desacetylcefotaxime with two aminoglycosides against gram-negative pathogens: An example of interactive synergy

DIAGN MICROBIOLINFECTDIS 1989;12:51-55

51

Activity of Cefotaxime/ Desacetylcefotaxime with Two Aminoglycosides Against GramNegative Pathogens: An Example of Interactive Synergy Stephen G. Jenkins

The susceptibility patterns of clinical Gram-negative isolates were determined to cefotaxime (CTX) and desacetylcefotaxime (dCTX) alone and in combination with gentamicin (GENT) or tobramycin (TOB) by an agar dilution technique. A constant ratio of 1:I (CTX to dCTX) was tested throughout the study. Isolates were challenged with subinhibitory levels of TOB or GENT in combination with clinically achievable levels of CTX, dCTX and CTX/dCTX to examine the interactions of the agents. Results of this study demonstrate that CTX!dCTX interacts synergistically with aminoglycosides against many Gram-negative pathogens. Synergy (defined as a fourfold or greater decrease in minimum inhibitory concentration (MIC) when CTX!dCTX was compared to CTX/dCTX/TOB) was demonstrable for 55% of isolates tested. Similarly, 45% were

synergistically inhibited by CTX/dCTX/GENT. Additivism (a 2-fold decrease in MIC with the same comparisons) was evident for an additional 18 isolates for CTX/dCTX/TOB and 19 with CTX/dCTX/GENT. When data for Pseudomonas spp. were excluded from the analysis, synergy or additivism was evident with CTX/dCTX/ TOB for 88% of the organisms tested and 72% with CTX/d CTX/GENT. Synergistic synergy for CTX!dCTX/TOB (an 8to greater than 16-fold decrease in MIC for CTX) was demonstrable for 35 and 32 of 82 nonspeudomonal isolates respectively with the TOB and GENT combinations. Ninety nine percent of the nonspeudomonal isolates were inhibited by less than 4 i~g/ml of CTX, 4 I~g/ml of dCTX and O. 12 p,g/ml of TOB, or 0.25 t~g/ml of GENT, respectively.

INTRODUCTION

noglycoside against many different bacterial species (Schrinner et al., 1981). CTX has also been shown to interact synergistically with its major metabolite, desacetylcefotaxime (dCTX) against many clinical aerobic and anaerobic bacterial isolates (Jones 1984; Neu 1982). Since nearly equal concentrations of CTX and dCTX are present in cerebrospinal fluid, bile, urine, and peritoneal fluid 1 hr after intravenous administration (Novick 1982; Ohkura et al., 1982), this study examined the interactions of CTX and dCTX alone and in combination with gentamicin (GENT) or tobramycin (TOB) against common Gramnegative bacterial pathogens.

Several potential benefits of the clinical use of antibiotic combinations have been advanced. These include expansion of the spectrum of either agent alone allowing treatment of polymicrobic infections, prevention of emergence of antibiotic resistant organisms, reducing the potential for toxicity with aminoglycosides and other agents, and with demonstrated in vitro synergistic or additive effects, more effective treatment of bacteremia in neutropenic patients (Klastersky et al., 1973, 1980). Cefotaxime (CTX) has been reported to act synergistically with an amiFrom the Baptist Medical Center, Service of Pathology,Jacksonville, Florida. Address reprint requests to: Stephen G. Jenkins, Ph.D., Baptist Medical Center, Service of Pathology,800 Prudential Drive, Jacksonville, FL 32207. Received September 17, 1988; revised and accepted September 30, 1988. © 1989ElsevierScience PublishingCo., Inc. 655 Avenue of the Americas, New York, NY 10010 0732-8893/89/$3.50

MATERIALS A N D M E T H O D S Bacterial Strains A total of 104 clinical isolates were tested in the TOB combination studies and 113 isolates in the GENT analyses. In addition, Pseudomonas aeruginosa ATCC

52

27853, Escherichia coli ATCC 25922, Enterococcus (Streptococcus) faecalis ATCC 29212, Staphylococcus aureus ATCC 29213, and E. coli ATCC 35218 were included in each run as controls. The number of isolates of each species in the TOB and GENT studies were, respectively: Morganella morganii (8,8); Acinetobacter calcoaceticus var. anitratus (9,8); Xanthomonas ( Pseudomonas ) maltophilia (8,10); Pseudomonas cepacia (6,5); Serratia marcescens (8,8); Providencia stuartii (6,6); Providencia alcalifaciens (2,2); Proteus vulgaris (8,4); P aeruginosa (8,8); Citrobacter freundii (9,9); Citrobacter diversus (8,8); Enterobacter aerogenes (8,8); Enterobacter cloacae (8,9); Klebsiella pneumoniae (8,8); Proteus mirabilis (0,3); and E. coli (0,9).

Antimicrobial Agents CTX and dCTX were provided by Hoechst-Roussel Pharmaceuticals (Somerville, NJ), TOB by Eli Lilly and Co. (Indianapolis, IN), and GENT by Schering Corp. (Kenilworth, NJ) as laboratory standard powders.

Medium Mueller-Hinton agar (BBL Microbiology Systems, (Cockeysville, MD) was used in all tests.

MIC Determinations All tests were run using an agar dilution technique following the guidelines of the National Committee for Clinical Laboratory Standards (NCCLS) (1985). Individual MIC results used in the data analysis represent the mode values of three separate determinations. MICs were first determined individually for CTX, dCTX, GENT, and TOB, and for CTX in combination with dCTX (1:1). CTX and dCTX were then tested in combination with subinhibitory (0.12, 0.25, and 0.5 ixg/ml) concentrations of GENT and TOB. In the final assays CTX and dCTX were tested in combination (1:1 ratio) with subinhibitory levels of GENT and TOB.

Data Analysis Synergy was defined as a four-fold or greater decrease in MIC when CTX, dCTX, or the combination of the two was combined with a subinhibitory concentration of aminoglycoside as compared to values for the agents alone. Additivism was defined as a two-fold drop in MIC in similar comparisons; indifference as no change in MIC values, and antagonism as a four-fold or greater increase in MIC in the combination over the individual assay results.

S.G. Jenkins

RESULTS CTX, dCTX, TOB, and GENT all showed significant activity against most of the Gram-negative isolates tested, with the exception of Pseudomonas spp. (Table 1). Of the 86 members in the Enterobacteriaceae tested, 85 were susceptible to 8 txg/ml or less of CTX as were 69 to 8 txg/ml or less of dCTX. dCTX was more active than CTX against five of the six P. cepacia isolates tested, the only organism more susceptible to the metabolite than to the parent compound. CTX acted synergistically with dCTX against 24 of 117 isolates tested and demonstrated additivism with an additional 24 (Table 2). Partial synergy was not assessed. Antagonism between CTX and dCTX was demonstrable for seven strains (three strains of

Morganella morganii), but MICs were not elevated above the CTX breakpoint for susceptibility (NCCLS, 1985). Synergy between CTX and TOB was evident with 32% of the isolates (Table 3) tested as compared to 52% for CTX and GENT. Similarly, synergy was demonstrable with dCTX and GENT for 46% of the isolates versus 41% for the TOB combination. When the three agents were tested together, however, synergy was demonstrable versus 55% of the isolates with CTX/dCTX/TOB, whereas 45% were synergistically inhibited by the GENT combination. When data for the Enterobacteriaceae were examined separately, synergy or additivism was demonstrable for 93% of the isolates with the TOB combination and for 77% of the isolates with the GENT combination. Although antagonism was seen with seven isolates using the TOB combination, the only organism for which the antagonism resulted in an increase in MIC above the CTX breakpoint for susceptibility was P. cepacia. Likewise, antagonism was evident for 11 isolates with the CTX/dCTX/GENT combination, but this resulted in resistance only for P. cepacia and X.

maltophilia. "Synergistic synergy," defined as a 16-fold or greater decrease in MIC for CTX alone as compared to the three agent combination, was demonstrable for 23% of all isolates with TOB and 24% of isolates with GENT. Interactive synergy, defined as an 8fold decrease in MIC in the same comparisons, was evident for an additional 14% of isolates with TOB and 11% with GENT (Table 4). Concentrations of CTX/dCTX required to inhibit most isolates of Enterobacteriaceae were in the nanogram/ml range when combined with subinhibitory concentrations of aminoglycosides (Table 5).

DISCUSSION The high potency of dCTX against many Gram-negative bacteria is well recognized (Schrinner et al., 1984; Wise et al., 1980). In part, this is due to the

Cefotaxime/Metabolite/AminoglycosideSynergy

TABLE 1.

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Activity of Cefotaxime (CTX), Desacetylcefotaxime (dCTX), Tobramycin, and Gentamicin against Gram-negative Bacteria MIC Range (p~g/ml)

Organism (no.)

CTAXa

DESb

Morganella morganii (8) Acinetobacter antitratus (9) Xanthomonas maltophilia (10) Pseudomonas cepacia (6) Serratia marcescens (8) Providencia spp. (8) Proteus vulgaris (8) Pseudomonas aeruginosa (8) Citrobacter freundii (9) Citrobacter diversus (8) Enterobacter aerogenes (8) Enterobacter cloacae (9) Klebsiella pneumoniae (8) Proteus mirabilis (3) Escherichia coli (9)

.015-. 25 4-64 16-256 4-32 0.25-2 .0078-4 .03-5 16->256 .06-64 .06-1

TOBc

2-32 4-> 256 16->256 4-8 2-16 .06-16 .06-8 >256 1->256 .5-32

.12-8

.25-2 .5-4 2->256 128-256 2->256 .25-4 .5-1 0.5-4 0.5-2 .25-1

1-128

.12-8 .03-.06 .015-.25 .12-.5

.5-1

4-32 .12-.25 .5-16 .25-1

GENTd .25-8 .25-8 1-256 1-256 0.25-128 .5-16 .25 1-16 .25-1 .25-.5 .25-1

.5 .25-.5 NT NT

.25-1 .5-1 .25-2 .5-1

NT, not tested. a, CTX. t,, dCTX. ", tobramycin. '~, gentamicin.

increased stability of the metabolite to beta-lactamases over that of the parent c o m p o u n d , CTX (Labia and Marand, 1984). The in vitro interaction of these two compounds is reinforced by data generated from serum bactericidal tests (Reller, 1984). Pharmacokinetic data indicate that concentrations of dCTX in

TABLE 2.

Cefotaxime/Desacetylcefotaxime Interaction Against Gram-negative Bacteria

Organism Morganella morganii Acinetobacter antitratus Xanthomonas maltophilia Pseudomonas aeruginosa Pseudomonas cepacia Serratia marcescens Providencia alcalifaciens Providencia stuartii Proteus vulgaris Klebsiella pneumoniae Citrobacter freundii Citrobacter diversus Enterobacter aerogenes Enterobacter cloacae Proteus mirabilis Escherichia coli

Totals

serum approximate those of CTX 2 hr following i.v. infusion and generally surpass those of CTX thereafter (Quintiliani et al., 1984). Since CTX acts synergistically with aminoglycosides (Glew and Paruk, 1984) as well as with its metabolite against Gramnegative isolates including multiply-resistant m e m -

No.

Synergy

Additivism

Indifference

Antagonism

8 9

4 1

0 6

1 2

3a 0

10

0

2

8

0

8 6 8 2 6 8 8 9 8 8 8 3 8

0 3 1 1 0 5 0 1 0 0 1 0 7

0 2 4 0 3 0 1 0 1 3 1 0 1

8 1 3 1 3 3 7 8 6 5 5 1 0

0 0 0 0 0 0 0 0 1a 0 Ia 2~ 0

117

24

24

62

7a

~MICs not elevated above cefotaxime breakpoint for susceptibility.

54

S.G. Jenkins

TABLE 3.

Interactions of Cefotaxime (CTX) Desacetylcefotaxime (dCTX) and Tobramycin Percent

Agents

Synergy

Additivism

Indifference

Antagonism

CTX/Tobramycin CTX/Gentamicin dCTX/Tobramycin dCTX/Gentamicin CTX/dCTX CTX/dCTX/Tobramycin CTX/dCTX/Gentamicin

32 52 41 46 20 55 45

26 21 22 13 20 17 17

38 24 31 35 54 21 27

4 3 6 6 6 7'~ 11b

"Pseudomonascepaciawas the onlyorganismfor whichthe demonstrableantagonismraised the MIC asbove the cefotaximebreakpoint for susceptibility.

bXanthomonasmaltophiliaand Pseudomascepaciawere the only organisms for which the demonstrable antagonism raised the MIC above the CTX breakpoint for susceptibility. bets of the family Enterobacteriaceae, this study investigated the interaction of the three antibiotics against such organisms. As expected, all three agents demonstrated potent antimicrobial activity on an individual basis. The more minimal activity of CTX against the pseudomonal isolates was due, in part, to intentional selection of resistant isolates for the interaction studies. In addition CTX acted synergistically with dCTX and with the aminoglycosides studied against many of the bacteria tested. The fact that dCTX, at concentrations well below those clinically achievable, demonstrated interactive activity with aminoglycosides was of particular interest and suggested that a complex interaction between CTX, its metabolite, and aminoglycosides may exist and have clinical importance. The synergistic activity of CTX, dCTX, and aminoglycosides was particularly evident with members of the Enterobacteriaceae. Further studies with CTX susceptible strains of P. aeruginosa would help to clarify whether such interactions are demonstrable with pseudomonal isolates as well. The activity of dCTX against P. cepacia isolates appeared to be obviated in most cases when CTX and an aminoglyTABLE 4.

coside were concomitantly tested against such isolates. This may be of import in the possible therapy of cystic fibrosis patients in whom P. cepacia represents a significant pathogen in pulmonary exacerbations of their disease (Isles et al., 1982). The terms "synergistic synergy" and "interactive synergy" attempt to describe the enhanced affects of the metabolite over and above those normally demonstrated with CTX and an aminoglycoside. The subinhibitory levels of aminoglycoside required for such interactions may imply that doses of TOB or GENT below those usually used might be utilized in some settings in turn decreasing the likelihood of nephro- and/or ototoxicity during such therapy. The

extremely low levels of CTX/dCTX combined with an aminoglycoside required for inhibition of most enteric bacilli would support extended CTX dosing schedules for infections caused by such bacteria. In summary, CTX and dCTX alone and in combination demonstrate synergistic in vitro activity with aminoglycosides against many Gram-negative bacteria, particularly members of the Enterobacteriaceae. The clinical significance of such "interactive synergy" is yet to be determined but certainly warrants further investigation.

Interaction of Cefotaxime/Desacetylcefotaxime with Tobramycin or Gentamicin (Gram-negative Clinical Isolates) % of Isolates Interaction

Tobramycin

Gentamicin

Synergistic Synergy (>16-fold decrease in MIC) Interactive synergy (8-fold decrease in MIC) Synergy (4-fold decrease in MIC) Additivism (2-fold decrease in MIC) Indifference (no change in MIC) Antagonism (>4-fold increase in MIC)

23 14 18 17 21 7

24 11 10 17 27 11

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Cefotaxime/Metabolite/Aminoglycoside S y n e r g y

TABLE 5.

Interaction of C e f o t a x i m e / D e s a c e t y l c e f o t a x i m e / A m i n o g l y c o s i d e s against Enterobacteriaceae MIC Range (all values in ~g/ml)

Organism

Morganella morganii Providencia alcalifaciens Providencia stuartii Serratia marcescens Proteus vulgaris Proteus mirabilis Citrobacter freu ndii Citrobacter diversus Enterobacter aerogenes Enterobacter cloacae Klebsiella pneumoniae Escherichia coli

CTAX/DES/TOB

CTAX/DES/GENT

~.0009-.06 ~.0009-.03 .015-2 .03-1 ~.0009M ND .03-32 .0039-. 12 .03-2 .015-.5 .0078-.015 ND

~.0009-.06 .015 .015-1 .015-1 .0019-.06 .0019-.015 ~. 0009-. 25 .015-. 125 ~.0009M ~.0009-.25 ~.0009-.25 .015-.25

aAll values represent concentration of cefotaxime (CTX) alone; desacetylcefotaxime (dCTX) present at equal concentration and gentamicin (GENT) or tobramycin (TOB) present at subinhibitory level. ND, no data.

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