ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Nov. 1990, p. 2256-2259
0066-4804/90/112256-04$02.00/0 Copyright C) 1990, American Society for Microbiology
Vol. 34, No. 11
In Vitro Activities of Cefoperazone and Sulbactam Singly and in Combination against Cefoperazone-Resistant Members of the Family Enterobacteriaceae and Nonfermenters ROBERT J. FASS,1* WILLIAM W. GREGORY,2 RICHARD F. D'AMATO,3 JOHN M. MATSEN,4 DONALD N. WRIGHT,4'5 AND LOWELL S. YOUNG6t Division of Infectious Diseases, Department of Internal Medicine, The Ohio State University College of Medicine, Columbus, Ohio 432101; Department of Clinical and Scientific Affairs, Pfizer Inc., New York, New York 100172; Division of Microbiology, Department of Pathology, and Department of Central Infection Control and Environmental Health, Catholic Medical Center of Brooklyn and Queens, Inc., Jamaica, New York 114323; Department of Pathology, University of Utah Medical Center, Salt Lake City, Utah 841324; Department of Microbiology, Brigham Young University, Provo, Utah 846025; and Division of Infectious Diseases, Department of Internal Medicine, University of California, Los Angeles, California 900246 Received 30 January 1990/Accepted 10 August 1990
Among 28,000 isolates of the family Enterobacteriaceae and nonfermenters isolated at multiple medical centers, 1,084 (4%) were resistant to cefoperazone (MIC, .64 ,ug/ml) and 1,711 (6%) exhibited cefoperazone MICs of 2 to 32 ,ug/ml. Ninety-six percent of these 2,795 isolates produced j8-lactamase, as determined by the nitrocefin test. Sulbactam alone (8 ,ug/ml) was inactive against 99.6% of the isolates other than Acinetobacter calcoaceticus and Pseudomonas cepacia. Sulbactam enhanced the activity of cefoperazone against 56% of the isolates of the family Enterobacteriaceae and 44% of the nonfermenters. In the presence of sulbactam concentrations of c8 ,ug/ml, 65% of the cefoperazone-resistant isolates had reductions in cefoperazone MICs of .2 log2 dilution steps and were susceptible to c32 ,ug/ml. Antagonism was not observed.
Cefoperazone is
an
extended-spectrum cephalosporin;
ville, Md.). No more than one isolate of the same species from any given patient was included. Among the 28,000 isolates, 2,795 with cefoperazone MICs of .2 ptg/ml were tested for their susceptibilities to cefoperazone, sulbactam, and the combination of cefoperazonesulbactam in a checkerboard microdilution test. Eighty-well microdilution plates manufactured specifically for this study (Prepared Media Laboratories, Tualatin, Oreg.) were delivered frozen to participating laboratories and stored at -20°C until use. Each plate contained wells with cefoperazone in doubling dilutions over the concentration range of 1.0 to 256 ,ugIml, sulbactam in doubling dilutions over the range of 0.25 to 16 ,ug/ml, and all possible combinations of cefoperazone and sulbactam within those ranges. Antibiotic dilutions were made in cation-supplemented Mueller-Hinton broth. After thawing, the plates were inoculated with disposable inoculators so that the final inoculum in each well was 1 x 105 to 2 x 105 CFU/ml. Microdilution plates were incubated at 35°C for 15 to 18 h, and MIC determinations were made with the aid of a backlighted MIC panel reader; results were recorded on a standardized form for computer entry. For quality control, the susceptibility of Acinetobacter anitratus S2 (ATCC 43498; cefoperazone MIC, 16 to 64 jig/ml; sulbactam MIC, 0.5 to 2 jig/ml) was tested daily. Isolates were grouped according to cefoperazone MIC. One group included organisms for which MICs were .64 ,ug/ml, and the second group included organisms for which MICs were in the range of 2 to 32 ,ug/ml. Isolates with cefoperazone MICs of .64 ,ug/ml were considered resistant. Enchancement was defined as a reduction in the cefoperazone MIC by at least 2 log2 dilution steps in the presence of -8 ,ug of sulbactam per ml compared with the MIC of cefoperazone alone. For the cefoperazone-resistant strains (MICs, .64 ,ug/ml), the percentage of strains for which there was enhancement plus conversion from the resistant inter-
most staphylococci, streptococci, members of the family
Enterobacteriaceae, nonfermenters, and anaerobes are susceptible to concentrations observed in vivo with recommended dosing regimens (1, 7). As a piperazinyl derivative, it has structural characteristics similar to those of the aminopenicillin piperacillin and is more active against Pseudomonas aeruginosa and enterococci than the iminomethoxyaminothiazolyl cephalosporins cefotaxime, ceftizoxime, and ceftriaxone or the oxacephem moxalactam (1, 6, 7, 15). However, cefoperazone is less consistently active against members of the family Enterobacteriaceae than these compounds and is hydrolyzed by certain ,B-lactamases from gram-negative bacilli at faster rates in cell-free systems (7). Sulbactam (penicillanic acid sulfone; CP-45,899) is a derivative of the basic penicillin nucleus (2). Sulbactam inhibits a broad spectrum of P-lactamases produced by gram-positive and gram-negative organisms (4, 5, 9, 10, 14, 16-19), but its intrinsic antibacterial activity is limited. In this study, investigators at medical centers in diverse geographic locations of the United States screened recent clinical isolates and determined their susceptibilities to cefoperazone and sulbactam, singly and in combination. Laboratories from participating institutions isolated bacterial pathogens from clinical material and tested approximately 28,000 isolates of the family Enterobacteriaceae and nonfermenters for susceptibility to cefoperazone in vitro (12). Each isolate was identified to the species level by standard methods (11), and 1-lactamase production was determined by a rapid chromogenic cephalosporin (nitrocefin) test (Cefinase; BBL Microbiology Systems, CockeysCorresponding author. t Present address: Kuzell Institute for Arthritis and Infectious Diseases, San Francisco, CA 94155. *
2256
TABLE 1. Sulbactam-enhanced activity of cefoperazone and geometric mean cefoperazone MICs in the presence or absence of sulbactam for 2,795 clinical isolates for which cefoperazone MICs were .2 ,ug/ml
No.
Organism
Of strains
tested
Geometric mean Enhance- cefoperazone MIC (pLg/mi):
mnt
mbere
s(t ofi
stan)
sul-
357 421 36 76 246 103 139 219 18 24 294 100 90 33 105 14 520
99 81 75 68 68 86 60 50 50 50 44 23 28 27
30.8 42.8 45.3 16.6 38.5 40.8 71.8 59.7 50.8 5.3 36.5 18.4 24.6 28.8
27 36 10
16.0 6.2 16.9
TABLE 2. Frequency of sulbactam-enhanced cefoperazone activity among 1,084 cefoperazone-resistant isolates and 1,711 isolates for which cefoperazone MICs were 2 to 32 ,ug/ml Cefoperazoneresistant isolates (MIC, .64 ,ug/ml)
Ac-
_______ty__ Withtivt With indexa out bactam
Acinetobacter calcoaceticusc Escherichia coli Proteus mirabilis Morganella morganii Klebsiella pneumoniae Enterobacter aerogenes Citrobacterfreundii Enterobacter cloacae Citrobacter diversus Proteus vulgaris Serratia marcescens Klebsiella oxytoca Xanthomonas maltophilia Pseudomonas cepaciad Providencia stuartii Providencia rettgeri Pseudomonas aeruginosa
2257
NOTES
VOL. 34, 1990
% Conversion from cefopera-
Organism
sul-
bactamb 1.1 2.9 3.1 1.9 5.2 5.7 19.3 16.9 14.8 1.6 12.1 7.9 10.8 13.0 8.5 3.4 12.5
No.
28.0 14.8 14.6 8.7 7.4 7.2 3.7 3.5 3.4 3.3 3.0 2.3 2.3 2.2 1.9 1.8 1.4
a Ratio of geometric mean MICs of cefoperazone to geometric mean MICs of the cefoperazone component of cefoperazone and sulbactam in combination. b Sulbactam was present at 8 pg/ml. c Sulbactam alone (8 ,ug/ml. The frequency with which sulbactam enhanced the activity of cefoperazone and the geometric mean MICs of cefoperazone without and with sulbactam for the 2,795 study organisms are given in Table 1. Overall, sulbactam enhanced the activity of cefoperazone against 56% of the Enterobacteriaceae and 44% of the nonfermenters. No instances of antagonism were observed. Table 2 shows the frequency of enhancement by group; for the 1,084 cefoperazone-resistant isolates (MICs, .64 ,ug/ml), the percentage of the isolates that were converted from the resistant interpretive category to the susceptible category by the addition of sulbactam is included in the definition of enhancement. Enhancement was dependent on the concen-
Isolates for which cefoperazone MICs were 2 to 32
zone-resistant to susceptible
No.
interpretive
Morganella morganii Providencia rettgeri Acinetobacter calcoaceticus Escherichia coli Proteus mirabilis Klebsiella pneumoniae Enterobacter aerogenes Citrobacter freundii Providencia stuartii Serratia marcescens Enterobacter cloacae Proteus vulgaris Xanthomonas maltophilia Citrobacter diversus Pseudomonas aeruginosa Pseudomonas cepacia Klebsiella oxytoca
pg/ml % Exhibiting increased sustib
cefopeiat
category by
zone in presence of
sulbactama
sulbaCtaMb
22 1 69
100 100 97
54 13 288
59 31 97
199 17 125 40 100 18 135 136 2 37 10 121 11 41
91 88 86 78 61 56 53 52 50 49 40 29 27 22
222 19 121 63 39 87 159 83 22 53 8 399 22 59
64 63 45 87 59 17 28 42 55 19 75 5.8 27 32
a Percentage of cefoperazone-resistant (MIC, 264 ,ug/ml) isolates converted to a cefoperazone MIC of -32 p.g/ml that was at least 2 log2 dilution steps lower in the presence of -8 ,ug of sulbactam per ml. b Percentage of isolates for which cefoperazone MICs were 2 to 32 pLg/ml and for which sulbactam (c8 ,ug/ml) enhanced the activity of cefoperazone within the susceptible interpretive category by reducing the cefoperazone MIC by at least 2 log2 dilution steps.
tration of sulbactam, and the effect of any given concentration of sulbactam varied with the bacterial species, as illustrated in Fig. 1. Twenty-five of the cefoperazone-resistant isolates did not produce detectable P-lactamase, as follows: Acinetobacter calcoaceticus (n = 8); Escherichia coli (n = 4); Pseudomonas aeruginosa (n = 3); Xanthomonas maltophilia (n = 3); Citrobacterfreundii (n = 2); Providencia stuartii (n = 2); and Klebsiella pneumoniae, Klebsiella oxytoca, and Enterobacter cloacae (n = 1 each). Nevertheless, the combination exhibited enhanced activity for 12 of the 18 (67%) isolates in this subgroup that were not susceptible to sulbactam alone. Cefoperazone is very active against most strains of the family Enterobacteriaceae and Pseudomonas aeruginosa (1, 5, 6, 7, 15); median MICs are typically .1 and 4 to 8 ,ug/ml, respectively, and a susceptibility breakpoint of c32 ,ug/ml is generally accepted. Many bacterial species, those that are both susceptible and resistant to cefoperazone, produce P-lactamase enzymes (3-5, 8, 13, 15). Although cefoperazone is vulnerable to the hydrolytic activity of some of these enzymes, they are only partially responsible for cefoperazone resistance (3, 13), and other mechanisms may contribute to the resistance phenotype. The concomitant administration of a 13-lactamase inhibitor such as sulbactam would be expected to enhance the activity of cefoperazone against organisms if the following four conditions were present: (i) the organisms produced ,-lactamase, (ii) the enzyme hydrolyzed the active antibiotic (cefoperazone), (iii) the primary
2258
ANTIMICROB. AGENTS CHEMOTHER.
NOTES 100
NI
d
b C
10
a
a10_
so a
610
a
0
-I%
4
410
4
2
2
2
so 7
w
z 0 N
w a.
0 IL.
0.5 0.25
0 0
2.0 1.0
0
0.25
4.0
2.0
0.5
1.0
8.0 4.0
16
0.5 0.25
10
1.0
0
8.0
2.0
16
4.0
e 80
so _
a0
aso
60
Cfoc-
410
4so
40
aVIw
210
2to0
20
w
0
p
0.5
0
0.25
z uJ C.
a.Lu
2.0 1.0
0.5
8.0 4.0
0.25
16
100
2.0 1.0
4.0
0
0 16
80
8 10
60
60
6 00
40h
410
20
20
20o-
2.0
8.0
4.0
1.0
0 16
k
II
80
-
0.5 0.25
-
01
I
I
I
_
8.0
100,
-i
4
0.5 0.25
8.0 4.0
16
0.25
4.0
2.0
1.0
8.0 4.0
I 1116
w
Z 100
60
0
I I
0
16
I
60
0
8.0
2.0
1.0
I
0.5
0.25
p.1 0
2.0
1.0
9
a0
SP
0.5 0.25
100r
100
(U) 0 m
1
p
0
2.0
0.5
0.25
1.0
SULBACTAM
0
8.0 4.0
0.5
0.25
16
2.0 1.0
1.0
4.0
CON4CENTRAT1N (Pg/mi)
8.0 4.0
16
SULBACTAM CONCENTRATION (pg/mi) FIG. 1. Effect of sulbactam concentration on cefoperazone MICs for 1,025 cefoperazone-resistant (MIC, .64 ,ug/ml) isolates (a through k). Datum points represent the percentage of total cefoperazone-resistant strains of each species for which the indicated concentrations of sulbactam reduced the cefoperazone MICs by .2 log2 dilution steps to a final cefoperazone MIC of s32 ,ug/ml. The following species are represented: Morganella morganii, 22 isolates (a); Escherichia coli, 199 isolates (b); Klebsiella pneumoniae, 125 isolates (c); Enterobacter aerogenes, 40 isolates (d); Citrobacterfreundii, 100 isolates (e); Enterobacter cloacae, 136 isolates (f); Serratia marcescens, 135 isolates (g); Xanthomonas maltophilia, 37 isolates (h); Pseudomonas aeruginosa, 121 isolates (i); Klebsiella oxytoca, 41 isolates (j); and Acinetobacter calcoaceticus, 69 isolates (k). The cumulative percentage of cefoperazone-resistant Acinetobacter isolates inhibited by sulbactam alone is shown in panel 1.
mechanism of resistance was due to P-lactamase production, and (iv) the inhibitor (sulbactam) inactivated the enzyme. The inhibitor could also enhance the activity of cefoperazone if it had significant intrinsic activity against the organisms, as is the case with Neisseria gonorrhoeae, Acinetobacter calcoaceticus, and Pseudomonas cepacia, or if it had
direct action (complementary to P-lactamase inhibition) on the penicillin-binding proteins of inhibitor-resistant organisms. In this study, the net effect of these various interactions on organisms from diverse geographic locations was determined with a large sample of isolates resistant to cefoperazone or for which cefoperazone MICs were 2 to 32 a
VOL. 34, 1990
,ug/ml; these isolates represented 4 and 6%, respectively, of all organisms screened. It is possible that the observed distribution of MICs might vary with the testing method. For example, if a broth macrodilution method had been used, the greater total number of organisms in a given inoculum would increase the chance of detecting an elevated MIC for a small portion of the population tested. Sulbactam-mediated enhancement of the activity of cefoperazone was observed more frequently against members of the family Enterobacteriaceae than against Pseudomonas species. It is likely that many of the former produced P-lactamase that was inhibited by sulbactam at the concentrations tested, whereas the latter did not. However, P-lactamase production, as measured by the nitrocefin test, was not a prerequisite for enhancement. The apparent synergistic effect of cefoperazone and sulbactam against Acinetobacter calcoaceticus and some strains of Pseudomonas cepacia, organisms which are typically more resistant to cefoperazone than members of the family Enterobacteriaceae and Pseudomonas aeruginosa are, was more likely due to the intrinsic activity of sulbactam than to P-lactamase inhibition. In addition, sulbactam increased the observed activity of cefoperazone against some of the ,-lactamase-negative isolates, although it did not potentiate activity against all ,-lactamase-positive isolates. This suggests that alternate mechanisms, such as porin restrictions or alterations of the cefoperazone or sulbactam target(s), contribute to cefoperazone resistance in some organisms. The reduction in the MIC observed for the small number of cefoperazone-resistant, 3-lactamase-negative isolates may be the result of classical synergy of the two B-lactams or may reflect the limits of nitrocefin in detecting ,-lactamase(s). Induction of inapparent chromosomal ,B-lactamases was not attempted in this study. The data presented here suggest that sulbactam increases the activity of cefoperazone against the Enterobacteriaceae and nonfermenters to various degrees in a species- and concentration-dependent manner and converts many cefoperazone-resistant strains into the susceptible range. Pfizer Inc. provided support for this work. We thank J. M. Ambrosi for assistance with computer programming and data analysis. LITERATURE CITED 1. Barry, A. L., and R. N. Jones. 1987. Bacterial antibiotic resistance before and after clinical application in the United States. Bull. N.Y. Acad. Med. 63:217-230. 2. English, A. R., J. A. Retsema, A. E. Girard, J. E. Lynch, and W. E. Barth. 1978. CP-45,899, a P-lactamase inhibitor that extends the antibacterial spectrum of beta-lactams: initial bacteriological characterization. Antimicrob. Agents Chemother. 14:414-419. 3. Fass, R. J. 1981. Inconsistency of synergy between the P-lactamase inhibitor CP-45,899 and beta-lactam antibiotics against multiply drug-resistant Enterobacteriaceae and Pseudomonas
NOTES
2259
species. Antimicrob. Agents Chemother. 19:361-363. 4. Fu, K. P., and H. C. Neu. 1980. Synergistic activity of cefoperazone in combination with beta-lactamase inhibitors. J. Antimicrob. Chemother. 7:287-292. 5. Fuchs, P. C., R. N. Jones, and A. L. Barry. 1987. Effect of beta-lactamase inhibitors on the antimicrobial activity of cefoperazone, cefotaxime, and ceftizoxime against aerobic and anaerobic beta-lactamase producing bacteria. Diagn. Microbiol. Infect. Dis. 8:61-65. 6. Jones, R. N. 1984. Changing patterns of resistance to new beta-lactam antibiotics. Am. J. Med. 77(Suppl. 1B):29-34. 7. Jones, R. N., and A. L. Barry. 1983. Cefoperazone: a review of its antimicrobial spectrum, beta-lactamase stability, enzyme inhibition, and other in vitro characteristics. Rev. Infect. Dis.
5(Suppl. 1):S108-5126. 8. Jones, R. N., A. L. Barry, R. R. Packer, W. W. Gregory, and C. Thornsberry. 1987. In vitro antimicrobial spectrum, occurrence of synergy, and recommendations for dilution susceptibility testing concentrations of the cefoperazone-sulbactam combination. J. Clin. Microbiol. 25:1725-1729. 9. Labia, R., A. Morand, V. Lelievre, D. Mattioni, and A. Kazmierczak. 1986. Sulbactam: biochemical factors involved in its synergy with ampicillin. Rev. Infect. Dis. 8(Suppl. 5):S496S502. 10. Labia, R., A. Morand, K. Tiwari, J. S. Pitton, D. Sirot, and J. Sirot. 1988. Kinetic properties of two plasmid-mediated betalactamases from Klebsiella pneumoniae with strong activity against third-generation cephalosporins. J. Antimicrob. Chemother. 21:301-307. 11. Lennette, E. H., A. Balows, W. J. Hausler, Jr., and H. J. Shadomy (ed.). 1986. Manual of clinical microbiology, 4th ed. American Society for Microbiology, Washington, D.C. 12. National Committee for Clinical Laboratory Standards. 1987. Performance standards for antimicrobial disk susceptibility tests, 3rd ed. Approved standard M2-A3-S2. National Committee for Clinical Laboratory Standards, Villanova, Pa. 13. Neu, H. C. 1985. Contribution of beta-lactamases to bacterial resistance and mechanisms to inhibit beta-lactamases. Am. J. Med. 79(Suppl. 5B):2-12. 14. Neu, H. C. 1985. The role of beta-lactamase inhibitors in chemotherapy. Pharmacol. Ther. 30:1-18. 15. Neu, H. C., K. P. Fu, N. Aswapokee, P. Aswapokee, and K. Kung. 1979. Comparative activity and P-lactamase stability of cefoperazone, a piperazine cephalosporin. Antimicrob. Agents Chemother. 16:150-157. 16. Retsema, J. A., A. R. English, and A. E. Girard. 1980. CP-45,899 in combination with penicillin or ampicillin against penicillinresistant Staphylococcus, Haemophilus influenzae, and Bacteroides. Antimicrob. Agents Chemother. 17:615-622. 17. Wise, R., J. M. Andrews, and K. A. Bedford. 1980. Clavulanic acid and CP-45,899: a comparison of their in vitro activity in combination with penicillins. J. Antimicrob. Chemother. 6:197206. 18. Yokota, T., E. Azuma, and E. Suzuki. 1984. Antibacterial activity of the combination drug of sulbactam with cefoperazone. Chemotherapy (Tokyo) 32(Suppl. 4):1-10. 19. Yokota, T., R. Sekiguchi, E. Azuma, and E. Suzuki. 1984. Sulbactam: permanent inactivation of various types of betalactamases and the affinity to penicillin-binding proteins in bacteria. Chemotherapy (Tokyo) 32(Suppl. 4):11-19.
0066-4804/90/112256-04$02.00/0 Copyright C) 1990, American Society for Microbiology
Vol. 34, No. 11
In Vitro Activities of Cefoperazone and Sulbactam Singly and in Combination against Cefoperazone-Resistant Members of the Family Enterobacteriaceae and Nonfermenters ROBERT J. FASS,1* WILLIAM W. GREGORY,2 RICHARD F. D'AMATO,3 JOHN M. MATSEN,4 DONALD N. WRIGHT,4'5 AND LOWELL S. YOUNG6t Division of Infectious Diseases, Department of Internal Medicine, The Ohio State University College of Medicine, Columbus, Ohio 432101; Department of Clinical and Scientific Affairs, Pfizer Inc., New York, New York 100172; Division of Microbiology, Department of Pathology, and Department of Central Infection Control and Environmental Health, Catholic Medical Center of Brooklyn and Queens, Inc., Jamaica, New York 114323; Department of Pathology, University of Utah Medical Center, Salt Lake City, Utah 841324; Department of Microbiology, Brigham Young University, Provo, Utah 846025; and Division of Infectious Diseases, Department of Internal Medicine, University of California, Los Angeles, California 900246 Received 30 January 1990/Accepted 10 August 1990
Among 28,000 isolates of the family Enterobacteriaceae and nonfermenters isolated at multiple medical centers, 1,084 (4%) were resistant to cefoperazone (MIC, .64 ,ug/ml) and 1,711 (6%) exhibited cefoperazone MICs of 2 to 32 ,ug/ml. Ninety-six percent of these 2,795 isolates produced j8-lactamase, as determined by the nitrocefin test. Sulbactam alone (8 ,ug/ml) was inactive against 99.6% of the isolates other than Acinetobacter calcoaceticus and Pseudomonas cepacia. Sulbactam enhanced the activity of cefoperazone against 56% of the isolates of the family Enterobacteriaceae and 44% of the nonfermenters. In the presence of sulbactam concentrations of c8 ,ug/ml, 65% of the cefoperazone-resistant isolates had reductions in cefoperazone MICs of .2 log2 dilution steps and were susceptible to c32 ,ug/ml. Antagonism was not observed.
Cefoperazone is
an
extended-spectrum cephalosporin;
ville, Md.). No more than one isolate of the same species from any given patient was included. Among the 28,000 isolates, 2,795 with cefoperazone MICs of .2 ptg/ml were tested for their susceptibilities to cefoperazone, sulbactam, and the combination of cefoperazonesulbactam in a checkerboard microdilution test. Eighty-well microdilution plates manufactured specifically for this study (Prepared Media Laboratories, Tualatin, Oreg.) were delivered frozen to participating laboratories and stored at -20°C until use. Each plate contained wells with cefoperazone in doubling dilutions over the concentration range of 1.0 to 256 ,ugIml, sulbactam in doubling dilutions over the range of 0.25 to 16 ,ug/ml, and all possible combinations of cefoperazone and sulbactam within those ranges. Antibiotic dilutions were made in cation-supplemented Mueller-Hinton broth. After thawing, the plates were inoculated with disposable inoculators so that the final inoculum in each well was 1 x 105 to 2 x 105 CFU/ml. Microdilution plates were incubated at 35°C for 15 to 18 h, and MIC determinations were made with the aid of a backlighted MIC panel reader; results were recorded on a standardized form for computer entry. For quality control, the susceptibility of Acinetobacter anitratus S2 (ATCC 43498; cefoperazone MIC, 16 to 64 jig/ml; sulbactam MIC, 0.5 to 2 jig/ml) was tested daily. Isolates were grouped according to cefoperazone MIC. One group included organisms for which MICs were .64 ,ug/ml, and the second group included organisms for which MICs were in the range of 2 to 32 ,ug/ml. Isolates with cefoperazone MICs of .64 ,ug/ml were considered resistant. Enchancement was defined as a reduction in the cefoperazone MIC by at least 2 log2 dilution steps in the presence of -8 ,ug of sulbactam per ml compared with the MIC of cefoperazone alone. For the cefoperazone-resistant strains (MICs, .64 ,ug/ml), the percentage of strains for which there was enhancement plus conversion from the resistant inter-
most staphylococci, streptococci, members of the family
Enterobacteriaceae, nonfermenters, and anaerobes are susceptible to concentrations observed in vivo with recommended dosing regimens (1, 7). As a piperazinyl derivative, it has structural characteristics similar to those of the aminopenicillin piperacillin and is more active against Pseudomonas aeruginosa and enterococci than the iminomethoxyaminothiazolyl cephalosporins cefotaxime, ceftizoxime, and ceftriaxone or the oxacephem moxalactam (1, 6, 7, 15). However, cefoperazone is less consistently active against members of the family Enterobacteriaceae than these compounds and is hydrolyzed by certain ,B-lactamases from gram-negative bacilli at faster rates in cell-free systems (7). Sulbactam (penicillanic acid sulfone; CP-45,899) is a derivative of the basic penicillin nucleus (2). Sulbactam inhibits a broad spectrum of P-lactamases produced by gram-positive and gram-negative organisms (4, 5, 9, 10, 14, 16-19), but its intrinsic antibacterial activity is limited. In this study, investigators at medical centers in diverse geographic locations of the United States screened recent clinical isolates and determined their susceptibilities to cefoperazone and sulbactam, singly and in combination. Laboratories from participating institutions isolated bacterial pathogens from clinical material and tested approximately 28,000 isolates of the family Enterobacteriaceae and nonfermenters for susceptibility to cefoperazone in vitro (12). Each isolate was identified to the species level by standard methods (11), and 1-lactamase production was determined by a rapid chromogenic cephalosporin (nitrocefin) test (Cefinase; BBL Microbiology Systems, CockeysCorresponding author. t Present address: Kuzell Institute for Arthritis and Infectious Diseases, San Francisco, CA 94155. *
2256
TABLE 1. Sulbactam-enhanced activity of cefoperazone and geometric mean cefoperazone MICs in the presence or absence of sulbactam for 2,795 clinical isolates for which cefoperazone MICs were .2 ,ug/ml
No.
Organism
Of strains
tested
Geometric mean Enhance- cefoperazone MIC (pLg/mi):
mnt
mbere
s(t ofi
stan)
sul-
357 421 36 76 246 103 139 219 18 24 294 100 90 33 105 14 520
99 81 75 68 68 86 60 50 50 50 44 23 28 27
30.8 42.8 45.3 16.6 38.5 40.8 71.8 59.7 50.8 5.3 36.5 18.4 24.6 28.8
27 36 10
16.0 6.2 16.9
TABLE 2. Frequency of sulbactam-enhanced cefoperazone activity among 1,084 cefoperazone-resistant isolates and 1,711 isolates for which cefoperazone MICs were 2 to 32 ,ug/ml Cefoperazoneresistant isolates (MIC, .64 ,ug/ml)
Ac-
_______ty__ Withtivt With indexa out bactam
Acinetobacter calcoaceticusc Escherichia coli Proteus mirabilis Morganella morganii Klebsiella pneumoniae Enterobacter aerogenes Citrobacterfreundii Enterobacter cloacae Citrobacter diversus Proteus vulgaris Serratia marcescens Klebsiella oxytoca Xanthomonas maltophilia Pseudomonas cepaciad Providencia stuartii Providencia rettgeri Pseudomonas aeruginosa
2257
NOTES
VOL. 34, 1990
% Conversion from cefopera-
Organism
sul-
bactamb 1.1 2.9 3.1 1.9 5.2 5.7 19.3 16.9 14.8 1.6 12.1 7.9 10.8 13.0 8.5 3.4 12.5
No.
28.0 14.8 14.6 8.7 7.4 7.2 3.7 3.5 3.4 3.3 3.0 2.3 2.3 2.2 1.9 1.8 1.4
a Ratio of geometric mean MICs of cefoperazone to geometric mean MICs of the cefoperazone component of cefoperazone and sulbactam in combination. b Sulbactam was present at 8 pg/ml. c Sulbactam alone (8 ,ug/ml. The frequency with which sulbactam enhanced the activity of cefoperazone and the geometric mean MICs of cefoperazone without and with sulbactam for the 2,795 study organisms are given in Table 1. Overall, sulbactam enhanced the activity of cefoperazone against 56% of the Enterobacteriaceae and 44% of the nonfermenters. No instances of antagonism were observed. Table 2 shows the frequency of enhancement by group; for the 1,084 cefoperazone-resistant isolates (MICs, .64 ,ug/ml), the percentage of the isolates that were converted from the resistant interpretive category to the susceptible category by the addition of sulbactam is included in the definition of enhancement. Enhancement was dependent on the concen-
Isolates for which cefoperazone MICs were 2 to 32
zone-resistant to susceptible
No.
interpretive
Morganella morganii Providencia rettgeri Acinetobacter calcoaceticus Escherichia coli Proteus mirabilis Klebsiella pneumoniae Enterobacter aerogenes Citrobacter freundii Providencia stuartii Serratia marcescens Enterobacter cloacae Proteus vulgaris Xanthomonas maltophilia Citrobacter diversus Pseudomonas aeruginosa Pseudomonas cepacia Klebsiella oxytoca
pg/ml % Exhibiting increased sustib
cefopeiat
category by
zone in presence of
sulbactama
sulbaCtaMb
22 1 69
100 100 97
54 13 288
59 31 97
199 17 125 40 100 18 135 136 2 37 10 121 11 41
91 88 86 78 61 56 53 52 50 49 40 29 27 22
222 19 121 63 39 87 159 83 22 53 8 399 22 59
64 63 45 87 59 17 28 42 55 19 75 5.8 27 32
a Percentage of cefoperazone-resistant (MIC, 264 ,ug/ml) isolates converted to a cefoperazone MIC of -32 p.g/ml that was at least 2 log2 dilution steps lower in the presence of -8 ,ug of sulbactam per ml. b Percentage of isolates for which cefoperazone MICs were 2 to 32 pLg/ml and for which sulbactam (c8 ,ug/ml) enhanced the activity of cefoperazone within the susceptible interpretive category by reducing the cefoperazone MIC by at least 2 log2 dilution steps.
tration of sulbactam, and the effect of any given concentration of sulbactam varied with the bacterial species, as illustrated in Fig. 1. Twenty-five of the cefoperazone-resistant isolates did not produce detectable P-lactamase, as follows: Acinetobacter calcoaceticus (n = 8); Escherichia coli (n = 4); Pseudomonas aeruginosa (n = 3); Xanthomonas maltophilia (n = 3); Citrobacterfreundii (n = 2); Providencia stuartii (n = 2); and Klebsiella pneumoniae, Klebsiella oxytoca, and Enterobacter cloacae (n = 1 each). Nevertheless, the combination exhibited enhanced activity for 12 of the 18 (67%) isolates in this subgroup that were not susceptible to sulbactam alone. Cefoperazone is very active against most strains of the family Enterobacteriaceae and Pseudomonas aeruginosa (1, 5, 6, 7, 15); median MICs are typically .1 and 4 to 8 ,ug/ml, respectively, and a susceptibility breakpoint of c32 ,ug/ml is generally accepted. Many bacterial species, those that are both susceptible and resistant to cefoperazone, produce P-lactamase enzymes (3-5, 8, 13, 15). Although cefoperazone is vulnerable to the hydrolytic activity of some of these enzymes, they are only partially responsible for cefoperazone resistance (3, 13), and other mechanisms may contribute to the resistance phenotype. The concomitant administration of a 13-lactamase inhibitor such as sulbactam would be expected to enhance the activity of cefoperazone against organisms if the following four conditions were present: (i) the organisms produced ,-lactamase, (ii) the enzyme hydrolyzed the active antibiotic (cefoperazone), (iii) the primary
2258
ANTIMICROB. AGENTS CHEMOTHER.
NOTES 100
NI
d
b C
10
a
a10_
so a
610
a
0
-I%
4
410
4
2
2
2
so 7
w
z 0 N
w a.
0 IL.
0.5 0.25
0 0
2.0 1.0
0
0.25
4.0
2.0
0.5
1.0
8.0 4.0
16
0.5 0.25
10
1.0
0
8.0
2.0
16
4.0
e 80
so _
a0
aso
60
Cfoc-
410
4so
40
aVIw
210
2to0
20
w
0
p
0.5
0
0.25
z uJ C.
a.Lu
2.0 1.0
0.5
8.0 4.0
0.25
16
100
2.0 1.0
4.0
0
0 16
80
8 10
60
60
6 00
40h
410
20
20
20o-
2.0
8.0
4.0
1.0
0 16
k
II
80
-
0.5 0.25
-
01
I
I
I
_
8.0
100,
-i
4
0.5 0.25
8.0 4.0
16
0.25
4.0
2.0
1.0
8.0 4.0
I 1116
w
Z 100
60
0
I I
0
16
I
60
0
8.0
2.0
1.0
I
0.5
0.25
p.1 0
2.0
1.0
9
a0
SP
0.5 0.25
100r
100
(U) 0 m
1
p
0
2.0
0.5
0.25
1.0
SULBACTAM
0
8.0 4.0
0.5
0.25
16
2.0 1.0
1.0
4.0
CON4CENTRAT1N (Pg/mi)
8.0 4.0
16
SULBACTAM CONCENTRATION (pg/mi) FIG. 1. Effect of sulbactam concentration on cefoperazone MICs for 1,025 cefoperazone-resistant (MIC, .64 ,ug/ml) isolates (a through k). Datum points represent the percentage of total cefoperazone-resistant strains of each species for which the indicated concentrations of sulbactam reduced the cefoperazone MICs by .2 log2 dilution steps to a final cefoperazone MIC of s32 ,ug/ml. The following species are represented: Morganella morganii, 22 isolates (a); Escherichia coli, 199 isolates (b); Klebsiella pneumoniae, 125 isolates (c); Enterobacter aerogenes, 40 isolates (d); Citrobacterfreundii, 100 isolates (e); Enterobacter cloacae, 136 isolates (f); Serratia marcescens, 135 isolates (g); Xanthomonas maltophilia, 37 isolates (h); Pseudomonas aeruginosa, 121 isolates (i); Klebsiella oxytoca, 41 isolates (j); and Acinetobacter calcoaceticus, 69 isolates (k). The cumulative percentage of cefoperazone-resistant Acinetobacter isolates inhibited by sulbactam alone is shown in panel 1.
mechanism of resistance was due to P-lactamase production, and (iv) the inhibitor (sulbactam) inactivated the enzyme. The inhibitor could also enhance the activity of cefoperazone if it had significant intrinsic activity against the organisms, as is the case with Neisseria gonorrhoeae, Acinetobacter calcoaceticus, and Pseudomonas cepacia, or if it had
direct action (complementary to P-lactamase inhibition) on the penicillin-binding proteins of inhibitor-resistant organisms. In this study, the net effect of these various interactions on organisms from diverse geographic locations was determined with a large sample of isolates resistant to cefoperazone or for which cefoperazone MICs were 2 to 32 a
VOL. 34, 1990
,ug/ml; these isolates represented 4 and 6%, respectively, of all organisms screened. It is possible that the observed distribution of MICs might vary with the testing method. For example, if a broth macrodilution method had been used, the greater total number of organisms in a given inoculum would increase the chance of detecting an elevated MIC for a small portion of the population tested. Sulbactam-mediated enhancement of the activity of cefoperazone was observed more frequently against members of the family Enterobacteriaceae than against Pseudomonas species. It is likely that many of the former produced P-lactamase that was inhibited by sulbactam at the concentrations tested, whereas the latter did not. However, P-lactamase production, as measured by the nitrocefin test, was not a prerequisite for enhancement. The apparent synergistic effect of cefoperazone and sulbactam against Acinetobacter calcoaceticus and some strains of Pseudomonas cepacia, organisms which are typically more resistant to cefoperazone than members of the family Enterobacteriaceae and Pseudomonas aeruginosa are, was more likely due to the intrinsic activity of sulbactam than to P-lactamase inhibition. In addition, sulbactam increased the observed activity of cefoperazone against some of the ,-lactamase-negative isolates, although it did not potentiate activity against all ,-lactamase-positive isolates. This suggests that alternate mechanisms, such as porin restrictions or alterations of the cefoperazone or sulbactam target(s), contribute to cefoperazone resistance in some organisms. The reduction in the MIC observed for the small number of cefoperazone-resistant, 3-lactamase-negative isolates may be the result of classical synergy of the two B-lactams or may reflect the limits of nitrocefin in detecting ,-lactamase(s). Induction of inapparent chromosomal ,B-lactamases was not attempted in this study. The data presented here suggest that sulbactam increases the activity of cefoperazone against the Enterobacteriaceae and nonfermenters to various degrees in a species- and concentration-dependent manner and converts many cefoperazone-resistant strains into the susceptible range. Pfizer Inc. provided support for this work. We thank J. M. Ambrosi for assistance with computer programming and data analysis. LITERATURE CITED 1. Barry, A. L., and R. N. Jones. 1987. Bacterial antibiotic resistance before and after clinical application in the United States. Bull. N.Y. Acad. Med. 63:217-230. 2. English, A. R., J. A. Retsema, A. E. Girard, J. E. Lynch, and W. E. Barth. 1978. CP-45,899, a P-lactamase inhibitor that extends the antibacterial spectrum of beta-lactams: initial bacteriological characterization. Antimicrob. Agents Chemother. 14:414-419. 3. Fass, R. J. 1981. Inconsistency of synergy between the P-lactamase inhibitor CP-45,899 and beta-lactam antibiotics against multiply drug-resistant Enterobacteriaceae and Pseudomonas
NOTES
2259
species. Antimicrob. Agents Chemother. 19:361-363. 4. Fu, K. P., and H. C. Neu. 1980. Synergistic activity of cefoperazone in combination with beta-lactamase inhibitors. J. Antimicrob. Chemother. 7:287-292. 5. Fuchs, P. C., R. N. Jones, and A. L. Barry. 1987. Effect of beta-lactamase inhibitors on the antimicrobial activity of cefoperazone, cefotaxime, and ceftizoxime against aerobic and anaerobic beta-lactamase producing bacteria. Diagn. Microbiol. Infect. Dis. 8:61-65. 6. Jones, R. N. 1984. Changing patterns of resistance to new beta-lactam antibiotics. Am. J. Med. 77(Suppl. 1B):29-34. 7. Jones, R. N., and A. L. Barry. 1983. Cefoperazone: a review of its antimicrobial spectrum, beta-lactamase stability, enzyme inhibition, and other in vitro characteristics. Rev. Infect. Dis.
5(Suppl. 1):S108-5126. 8. Jones, R. N., A. L. Barry, R. R. Packer, W. W. Gregory, and C. Thornsberry. 1987. In vitro antimicrobial spectrum, occurrence of synergy, and recommendations for dilution susceptibility testing concentrations of the cefoperazone-sulbactam combination. J. Clin. Microbiol. 25:1725-1729. 9. Labia, R., A. Morand, V. Lelievre, D. Mattioni, and A. Kazmierczak. 1986. Sulbactam: biochemical factors involved in its synergy with ampicillin. Rev. Infect. Dis. 8(Suppl. 5):S496S502. 10. Labia, R., A. Morand, K. Tiwari, J. S. Pitton, D. Sirot, and J. Sirot. 1988. Kinetic properties of two plasmid-mediated betalactamases from Klebsiella pneumoniae with strong activity against third-generation cephalosporins. J. Antimicrob. Chemother. 21:301-307. 11. Lennette, E. H., A. Balows, W. J. Hausler, Jr., and H. J. Shadomy (ed.). 1986. Manual of clinical microbiology, 4th ed. American Society for Microbiology, Washington, D.C. 12. National Committee for Clinical Laboratory Standards. 1987. Performance standards for antimicrobial disk susceptibility tests, 3rd ed. Approved standard M2-A3-S2. National Committee for Clinical Laboratory Standards, Villanova, Pa. 13. Neu, H. C. 1985. Contribution of beta-lactamases to bacterial resistance and mechanisms to inhibit beta-lactamases. Am. J. Med. 79(Suppl. 5B):2-12. 14. Neu, H. C. 1985. The role of beta-lactamase inhibitors in chemotherapy. Pharmacol. Ther. 30:1-18. 15. Neu, H. C., K. P. Fu, N. Aswapokee, P. Aswapokee, and K. Kung. 1979. Comparative activity and P-lactamase stability of cefoperazone, a piperazine cephalosporin. Antimicrob. Agents Chemother. 16:150-157. 16. Retsema, J. A., A. R. English, and A. E. Girard. 1980. CP-45,899 in combination with penicillin or ampicillin against penicillinresistant Staphylococcus, Haemophilus influenzae, and Bacteroides. Antimicrob. Agents Chemother. 17:615-622. 17. Wise, R., J. M. Andrews, and K. A. Bedford. 1980. Clavulanic acid and CP-45,899: a comparison of their in vitro activity in combination with penicillins. J. Antimicrob. Chemother. 6:197206. 18. Yokota, T., E. Azuma, and E. Suzuki. 1984. Antibacterial activity of the combination drug of sulbactam with cefoperazone. Chemotherapy (Tokyo) 32(Suppl. 4):1-10. 19. Yokota, T., R. Sekiguchi, E. Azuma, and E. Suzuki. 1984. Sulbactam: permanent inactivation of various types of betalactamases and the affinity to penicillin-binding proteins in bacteria. Chemotherapy (Tokyo) 32(Suppl. 4):11-19.
