H. Grimm

Interpretive Criteria of Antimicrobial Disk Susceptibility Tests with Flomoxef S u m m a r y : 320 recently isolated pathogens, 20 strains from each of 16 species, were investigated using MuellerHinton agar and DIN as well as NCCLS standards. The geometric mean of the agar dilution MICs of flomoxef were 0.44 mg/1 for Staphylococcus aureus, 0.05 mg/1 (Klebsiella oxytoca) to 12.6 mg/1 (Enterobacter spp.) for enterobacteriaceae, 33.1 rag/1 for Acinetobacter anitratus, 64 mg/1 for Enterococcus faecalis, and more than 256 mg/1 for Pseudomonas aeruginosa. For disk susceptibility testing of flomoxef a 30 ~tg disk loading and the following interpretation of inhibition zones using the DIN method were recommended: resistant - up to 22 mm (corresponding to MICs of 8 mg/1 or more), moderately susceptible - 23 to 29 mm (corresponding to MICs from 1 to 4 mg/l), and susceptible - 30 mm or more (corresponding to MICs of 0.5 mg/l or less). The respective values for the NCCLS method using the American high MIC breakpoints are: resistant - up to 14 mm (corresponding to MICs of 32 mg/1 or more), moderately susceptible - 15 to 17 mm (corresponding to MICs of 16 mg/1), and susceptible - 18 mm or more (corresponding to MICs of 8 mg/1 or less).

Introduction The parenteral cephalosporin flomoxef (formerly 6315-S) is a new oxacephem antibiotic with an in vitro activity comparable to that of cefotaxime against most enterobacteriaceae and staphylococci. It is superior to cefotaxime against Proteus vulgaris and Bacteroides fragilis [1]. The pharmacokinetic data of flomoxef are similar to those of cefotiam [2]. We presented the first recommendations for the interpretation of the susceptibility testing of flomoxef at the 27th Interscience Conference on Antimicrobial Agents and Chemotherapy, New York, 1987 [3]. Up to now no detailed regression data for flomoxef are available in the literature. The aim of the following study is to evaluate the correlation between MICs and zone diameters, and to propose interpretive criteria for the flomoxef agar diffusion susceptibility test.

Materials and Methods 320 bacterial strains freshly isolated from clinical specimens were used for this study comprising 20 strains each of: Escherichia

coli, Citrobacter freundii, Citrobacter diversus, Klebsiella pneumoniae, Klebsietla oxytoca, Enterobacter spp., Serratia marcescens, Proteus mirabilis, P. vulgaris, Morganella morganii, Proteus rettgeri, Providencia spp., Pseudomonas aeruginosa, Acinetobacter anitratus, Staphylococcus aureus and Enterococcus S 258

Zusammenfassung: Interpretationskriterien fiir den AgardOfusions-Bliittchentest mit Flomoxef. Unter Anwendung der DIN- und NCCLS-Methoden wurden auf Mueller-Hinton Agar 320 frisch isolierte Bakterienkulturen (16 Spezies, je 20 St~imme) untersucht. In der Agardilution betrug die Flomoxef-MHK im geometrischen Mittel ftir Staphylococcus aureus 0,44 mg/l, ftir Enterobakterien 0,05 (Klebsiella oxytoca) bis 12,6 mg/1 (Enterobacter spp.), ftir Acinetobacter anitratus 33,1 mg/1, ftir Enterococcusfaecalis 64 mg/1 und fur Pseudomonas aeruginosa tiber 256 mg/l. FUr den Agardiffusionstest werden mit 30 ~tg Flomoxef beladene Disks und folgende Interpretation des mit der DIN-Methode gewonnenen Hemmhofs empfohlen: resistent bis 22 mm (entsprechend MHK 8 mg/1 oder mehr), m ~ i g sensibel 23 bis 29 mm (entsprechend MHK 1-4 mg/1), und sensibel 30 mm oder mehr (entsprechend M H K 0,5 mg/1 oder weniger). Die jeweiligen Werte fiJr die NCCLS-Methode und die hrheren amerikanischen MHK-Grenzwerte betragen: resistent bis 14 mm (entsprechend MHK 32 mg/1 oder mehr), maSig sensibel 15 bis 17 mm (entsprechend MHK 16 mg/1) und sensibel 18 mm oder mehr (entsprechend MHK 8 mg~ oder weniger).

faecalis. Regarding better correlation statistics strains with reduced susceptibility against cefotaxime were preferred. MIC determinations were made using the agar dilution method according to the NCCLS recommendations [4] with a multipoint inoculator and an inoculum of 104 CFU/spot. The disks for the agar diffusion test loaded with 5 p.g, 10 ~tg and 30 pg of flomoxef were freshly manufactured in our laboratory. Inhibition zones were obtained by using the NCCLS [5] as well as the DIN-58940 [6] methods. Mueller-Hinton agar (Oxoid CM 337) served as nutrient medium in all cases. The correlations between zone diameters and MIC values were determined by regression line analyses according to the method of the fewest squares where "y" represented the zone diameter and "'x" a function of the MIC (x = log2 MIC+9). Results and Discussion The species-dependent distribution of MIC values of flomoxef is shown in Table 1. The geometric mean of agar dilution MICs of flomoxef were 0.44 mg/l for S. aureus, 0.05 mg/1 (K. o.tytoca) to 12.6 rag/1 (Enterobacter spp.) for enterobacteriaceae, 33,1 mg~ for A. anitratus, 64 rag/1 for E.faecalis, and more than 256 mg/1 for P. aeruginosa. The values for the enterobacteriaceae do not reflect the real

Dr. H. Grimm,Institut fiirMikrobiologieund KlinischeChemie(LaborDr. Gaertner), Hoyerstr.51, W-7987 Weingarten,Germany.

Infection 19 (1991) Suppl. 5 © MMV Medizin Verlag GmbH Miinchen,Mfinchen1991

I t . G r i m m : Susceptibility Testing o f F l o m o x e f

Table 1 : In-vitro activity of flomoxef against aerobic pathogens.

Escherichia coli

6

4

-

-

-

5

Citrobacterfreundii

1

5

6

-

-

1

Citrobacter diversus

9

10

-

-

1

-

1

-

2

12

2

Klebsiella oxytoca

13

6

.

spp.

-

Klebsiellapneumoniae Enterobacter

Serratia marcescens

.

.

-

-

19

-

-

-

Morganella morganii

.

Proteus rettgeri

-

.

.

1

.

2

1

.

2

1

3

7

1

-

-

11

8

.

Proteus mirabilis

spp.

.

1 .

Proteus vulgaris

Providencia

.

.

5

6

12

-

6

1

3 -

1

11

1

2

.

1

-

.

-

Pseudomonas aeruginosa

.

.

.

.

.

.

.

Staphylococcus aureus

-

Enterobactet:faecalis

.

.

.

11

susceptibility rate of flomoxef because of the preference for s t r a i n s w i t h r e d u c e d s u s c e p t i b i l i t y t o t h i r d g e n e r a t i o n cep h a l o s p o r i n s i n t h i s s t u d y . T h i s s e l e c t i o n w a s n e c e s s a r y to have a representative number of strains in each MIC step in o r d e r t o g e t a b e t t e r c o r r e l a t i o n statistic. T h e c o r r e l a t i o n b e t w e e n M I C s a n d z o n e d i a m e t e r s is h i g h e r using the low inoculum of the DIN method than with the h i g h i n o c u l u m o f t h e N C C L S m e t h o d . T h e c o r r e l a t i o n is c o r r e s p o n d i n g l y h i g h w i t h 10 ~tg a n d 3 0 ~tg d i s k s (r = -0.9 o r m o r e ) ; it is l o w e r w i t h 5 ktg d i s k s ( T a b l e 2). T h e s c a t t e r g r a m s f o r all s p e c i e s t e s t e d (Figures 1 a n d 2) show a distinct cluster of strains out of the usual range

.

.

.

.

.

2

-

1 1

.

.

. 4

1

2 6

.

7

.

. -

1

.

.

1

.

--

.

-

.

-

Acinetobacter anitratus

.

1 .

1

-

1

1

3 1

5 -

.

.

1

.

1 -

1 6

6

3 .

1

.

.

.

1 .

1 .

.

2

.

. .

.

.

.

12 .

. .

.

.

5

-

. .

-

20

.

.

.

. 2 0

~

-

a r o u n d t h e r e g r e s s i o n l i n e w i t h M I C s b e t w e e n 0 . 2 5 a n d 0.5 as w e l l as r e l a t i v e l y l a r g e i n h i b i t i o n z o n e s . T h e s e s t r a i n s are S. aureus. T h e e x c l u s i o n o f g r a m - p o s i t i v e c o c c i i n t h e reg r e s s i o n a n a l y s i s a m o u n t s to a c l e a r l y h i g h e r c o r r e l a t i o n (Figures 3 a n d 4, T a b l e 2). F o r t h a t r e a s o n t h e f o l l o w i n g calculated interpretive criteria for the flomoxef inhibition zones are for gram-negative rods only. The main problem of regression analyses in the early phase o f c l i n i c a l i n v e s t i g a t i o n o f a n e w a n t i b i o t i c is t h e s e l e c t i o n of MIC breakpoints. The similarity of pharmacokinetic data o f f l o m o x e f a n d c e f o t i a m [2] s u g g e s t s t h e u s e o f t h e s a m e MIC breakpoints for both antibiotics. An additional prob-

Table 2: Statistical data of regression line analyses with flomoxef.

DIN 5 }.tg

All GNR

243 223

- 0.8892 - 0.9452

x = - 0.33y + 16.47 x = - 0.37y + 17.13

22.6 22.0

19.6 19.3

16.6 16.6

13.5 13.9

10.5 11.2

10 ~tg

All GNR

265 245

- 0.9267 -0.9621

x = - 0.33y + 17.04 x = - 0 . 3 5 y + 17.51

24.4 24.3

21.3 21.5

18.3 18.6

15.3 15.7

12.2 12.9

30~tg

All GNR

285 254

-0.9329 - 0.9635

x = - 0 . 3 4 y + 18.67 x = - 0.38y + 19.88

28.4 28.6

25.5 26.0

22.6 23.4

19.6 20.7

16.7 18.1

All GNR

243 223

- 0.8619 - 0.9231

x = - 0.35y + 16.03 x = - 0.39y + 16.77

20.1 19.9

t 7.2 17.4

14.4 t4.8

11.5 12.2

8.7 9.7

10 ~tg

All GNR

262 242

- 0.8970 - 0.9450

x = - 0.35y + 16.65 x = - 0.38y + 17.32

21.9 21.9

19.0 19.3

16,1 16.6

13.3 14.0

10.4 11.4

30 I.tg

All GNR

281 251

- 0.9047 - 0.9462

x = - 0.36y + 18.19 x = - 0.41y + 19.52

25.5 25.7

22.8 23.2

20.0 20.8

17.2 18.3

14.4 15.9

NCCLS 5~g

*) On-scale values only; x = Log2 MHK + 9; y = zone diameter (rnm).

Infection 19 (1991) Suppl. 5

© MMV Medizin Verlag GmbH Miinchen, Miinchen 1991

S 259

H. Grimm:

Susceptibility

Zone diameter (mm) 4 8 __ 47__ 46 _ 4 5 __ 44_ 43 _ 42_ 41 _ 40_ 39_ 38 37 _ 36_ 35_ 34 _ 33 _ 32 _ 31 _ 30_ 29 _ 28 _ 27 _ 26_ 25 _ 24 _ 23_ 22_ 21 _ 20 _ 19 _ 18

Testing of Flomoxef

n = 2 8 5 ( w i t h o u t e x t r e m e values); r = - 0.9329; x = L o g 2 M I C + 9 = - 0 . 3 4 y + 18.67. 1 2 1

2 3 9 11 2 3

1 t 12 7 9 6 4

2 4 5 4 12 7

1

2

9

2'

3

4 2

6 6 1

_

_ 16 m 15 _ 14_ 13 _ 12 _ 17

11

_

10

_

1 1 2 2 1 3

9 _ 8

_

_ 6 _

7

I

I

I

I

t

I

.03

.06

.12

.25

.5

I 1

I

I 2

t

4

I 8

I

2

1

1

7 1 9

2 1

I

26

I

I

64 128 16 32 M i n i m u m I n h i b i t o r y C o n c e n t r a t i o n (mg/1)

Figure 1 : Scattergram of flomoxef MICs versus 30 lag disk inhibitory zone diameters obtained on Mueller-Hinton agar using the DIN method, all species tested.

lem is the big difference between MIC breakpoints recommended for the same antibiotic in different countries. In the USA these recommendations for cefotiam, mainly based on serum peak concentrations, are < 8 rag/1 for susceptibility and > 32 mg/1 for resistance [7]. The respective values in Germany are < 0.5 mg/1 and > 8 mg/l, mainly based on mean serum concentrations between two doses [8]. For the preliminary interpretation of flomoxef susceptibility testing both these MIC breakpoints are used in this study. Disks loaded with 5 gg of flomoxef are useless because of very small inhibition zones. For the NCCLS method a

S 260

10 gg loading is insufficiem as well. The 30 gg disks are optimal for the susceptibility testing of flomoxef with the NCCLS and DIN methods as well. The tentative criteria for the interpretation of the flomoxef inhibition zones using the method of DIN 58940 [6] and the German tow MIC breakpoints are: resistant-up to 22 mm (corresponding to MICs of 8 mg]l or more), moderately susceptible - 23 to 29 mm (corresponding to MICs from 1 to 4 mg/l), and susceptible - 30 mm or more (corresponding to MICs of 0.5 mg/l or less). The respective values for the NCCLS [5] method using the American high MIC

I n f e c t i o n 19 ( 1 9 9 1 ) S u t ~ l . 5

© M M V M e d i z i n V e r l a g G m b H M i m c h e n , M t i n c h e n 1991

H. G r i m m : Susceptibility Testing o f F l o m o x e f

Zone diameter (mm) 48 47 46 45 _ 44_ 43 _ 42 41 40 _ 39 _ 38 _ 37 36 35 _ 34_ 33 32 31 m 30m 29 _ 28 _ 27 26 25 _ 24_ 23 _ 22 _ 21 20m 19 _ 18 _ 17 16_ 15 _ 14 _ 13 _ 12 _ 11

n = 281 (without extreme values); r = - 0.9047; x = Log2 MIC + 9 = - 0 . 3 6 y + 18.19.

1 4 4 6 2 2 2

2 8 4 4

1

4

1

2 3 3 5 4

3 2

_

10 _ 9 _ 8 _ 7 _ 6 _

1

I

I

I

I

I

1

1

•03

.06

.12

.25

.5

1

I 2

I

1

4

8

I

I

1 5 3 tl

3 1 2

I

I

26

1

16 32 64 128 Minimum Inhibitory Concentration (mg/1)

Figure 2: Scattergram of flomoxef MICs versus 30 ~tg disk inhibitory zone diameters obtained on Mueller-Hinton agar using the NCCLS method, all species tested.

b r e a ~ o i n t s are: r e s i s t a n t - u p to 14 m m ( c o r r e s p o n d i n g t o M I C s o f 3 2 mg/1 o r m o r e ) , m o d e r a t e l y s u s c e p t i b l e - 15 t o 17 m m ( c o r r e s p o n d i n g t o M I C s o f 16 mg/1), a n d s u s c e p t i b l e - 18 m m o r m o r e ( c o r r e s p o n d i n g t o M I C s o f 8 m g / l o r less). Depending on MIC breakpoints customary in different countries, the interpretation of inhibition zones varies a

Infection 19 (1991) Suppl. 5

great deal not only for flomoxef but also for cefofiam and m a n y o t h e r a n t i b i o t i c s . T h i s s p e c i a l s i t u a t i o n is o f g r e a t importance when comparing percentages of resistance in different countries on the basis of disk test results. These results demonstrate the necessity to introduce uniform MIC b r e a k p o i n t s all o v e r t h e w o r l d .

© MMV Medizin Verlag GmbH Mtinchen, Miinchen 1991

S 261

H. Grimm:

Susceptibility Testing of Flomoxef

Zone diameter (mm) 48 __ 47 _ 46 _ 45 __ 44 _ 43 __ 42 __ 41 __ 40 __ 39 __ 38__ 37 __ 36~ 35__ 34~ 33 - 32 __ 31 __ 30__ 29 __ 28 ~ 27 __ 26__ 25 __ 24 __ 23 __ 22 __ 21 __ 20__ 19__ 18 __ 17 __ 16 __ 15 __ 14 __ 13 __ 12 __ 11 __ 10 __ 9 __ 8 __ 7 __ 6 __

n = 254 (without extreme values); r = - 0.9635; x = Log2 MIC + 9 = - 0.38 y + 19.88.

2

3\1

11 2 3

1

2

12

5

6

4

1

2 3

4 12

7.,,,

1 1 3 1 2

2 6

4 2

6

L"~_4_ ......... 4

5 3

1 1

1

7

2

1

1 1

1 1 4 3 5

1 1 2 2

1

2

1\

2 1 26

I

t

I

I

I

•03

.06

.12

.25

1 .5

I

I

1

I 2

I

4

8

I

I

I

1

I

16 32 64 128 M i n i m u m Inhibitory Concentration (mg/l)

Figure 3: Scattergram of flomoxef MtCs versus 30 ~g disk inhibitory zone diameters obtained on Mueller-Hinton agar using the DIN method, gram-negative rods only. References 1.

2.

3.

4.

Neu, H. C., Chin, N. X.: In vitro activity and ~-lactamase stability of a new difluoro oxacephem, 6315-S. Antimicrob. Agents Chemother. 30 (1986) 638-644. Saito, A , K a t o , Y., I s h i k a w a , K., O d a g a k i , E., S h i n o h a r a , M., Fukuhara, I., T o m i z a w a , M., N a k a y a m a , I., Sato, K., Yoshida, R.: 6315-S (Flornoxef). Chemotherapy (Tokyo) 35 (Suppl. 1) (1987) 523-540. G r i m m , H.: In vitro studies with 6315-S, a new oxacephem antibiotic: In vitro activity and regression line analyses. Program and abstracts of the 27th ICAAC, New York, 1987. American Society for Microbiology, Washington 1987, abstract no. 650, p. 2 t0. National C o m m i t t e e for Clinical Laboratory S t a n d a r d s : Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. N C C L S 8 No. 8 (1988) 95-117.

S 262

5.

National C o m m i t t e e for Clinical Laboratory Standards: Performance standards for antimicrobial disk susceptibility tests - Third edition. NCCLS 8 No. 7 (11988) 75-93.

6.

D e u t s c h e s I n s t i t u t fiir N o r m u n g e.V.: Methoden zur Ernpfindlichkeitsprtifung von bakterietlen Kramkheitserregern (aul~er Mykobakterien) gegen Chemotherapeutika. DIN 58940, Teil 3. Agar- Diffusionstest. Beuth Verlag, Berlin 1989, pp. 1-3.

7.

F u c h s , P. C., B a r r y , A. L., Jones, R. N., T h o r n s b e r r y , C.: Cefotiam susceptibility testing criteria. J. Clin. Microbiol. 22 (1985) 1045-1047.

8.

D e u t s c h e s I n s t i t u t ffir N o r m u n g e.V.: Methoden zur Empfindlichkeitspriifung von bakteriellen Krankheitserregern (auger Mykobakterich) gegen Chemotherapeutika. DIN 58940, Teil 4, Beiblatt 1. Liste der Grenzwerte und Tagesdosierungen von Chemotherapeutika sowie Bewertung derminimalen Hemmkonzentration (MHK). Beuth-Verlag, Berlin 1989, pp. t - 7 .

Infection 19 (1991) Suppl. 5

© M M V Medizin Veflag G m b H Mtinchen, Mtinchen 1991

tI. Grimm:

Zone diameter (mm) 48 46 45 __ 44__ 43 __ 42 __ 41 _ _ 40__ 39__ 38 __ 37 __ 36~ 35 _ 34 _ 33_ 32_ 31 _ 30_ 29_ 28 _ 27 _ 26_ 25 _ 24 _ 23 _ 22 _ 21 _ 20 _ 19 _ _

17

_ _ _ _ _

16 15 14 13 12 11

Testing of Flomoxef

n = 251 ( w i t h o u t e x t r e m e values); r = - 0.9462; x = L o g 2 M I C + 9 = - 0.41 y + 19.52.

47

18

Susceptibility

1

3 7 3 1

, 1 3 6 5 4 5 3

1 1

1 2 7 7

4 9 4 2

2

5 3

1

1

5 7 3 ~ 3

4 3 3

1

1 1

2~7

2

1

3

2 2

4 4 2

2

k

3

3 2

_ __

__ 9 __

10

8

3

__

7 __ 6 __

1

I

I

I

I

I

I

.03

.06

.12

.25

.5

I

I 1

I 2

4

I

I 8

I

I

2

26

I

I

16 32 64 128 M i n i m u m I n h i b i t o r y C o n c e n t r a t i o n (mg/1)

Figure 4: Scattergram of flomoxef MICs versus 30 ~g disk inhibitory zone diameters obtained on Mueller-Hinton agar using the NCCLS method, gram-negative rods only.

I n f e c t i o n 19 ( 1 9 9 1 ) Suppl. 5

© M M V M e d i z i n V e r l a g G m b H M f i n c h e n , M i J n c h e n 1991

S 263