desacetylcefotaxime

DIAGN MICROBIOLINFECTDIS 1989;12:81-85

81

Evaluation of Disk Susceptibility Testing of Cefotaxime/ Desacetylcefotaxime Peter C. Fuchs, Ronald N. Jones, and Arthur L. Barry

The susceptibility of 453 clinical bacterial isolates was determined by broth microdilution and disk diffusion methods to cefotaxime (CTX), desacetylcefotaxime (dCTX), and various combinations of CTX plus dCTX. With a 1:1 ratio of CTX/ dCTX, 97% of the minimal inhibitory concentrations were within one doubling dilution of those for CTX alone. With the

disk diffusion test, the 30/30 tzg disk of CTX/dCTX produced zone diameters very similar to those of the 30 ttg CTX disk, averaging 0.4 mm larger than the latter. Regression analysis demonstrated that the 30 tzg CTX disk was equally predictive for CTX and a 1:1 ratio of CTX/dCTX.

INTRODUCTION

with various combinations of CTX plus dCTX in an attempt to assess the merits of such testing compared to CTX alone.

Desacetylcefotaxime (dCTX) is the initial metabolite of cefotaxime (CTX) in the liver (Coombes, 1982). Unlike the metabolites of many antimicrobial agents, dCTX has been demonstrated to have significant in vitro antimicrobial activity of its own (Jones et al., 1982; Neu, 1982). Although this activity was somewhat less than that of CTX, it was found to act synergistically with CTX against a number of bacteria (Jones et al., 1982; Jones, 1984; Neu, 1982). This antimicrobial activity of DES together with its reported synergistic interaction with cefotaxime, and the longer half-life of DES (Doluisio, 1982) have prompted the suggestion that the dosing frequency of CTX might be reduced to q 8 hr or q 12 hr for more susceptible bacteria (Chin and Neu, 1984). Because of the interaction between these two compounds, and because the combination is being considered as a combination drug, we have evaluated the disk susceptibility test From the Department of Pathology, St. Vincent Hospital and Medical Center (P.C.F.), Portland, Oregon, Clinical Microbiology Institute (R.N.J., A.L.B.), Tualatin, Oregon. Address reprint requests to: Dr. Peter C. Fuchs, Department of Pathology, St. VincentHospital and Medical Center, Portland, OR 97225. ReceivedJune 26, 1988; accepted August 4, 1988. © 1989ElsevierSciencePublishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010 0732-8893/89/$3.50

MATERIALS AND METHODS CTX and dCTX were provided as standard powder by Hoechst-Roussel Pharmaceuticals, Inc., Somerville, NJ. CTX 30 p,g disks were supplied by Difco Laboratories, Detroit, MI. Disks containing 30 p,g of dCTX and disks with the following CTX/dCTX combinations were prepared in house: 15/15 p,g, 20/ 10 ~g, 25/5 ~g, and 30/30 p,g. A total of 453 aerobic clinical bacterial isolates (listed in Table 1) were tested simultaneously by both broth microdilution and disk diffusion methods. The broth microdilution procedure was performed as outlined by the National Committee for Clinical Laboratory Standards (NCCLS, 1985). CTX was diluted in cationsupplemented Mueller-Hinton broth to achieve a series of twofold concentrations ranging from 0.06 to 32 p~g/ml. A 1:1 ratio of CTX:dCTX was similarly prepared and diluted to achieve final concentrations of each com pound ranging from 0.06 to 32 p,g/ml. The 1:1 ratio was selected because this approximates the in vivo ratio of these two compounds in serum several hours after CTX administration (Doluisio, 1982) and the ratios outside the vascular compartment, e.g., in CSF in children with

82

P . C . F u c h s e t al.

TABLE 1.

Comparison of Cefotaxime (CTX) versus Cefotaxime/ Desacetylcefotaxime (dCTX) MICs and Zone Diameters and 30/30-p,g disks, Respectively

Organism

Citrobacter diversus Citrobacter freundii Enterobacter aerogenes Enterobacter agglomerans Enterobacter cloacae Escherichia coli Klebsiella oxytoca Klebsiella pneumoniae Morganella morganii Proteus mirabilis Proteus vulgaris Providencia rettgeri Providencia stuartii Serratia marcescens Total Enterobacteriaceae

No. 10 10 19 10 20 24 4 21 10

M e a n of Differences Between MIC a -

0.40 0.40 0.32 0.30 0.40 0.13 0 - 0.14 - 0.20

25 10 10 20 25 218

+ -

w i t h 30-~zg

M e a n of Differences Between Zone Diameters b + + + + + -

0.40 0.10 0.37 0.10 0.20 0.75 2.00 0.48 0.90

0

+ 1.00

0.40 0.20 0.35 0.40

+ + + +

0.80 1.10 1.35 0.68

- 0.22

+ 0.42

-

1.10 0.55 2.00 0.04 1.00 0 -0.17

+ 2.60 + 0.65 + 2.00 - 0.18 + 4.00 + 0.20 +0.17

5

0

0

Acinetobacter anitratus Branhamella catarrhalis Pseudomonas acidovorans Pseudomonas aeruginosa Pseudomonas cepacia Pseudomonas fluorescens Pseudomonas maltophilia Pseudomonas putida Pseudomonas stutzeri

10 20 3 49 4 5 6 5

- 0.60

0

Total N o n e n t e r i c s

107

- 0.36

+ 0.50

25

- 0.48

25 10 16 9 4 2

-

0.36 0.10 0.69 0.22 0.50 0

+ 0.24 + 0.72 0 + 0.75 + 0.44 + 0.25 - 1.50

3

0

+ 1.00

4

- 0.25

+ 1.50

98

- 0.39

+ 0.48

30

- 0.03

0

453

- 0.28

+ 0.43

Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus

aureus ( - )c aureus ( + )c aureus (MR) c epidermidis epidermidis (MR) c haemolyticus hominis saprophyticus warneri

Total s t a p h y l o c o c c i Total e n t e r o c o c c i Total--all organisms

aMean twofold dilutional difference in MICs of CTX/dCTX compared to CTX alone. bMean zone diameter difference (in ram) with disks containing 30/30 tag CTX/dCTX compared to those with 30 tag of CTX alone. c( _ ) = [3-1actamase-negative; ( + ) = [3-1actamase-positive; (MR) = methicillin-resistant. m e n i n g i t i s ( T r a n g , 1985). E a c h i n o c u l u m c o n t a i n e d approximately 5 x 105 o r g a n i s m s / m l , and the minimum inhibitory concentration (MIC) was interpreted as the lowest concentration completely i n h i b i t i n g v i s i b l e g r o w t h a f t e r 1 6 - 1 8 h r a t 35°C. T h e disk diffusion susceptibility testing procedure was t h a t o u t l i n e d b y t h e N C C L S ( N C C L S , 1984). M u e l -

ler-Hinton agar plates were swab-inoculated with organism suspensions adjusted to match the turbidity of a 0.5 BaSO4 standard. After aerobic incubations at 35°C for 16-18 hr, the inhibitory zone diameters around each disk were measured with calipers. Regression analysis was performed by the method of least squares.

Evaluation of dCTX Disk Tests

83

RESULTS A N D DISCUSSION The MICs of CTX a n d CTX/dCTX w e r e v e r y similar (Table 1). For 97% of isolates tested, the MICs of the two w e r e within o n e twofold concentration of each other. Three species (Pseudomonas acidovorans, Pseudomonas cepacia, a n d Acinetobacter anitratus) w e r e inhibited b y CTX/dCTX at />1 twofold concentration lower t h a n CTX alone. There w e r e 13 strains for w h i c h the CTX/dCTX MICs w e r e fourfold lower t h a n those of CTX alone. H o w e v e r , for five of these, the MIC of DES alone w a s within one twofold concentration of that of the combination. Therefore, at the 1:1 ratio tested, only eight instances of probable s y n e r g y w e r e observed. The isolates inhibited b y a p p a r e n t synergistic activity were: C. freundii (1), E. aerogenes (1), M. morganii (1), P. stuartii (2), A. anitratus (1), P. stutzeri (1), and P. acidovorans (1). Of these eight isolates, two had a change in susceptibility categories, f r o m m o d e r ately susceptible to susceptible. The m e a n reduction in cefotaxime MIC for all o r g a n i s m s tested b y the addition of equal a m o u n t s of dCTX w a s 0.28 twofold dilutions. For t w o strains of Proteus vulgaris, the MIC of CTX/dCTX w a s eightfold greater t h a n that of CTX alone. O n e of these w a s susceptible to CTX alone a n d resistant to the c o m b i n a t i o n . Similar antagon i s m with P. vulgaris has b e e n r e p o r t e d before

TABLE 2.

MIC CTX

CTX/dCTX

(Neu, 1982). Previous reports h a v e indicated CTX/ dCTX a n t a g o n i s m with s o m e strains of Morganella morganii (Jones et al., 1982; Neu, 1982), but this was not observed with any of the ten strains tested here. Such a n t a g o n i s m w o u l d a p p e a r to be infrequent, a n d to date has b e e n r e p o r t e d only for a f e w Enterobacteriaceae b e l o n g i n g to the Proteus a n d Morganella genera. Of the four CTX/dCTX combination disks tested, the 30/30 p,g disks yielded the fewest interpretive errors w h e n c o m p a r e d to the MICs of CTX/dCTX or CTX alone (Table 2). Figure 1 p r e s e n t s scatterg r a m s for: (A) CTX MICs v e r s u s CTX inhibitory z o n e diameters, (B) CTX/dCTX MICs v e r s u s CTX zone diameters, a n d (C) CTX/dCTX M I C s v e r s u s z o n e d i a m e t e r s with 30/30 ~g disks. The correlation coefficients of the latter t w o pairings are comparable to each other and s o m e w h a t better than for CTX MICs versus CTX disks (Table 2). I n t e r p r e t i v e error rates w e r e calculated o n the basis of N C C L S r e c o m m e n d e d b r e a k p o i n t s for CTX (NCCLS, 1984; NCCLS, 1985). For CTX/dCTX MIC categories, error rates with 30-p,g CTX disks or the 30/30-~g CTX/ dCTX disks w e r e identical. Thus, b o t h disks appear to be equally predictive of susceptibility to CTX/dCTX. C o n s e q u e n t l y , there w o u l d a p p e a r to be no merit in routinely testing susceptibility of clinical aerobic isolates to CTX/dCTX with a separate disk.

Correlation Coefficients a n d Error Rates for Cefotaxime (CTX) a n d CTX/dCTX Disk Tests v e r s u s MICs

Disk Diffusion Test CTX 30 ~g CTX/dCTX 30/30 ~g CTX/dCTX 25/5 ~g CTX/dCTX 20/10 ~g CTX/dCTX 15/15 p-g CTX 30 p,g CTX/dCTX 30/30 ~g CTX/dCTX 25/5 p,g CTX/dCTX 20/10 ~g CTX/dCTX 15/15 p,g

Correlation Coefficient

Very Major Errors ~ (%)

Major Errors ~ (%)

Minor Errors b (%)

Agreements (%)

0.82 0.81

0.2 0.2

0.2 0.2

7.3 7.1

92.3 92.5

0.84 0.87

0.2 0

0.4 0.7

8.4 14.8

90.9 84.5

0.85

0

0.7

19.0

80.3

0.85 0.86

0.4 0.4

0.2 0.4

9.3 8.6

90.1 90.7

0.87 0.90

0.4 0.2

0.4 0.7

10.4 17.3

88.7 81.8

0.90

0.2

0.9

21.2

77.6

"For the disk diffusion test the zone diameter breakpoints recommended for th e 30-~g CTX disk (NCCLS, 1984) were utilized for each of the combination disks. bVery major error = susceptible zone diameter and resistant MIC; major error = resistant zone diameter and susceptible MIC; minor error = moderately susceptible zone diameter and susceptible or resistant MIC, vice versa. CFor this table, the CTX/dCTX MICs were based on the CTX portion only.

84

P.C. Fuchs et al.

32

11

16

2

5 2

4 1 1 1

1 3

11

1

71114711

80

1

1 1 1 4 2

1

40 20

1

115832

1 6 1 4 4 4

0 25

I

t

53

3 6 3 3 3 2 2 1 1

013

2 2 4 4 0 0 8 4 1 3 1

~0~

1 3

>32

32

32

2

16

2

3

1

I

I

1 1 4 2 23

7' iS 15 ~I

1

51

8 1 1 41 32

I

1 31013511

80

2 3 2 3 2 3

4.0

1

1

12

2.0

2

22

21

37,30

1.0

12

1

2 2 2 4 8 7 , 0 4 3 1 1

0.3

11

4

0.2'3

1

3 4 1 4 1 0 1

2 4 1 3 3 4

013

242

3 3 3 4 9 3 2 1

_,0(~

1

22811~17,17,11121232

32

2

18

2

3

1

1

232

1

1

42 2

3781441 1 1 0 2 3

40

1

I

2.0

1

1

1

2

1 1 1

10

1

1

1

2 3 3 1 0 3 2 1

2

2

1

2 4 1 0 0 7 2 1 2 1

3

213

3 4 3 4 1 4

3 4 3 2 3 4 3 1 4

013

1 6 2 4 6 8 4 2

,OO6

I

271115~15141t12~ ~0

1~

20

Zone

Ratio of CTX:DES MICs

1

21

0 25

TABLE 3.

1

12

80

1:2 1:4 1:8 1:16

1

1 1 1 3 2 4 4 3 4 2 1 1

05

1:1

T I

401~

10

:z

2

32

2~

Diameter

The evidence from this study suggests the activity of CTX and DES are additive as follows: 1) For 96.0% of the isolates with on-scale MICs, the MIC of the combination drug (MIC expressed as concentration of CTX in the presence of equal amounts of DES) was the same or within 1 dilution of the lower individual MIC. When the MIC of CTX was the same as that of dCTX, the MIC of the combination was one-half that of each alonem e.g., if MIC of CTX and dCTX were 4.0 ~g/ml each, the MIC of the 1:1 combination was 2.0:2.0 p~g/ml. As the relative activity of dCTX (compared to CTX) decreased, the more closely did the MICs of the combination match those of cefotaxime alone (Table 4). For example, when the MIC of DES was 1/8 that of CTX, the MIC of CTX/dCTX equalled that of CTX alone 83% of the time. 2) For the 14 strains with identical on-scale MICs with both CTX and dCTX, the CTX 30-p,g disk zone diameters were essentially identical to those of the CTX/dCTX 15/ 15-p,g disks (Table 3). As the MIC of dCTX increased relative to the CTX MIC, the additive influence of dCTX decreased--such that when dCTX MICs were eightfold higher than CTX MICs, the CTX disk and CTX/dCTX 30/30-}zg disks gave identical zone diameters (Table 3). Because the proportion of organisms with equal CTX and dCTX

:~

~35

FIGURE 1. Scattergrams s h o w i n g the MIC/zone diameter correlation and regression for: (A) CTX MICs versus CTX (30 ~.g) z o n e diameters, (B) CTX MICs versus CTX/ dCTX (30/30 ~g) z o n e diameter, a n d (C) CTX/dCTX MICs and CTX/dCTX (30/30 ~g) z o n e diameters. Horizontal lines represent the N C C L S r e c o m m e n d e d MIC breakpoints for CTX (2), and the vertical lines represent the N C C L S reco m m e n d e d zone diameter breakpoints for 30-~,g CTX disks

(8).

(ram)

Effect of Relative CTX:dCTX MICs on Inhibitory Zone Diameters Around Different CTX/dCTX Disks Zone Diameters in m m

No. ~

CTX 30

14 17 56 54 12

26.1 27.4 28.6 28.3 27.6

CTX/dCTX-15/15 26.3 26.6 26.7 25.9 26.0

CTX/dCTX-30/30

( + 0.2) b

28.3 ( + 2.2) b

(-0.8) ( - 1.9) ( - 2.4) ( - 1.6)

28.5 29.3 28.6 27.4

(+1.1) ( + 0.7) ( + 0.3) (-0.2)

aOnly those strains for which all values were on scale are included. bValues in parentheses indicate the magnitude of differences between the mean zone diameter for CTX-30 and the mean zone diameter for the indicated combination disk.

Evaluation of dCTX Disk Tests

TABLE 4.

85

MICs is v e r y small, the overall correlation is m u c h better with the 30/30-p,g CTX/dCTX disks.

Effect of Relative Potency of dCTX C o m p a r e d to CTX on the MICs of the C o m b i n e d Drug C o m p a r e d to CTX Alone

CONCLUSION Twofold Concentration Changes in MIC of CTX:dCTX compared to CTX MIC Ratio of CTX:dCTX

No. a

1:0.5 1:1 1:2 1:4 1:8 1:16 1:32

4 14 17 56 54 12 5

+1

1 1

0 1 3 19 45 9 5

- 1

- 2

2 10 14 37 8 1

2 3

1

The data from this s t u d y indicate that the activities of CTX and dCTX are additive. This is readily a p p a r e n t w h e n the activities of the two individual drugs are equal, or nearly so. H o w e v e r , for the majority (93%) of the isolates in this s t u d y the activity of dCTX was 41/4 that of CTX. Consequently, the addition of equal quantities of dCTX a d d e d proportionately m u c h less additional antimicrobial activity against these isolates. The net aggregate effect of dCTX in the combination against all isolates in this study was a reduction of the CTX MIC by 0.28 twofold concentrations.

~Only those strains for which all values were on scale are included.

REFERENCES Chin N, Neu HC (1984) Cefotaxime and desacetylcefotaxime: an example of advantageous antimicrobial metabolism. Diagn MicrobioI Infect Dis 2(Suppl):215. Coombes JD (1982) Metabolism of cefotaxime in animals and humans. Rev Infect Dis 4(Suppl):S325. Doluisio JT (1982) Clinical pharmacokinetics of cefotaxime in patients with normal and reduced renal function. Rev Infect Dis 4(Suppl):S333. Jones RN (1984) The activity of cefotaxime and desacetylcefotaxime against Bacteroides species compared to 7methoxy cephems and other anti-anaerobe drugs. J Antimicrob Chemother 14(Suppl B):39. Jones RN, Barry AL, Thornsberry C (1982) Antimicrobial activity of desacetylcefotaxime alone and in combination with cefotaxime: Evidence of synergy. Rev Infect Dis 4(Suppl):S366. National Committee for Clinical Laboratory Standards (1984)

Performance standards for antimicrobial disk susceptibility tests. Approved Standard M2-A3. National Committee for Clinical Laboratory Standards, Villanova, PA. National Committee for Clinical Laboratory Standards (1985) Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved Standard M7-A. National Committee for Clinical Laboratory Standards, Villanova, PA. Neu HC (1982) Antibacterial activity of desacetylcefotaxime alone and in combination with cefotaxime. Rev Infect Dis 4(Suppl):S374. Trang JM, Jacobs RF, Kearns GL, Brown AL, Wells TG, Underwood FL, Kluza RD (1985) Cefotaxime and desacetylcefotaxime pharmacokinetics in infants and children with meningitis. Antimicrob Agents Chemother 28:791.