp. (4) 0.25-0.5 0.25 0.25-0.5 0.5

E. rhusiopathiae (8) 4-8 8 128 128

Lactobacillus sp. (1) 8 > 128

Nocardia spp. (2) 32 ->128 16-128

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TABLE 2. Mean zone diameters (mm) observed for 121 strains with three medium types and six disc types MHB medium PDM medium DBA medium

Teicoplanin: NeoSensitabs 25.8 26.5 28.8 PDM disc 21.9 23.0 25.0 SS disc 22.2 23.2 25.4

NeoSensitabs 27.2 28.2 31.4 PDM disc 22.0 23.0 25.9 SS disc 22.4 23.6 26.5

Vancomycin:

groups 1 and 2, but varied only slightly among MIC groups. The deviations for vancomycin increased with increasing MIC level for group B strains on MHB and DBA medium. On PDM medium, deviations varied only slightly among bacteria and MIC groups for both teicoplanin and vancomycin.

Comparison of MHB medium and M H medium

Differences between mean zone diameters observed on MHB medium and MH medium as related to MIC and bacteria groups are shown in Table 3. Addition of blood caused a significant reduction in the mean zone diameter for both teicoplanin and vancomycin in group B strains. This was seen in all MIC groups. For group C strains the addition of blood only affected the mean zone diameter

for teicoplanin in MIC group 1 and for vancomycin in MIC group 2.

Regression analysis A uniform description of the relationship

between MIC and zone diameter was not possible (Fig. 3), so the regression analyses were carried out separately for strains with MICs 11 pg/ml and strains with MICs 21 pg/ml. The slopes and the prediction errors were estimated for each combination of the three disc types and the three medium types.

Regression analysis for strains with MICs of 21 pg/ml

Slope values of the regression lines for teicoplanin were between -0.28 and -0.21 and for vancomycin between -0.36 and -0.26. For example, the slope value for the teicoplan-

Disc types

mm

I I

Fig. 1. Deviation between the mean zone diameter for each disc type and the mean of all zone diameters (0 line) as related to bacteria and MIC groups. Te- icoplanin (unbroken lines); vancomycin (broken lines).

0

0.w9.5 1.0-4.0 a.O-Ll26 1.0.4.0 0 . 0 . P l Z B -j ' O . W . 6 0 B

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JENSEN et ul.

Fig. 2. Deviation between the mean zone diameter for each medium type and the mean of all zone diameters (0 line) as related to bacteria and MIC groups. Teicoplanin (unbroken lines); vancomycin (broken lines).

Medium types

mm 5

4

3

2

1

0

-1

-2

-3 0.050.6 1.04.0 6 .o.a12a 0.06-0.5 1.04.0 8.0-ai2e MIC @g/ml) C 0 Bacteria groups

in PDM disc on PDM medium was -0.28, i.e. a reduction in zone diameter of 10 mm corresponded to an increase in MIC of 2.8 dilution steps. The best prediction of the MIC from the zone diameter was obtained with this disc type/medium type combination. The MIC could be predicted from a given zone diameter with an accuracy of f 2 x 0.99 = f 1.98 dilution steps. The poorest combination was PDM disc/MHB medium, which had an accuracy of k 2 x 1.08 = f 2.16 dilution steps. For vancomycin the best prediction of the MIC was seen with the combination SS disc/MHB medium (accuracy k 2 x 1.07 = k 2.14 dilution steps). The poorest was NeoSensitabdDBA medium with an accuracy of 2 x 1.56 = & 3.12 dilution steps. Any combination of PDM disc or SS disc and MHB medium or PDM medium had an accuracy better than k2.22 dilution steps.

Regression analysis f o r strains with MICs of 2 1 pg/ml

There was no correlation between MIC and zone diameter for any of the medium type/ disc type combinations, so prediction of the MIC was not possible based on the zone diameter.

Analysis b y the error-rate bounded method Data for teicoplanin are shown in Table 4.

The best results were obtained with the SS disc on MHB medium. The one very major error was observed with NeoSensitabs on MHB medium. Major errors were not observed on MHB medium only.

Data for vancomycin are shown in Table 5. The best results were obtained with the SS disc on MHB medium. Neither very major nor

TABLE 3. Differences between mean zone diameters (mm) observed on MHB medium and MH medium for 83 bacteria

MIC group 0.06-2 128 1) 0.06-0.5 2) 1.0-4.0 3) 8.0-2 128

Bacteria group B C B C B C B C Teicoplanin -2.0 -1.4 -2.1* -2.7* - 1.7* 0.8 - 1 . 1 Vancomycin -2.6 -0.8 -3 . l* -2.5* -1.1* -2.2* -0.3 * Statistically significant differences between the two medium types (P < 0.01).

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TUCOPUNIN

40-

30-

20-

10 -

0

0

- 0 0

8

f

40-

30-

20-

10 -

? f

D

0

e 0 0

0

D .

i , - l 0.26 1 4 I I 1 18 I 64 1 , > 128 I

bm

0

0.08

0

0 8 a

VANCOMYCN

I D

0.25 1 4 16 64 > 128

0

0.06

MIC CLlglml)

N , . 3 0 0 1 0 0 0 2 + + + a Fig. 3. Mean zone diameter determined Ibr each of 121 bacteria on three medium types with three disc types (mean of 9 zone diameters) plotted against the MIC for teicoplanin and vancomycin, respectively. Plot symbols indicate number of strains.

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JENSEN er al.

TABLE 4. Results of error-rate bounded analysis for teicoplanin zs” ZRb Number of strains (%)

Very major Major Indeterminate Intermediate errors errors st rains strains

MH medium ( N = 85) : NeoSensitabs 219 1 1 3 0 4 (4.7) 2 (2.4) 0 PDM disc (30 pg) 215 1 1 2 0 2 (2.4) 1 (1.2) 0 SS disc (30 pg) 215 112 0 2 (2.4) 1 (1.2) 0

NeoSensitabs 215 1 1 0 1 (0.8) 0 1 (0.8) 0 PDM disc (30 pg) 212 1 8 0 0 4 (3.3) 0 SS disc (30 pg) 212 1 1 1 0 0 0 0

NeoSensitabs 220 1 1 7 0 5 (3.8) 1 (0.8) 0 PDM disc (30 pg) 216 1 1 3 0 2 (1.5) 1 (0.8) 0 SS disc (30 pg) 215 1 1 3 0 5 (3.8) 1 (0.8) 0

NeoSensitabs 221 1 1 6 0 4 (3.1) 1 (0.8) 0 PDM disc (30 pg) 217 512 0 4 (3.1) 1 (0.8) 0 SS disc (30 pg) 218 1 1 2 0 4 (3.1) 1 (0.8) 0

MHB medium ( N = 122) :

PDM medium ( N = 132):

DBA medium ( N = 130):

a Zone diameter (mm) indicating susceptibility (MIC correlate 1 8 pg/ml). Zone diameter (mm) indicating resistance (MIC correlate 232 pg/ml).

TABLE 5 . Results of error-rate bounded analvsis for vancomvcin zsa ZRb Number of strains (%)

Very major Major Indeterminate Intermediate errors errors strains strains

MH medium ( N = 84) : NeoSensitabs 223 1 1 8 0 0 3 (3.6) 3 PDM disc (30 pg) 219 1 1 1 0 0 l(1.2) 6 SS disc (30 pg) 219 1 1 2 0 0 0 6

NeoSensitabs 2 2 1 1 1 7 0 0 5 (4.1) 2 PDM disc (30 pg) 217 1 1 2 0 0 1 (0.8) 5 SS disc (30 pg) 218 1 1 2 0 0 0 6

NeoSensitabs 223 1 1 8 0 0 4 (3.0) 2 PDM disc (30 pg) 219 1 9 0 0 2 (1.5) 4 SS disc (30 pg) 220 1 9 0 0 2 (1.5) 4

NeoSensitabs 224 1 2 1 0 0 5 (3.8) 0 PDM disc (30 pg) 219 1 1 1 0 0 5 (3.8) 0 SS disc (30 pg) 220 1 1 0 0 0 5 (3.8) 0

MHB medium ( N = 122) :

PDM medium ( N = 132):

DBA medium ( N = 130):

a Zone diameter (mm) indicating susceptibility (MIC correlate 1 4 pg/ml). Zone diameter (mm) indicating resistance (MIC correlate 2 32 pg/ml).

major errors were observed. No strains with MICs within the intermediate category could be correctly classified on DBA medium (five strains with MIC 16 pg/ml).

100

DISCUSSION

We have previously observed a poor correlation between MICs and zone diameters when strains

TEICOPLAMN AND VANCOMYCIN

belonging to bacteria groups B and C were tested against teicoplanin on DBA medium. The correlation was especially poor for group B strains (4). As we contended that the medium might be a critical factor, we have compared results obtained on DBA medium for the two bacteria groups with results obtained on two standard media used for routine disc susceptibility testing. The NCCLS recommends the use of 30 pg paper discs for susceptibility testing against teicoplanin and vancomycin (8). As we have previously investigated the effect of disc potency (4), we chose 30 pg paper discs for our experiments besides Neo- Sensi tabs. Almost equal differences between NeoSensitabs and the two other disc types were seen through all MIC and bacteria groups. This is consistent with the higher content of antibiotics in tablets than in paper discs (5). The very similar results obtained with the PDM disc and the SS disc (both 30 yg paper discs) support the reprodu- cibility of the method used.

The present study has clearly demonstrated the impact of the medium type on the zone diameter for teicoplanin and vancomycin. DBA medium consistently gave larger zone diameters compared to PDM and MHB medium. On MHB and DBA medium the deviations from the mean zone diameter varied among bacteria and MIC groups, although variations were less pronounced for teicoplanin than for vancomycin. On PDM medium, deviations from the mean zone diameter were relatively small and, more importantly, varied only slightly among bacteria and MIC groups. Thus, PDM medium may be a more reliable medium type for disc susceptibility testing against teicoplanin and vancomycin.

The medium type had only a slight effect (if any) on the results of regression analysis. It was not possible to predict the MIC from the zone diameter for strains with MICs I 1 pg/ml and it had a poor predictability for strains with MICs 2 1 kg/ml. Regression statistics have been deemed inappropriate because of the bimo- dal distribution of endpoints when Gram-nega- tive bacteria represented the category resistant to teicoplanin and vancomycin (1). In the present study the resistant category for both antibiotics was represented by Gram-positive bacteria (motile enterococci, Nocardiu, Lactobacil- lus, Erysipelothrix,) and there was a more even distribution of endpoints.

The effect of the medium type was clearly demonstrated by error-rate bounded analysis. The best result for teicoplanin (Table 4) was obtained on MHB medium with the SS disc, although definition of an intermediate range of zone diameters as recommended by Barry et ul. (1986) (1) was not possible. This could be achieved on MHB medium with the PDM disc, but at the expense of 3.3% indeterminate strains. Moreover, the result obtained on PDM medium with the PDM disc was favourable. Only one strain was intermediately susceptible (MIC 16 pg/ml) and this strain failed to grow on some of the medium types. Therefore, the ability of the medium types to correctly classify strains with intermediate susceptibility to teicoplanin could not be assessed. The results of the error- rate bounded analysis for vancomycin (Table 5 ) revealed that none of the strains with MICs within the intermediate range of susceptibility could be correctly classified on DBA medium. This could be achieved on MHB medium with the SS disc, whilst the results with the PDM disc were almost equally good.

The NCCLS recommends MH medium for routine disc susceptibility testing (7). For strains that fail to grow on MH medium, supplementation with 5% defibrinated sheep blood is recommended. Results of disc diffusion testing of enterococci against vancomycin on MH medium with 5% sheep blood have been found comparable to results on unsupplemented MH medium (10, 11). We observed, however, significantly smaller zone diameters on MHB medium than on MH medium for group B strains with both teicoplanin and vancomycin, irrespective of MIC group (Table 3). Our zone size interpretive breakpoints for the unsupplemented MH medium with PDM and SS discs (Tables 4 & 5) did not correspond to the NCCLS breakpoints for 30 pg paper discs (8). Using their criteria the error-rates did not change for teicoplanin, two Lactobacillus spp. still being classified as major errors. For vancomycin three of four enterococci (MIC 16 pg/ml) would be indeterminate with both disc types and Nocardiu spp. would be either indeterminate or classified as a very major error. These discrepancies are disturbing because a recent change in interpretive zone diameters for vancomycin was a re- sponse to the development of varying degrees of vancomycin resistance among enterococci (8).

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JENSEN er a/.

Although we used an inoculum not recommended by the NCCLS, this could hardly ex- plain these discrepancies because even a 1 0-fold increase in inoculum had no effect on disc diffusion results with Enterococcus species (10). Nevertheless, the results of the present study indicate that MH medium was not suitable for disc susceptibility testing against teicoplanin and vancomycin without blood supplementation.

In conclusion, performance standards for disc susceptibility testing of Gram-positive bacteria against teicoplanin and vancomycin are needed. Reliable zone size interpretive breakpoints for glycopeptide antibiotics may, however, not be established until sufficient numbers of clinically important Gram-positive bacteria with reduced susceptibility can be investigated. Until then we recommend that clinically important Gram- positive bacteria are tested with a dilution method against teicoplanin and vancomycin. By this approach more knowledge as to varying degrees of susceptibility to these antimicrobial agents will be gained, creating a basis for future investigations. This is supported by the obser- vation that plasmid-mediated glycopeptide resistance, although of high level in enterococci, may be expressed merely as intermediate resistance in other Gram-positive bacteria (3) and such resistance may not be detected by the disc diffusion method (12). In future studies the im- portance of blood supplementation of the test agar should be considered.

REFERENCES

1. Barry, A. L., Thornsberry, C. & Jones, R. N.: Evaluation of teicoplanin and vancomycin disk susceptibility tests. J. Clin. Microbiol. 23: 100-103, 1986.

2. Courvalin, II: Resistance of enterococci to glyco- peptides. Antimicrob. Agents Chemother. 34:

3. Gray, J. W & Pedler, S. J.: Antibiotic-resistant enterococci. J. Hosp. Infect. 21: 1-14, 1992.

4. Jensen, K. T , Schenheyder, H.. Pers, C. & Thom- sen, l! E: In vitro activity of teicoplanin and vancomycin against Gram-positive bacteria from human clinical and veterinary sources. APMIS

5 . Lauwers, S.. Philippe, J., Van Zeebroeck, A . , Pier- ad, D., Derde, M.-I! & Kaufman, L.: Quality control in antimicrobial disk susceptibility testing: A Belgian multicenter study. Eur. J. Clin. Micro- biol. Infect. Dis. 10: 652-656, 1991.

6. Metzler, C. M. & DeHaan, R. M.: Susceptibility tests of anaerobic bacteria: Statistical and clinical considerations. J. Infect. Dis. 130: 588- 594, 1974.

7. National Committee for Clinical Laboratory Stan- dards. Performance standards for antimicrobial disk susceptibility tests - fourth edition. Ap- proved Standards. NCCLS Document M2-A4. Villanova, PA: NCCLS 1990.

8. National Committee for Clinical Laboratory Stan- dards. Performance standards for antimicrobial disk susceptibility testing. Third Information Supplement. NCCLS Document M 100-S3 (ISBN 1-56238-136-9). NCCLS, 771 E. Lancaster Av- enue, Villanova, PA 19085, 1991.

9. Sahm, D. F , Kissinger, J., Gilmore, M. S., Murray, II R., Mulder, R., Solliahy, J. & Clarke, B.: In vitro susceptibility studies of vancomycin-resistant Enterococcus faecalis. Antimicrob. Agents Chemother. 33: 1588-1591, 1989.

10. Sahm, D. E & Olsen, L.: In vitro detection of enterococcal vancomycin resistance. Antimicrob. Agents Chemother. 34: 1846-1848, 1990.

11. Swenson, J. M., Hill, B. C. & Thornsberry, C.: Problems with the disk diffusion test for detection of vancomycin resistance in enterococci. J. Clin. Microbiol. 27: 2140-2142, 1989.

12. Woodford, N., Johnson, A. II & George, R. C.: Detection of glycopeptide resistance in clinical isolates of Gram-positive bacteria. J. Antimicrob. Chemother. 28: 483-490, 1991.

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