DIAGN MICROBIOLINFECTDIS 1991;14:147-155
147
ANTIMICROBIAL CLINICAL STUDIES
Antibacterial Activity of Cefoperazone and Cefoperazone Plus Sulbactam in a Neutropenic Site Model Claudine E. Fasching, Julia A. Moody, Leann M. Sinn, Jane Tenquist, Dale N. Gerding, and Lance R. Peterson
Efficacy of cefoperazone versus cefoperazone plus sulbactam was studied in a rabbit neutropenic site infection model against a broad range of clinical isolates including six isolates each of staphylococci, enterococci, pneumococci, Enterobacteriaceae, and Pseudomonas aeruginosa. Therapy of cefoperazone plus sulbactam demonstrated enhanced efficacy against the staphylococci, pseudomonads, and Enterobacteriaceae. The activity of cefoperazone against enterococci and pneumococci
was not enhanced or inhibited by the addition of sulbactam. Increased concentrations of cefoperazone found at the infection sites when sulbactam was added to the therapeutic regimen indicates that sulbactam provided a protection to cefoperazone from [3-1actamases produced by staphylococci and Enterobacteriaceae. The combination improved the efficacy of cefoperazone in this animal model.
INTRODUCTION
Sulbactam inhibits B-lactamases from Staphylococcus aureus and plasmid-mediated [3-1actamases from Enterobacteriaceae (Vu and Nikaido, 1985), and sulbactam has not been shown to routinely induce J3-1actamase production in strains of Enterobacteriaceae (Moosdeen et al., 1986). The combination of cefoperazone plus sulbactam has shown a synergistic action in vitro by reducing minimum inhibitory concentration (MIC) of cefoperazone 2- to 32-fold in strains susceptible to cefoperazone (Jones et al., 1985), and by extending the antimicrobial spectrum of cefoperazone (Jones et al., 1987). We evaluated the activity of cefoperazone and cefoperazone plus sulbactam against organisms that have previously been studied in a rabbit model, developed in our laboratory, which simulates a closed-space, neutropenic site of infection. The purpose of this study was (a) to compare the efficacy of cefoperazone to the combination of cefoperazone plus sulbactam in an in vivo model simulating a neutropenic infection site, (b) to compare results from in vitro susceptibility testing to in vivo quantitative bacterial reductions achieved through antimicrobial therapy, and (c) to investigate the occurrence of any resistance development during drug therapy.
Cefoperazone is a third-generation, extended spectrum cephalosporin that has broad antimicrobial activity including Pseudomonas aeruginosa and anaerobes (Jones and Barry, 1983). Hydrolysis of cefoperazone by most Gram-negative f3-1actamases is relatively slow (Matsubara et al., 1979). Reports that demonstrate hydrolysis of cefoperazone by TEM enzymes (Jones et al., 1987) and B-lactamase produced by Enterobacter cloacae (Fass, 1981) reveal that resistance to cefoperazone may be negated by the addition of an appropriate [3-1actamase inhibitor. From the Infectious Disease Section, Medical Service (C.E.F., J.A.M., L.M.S., J.T., D.N.G., L.R.P.), and the Microbiology Section, Laboratory Service (D.N.G., L.R.P.), Department of Veterans Affairs Medical Center, Minneapolis, Minnesota, USA. Present address of J.A. Moody: St. Paul-RamseyMedical Center, St. Paul, Minnesota. Address reprint requests to C. E. Fasching, VA Medical Center, One Veterans Drive, Infectious Disease Section (111F), Minneapolis, MN 55417, USA. Received 29 March 1990; revised and accepted 22 August 1990. © 1991 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010 0732-8893/91/$3.50
148
METHODS Bacteria The microorganisms tested comprised five different “infection groups” that were used in the subsequent data analysis. Enferococcus fuecuZis (EnFlO, EnF20, EnF21, and EnF30), Streptococcus avium (SAvl and SAv25), the pneumococci group of Pneumococcus (SPn5, SPnl7, SPn20, SPn25, SPn61, and SPnl31), the Enterobacteriaceae group of Escherichia coli (ECol), Cifrobucfer diversus (CiDlO), KZebsieZZupneumoniue (KPn85 and KPn95), Serrufiu murcescens (SeM157 and SeM230), the pseudomonads group of Pseudomonas
ueruginosu (PsA833, PsA845, PsA864, PsA876, PsA913, and PsA915), and the staphylococci group of SfuphyZococcus uureus (StA1295, StA1506, and StA1115), and SfuphyZococcus epidermidis (StE2841, StE1594, and StE3017). The staphylococci were selected on the basis of oxacillin (OX) and cephalothin (CE) susceptibility which were StA1295, OX MIC = 2 kg/ml and CE MIC = 2 pg/ml; StA1506, OX MIC = 16 pg/ml and CE MIC = 8 kg/ml; StA1115, OX MIC 2 128 pg/ml and CE MIC = 128 kg/ml; StE2841, OX MIC = 0.25 pg/ml and CE MIC = 0.25 kg/ml; StE1594, OX MIC 2 128 pg/ml and CE MIC = 4 Fgiml; and StE3017, OX MIC = 64 kg/ml and CE MIC = 64 @nl. Isolates tested were recovered from clinical specimens at the VA Medical Center, Minneapolis, and stored at - 70°C until used.
Animal Model The animal model was one previously described for the study to extravascular penetration of antibiotics (Peterson and Gerding 1978) and has been used to study the interaction of antimicrobials and bacteria in an in vivo model that is meant to simulate host neutropenia (Bamberger et al., 1986; Gerding et al., 1985; Moody et al., 1987; Peterson et al. 1984 and 1987). Extravascular spaces consisted of regenerated cellulose tubing (Union Carbide, Chicago, IL) that were tied at one end and occluded at the other with a cork through which tubing from a 21-gauge butterfly intermittent infusion set (Abbott Hospital Products, North Chicago, IL) was passed. These chambers allow for the free diffusion of molecules with molecular weights of 5.0 log10 CFU/ml from the growth control observed in the animal model correlated to a bactericidal action of the drug based on the number of organisms present in the original inoculum. Reductions between 5.0 and 3.0 log10 CFU/ml correspond to a bacteriostatic action of the therapy, and reductions of 43.0 log10 CFU/ml indicated growth at the infection site over the treatment period as compared to the original inoculum. We defined reductions of/>5.0 log~0 to indicate efficacy of the antimicrobial regimen.
Statistical Methods Statistical analysis was performed to determine confidence levels and significance values of the lOgl0 CFU/ml reductions seen in each infection group for the cefoperazone versus cefoperazone plus sulbactam treatment outcomes. These statistics were generated by a computer bootstrap analysis method (Diaconis and Efron, 1983) in which the distribution of outcome measurements was generated by repeated sampling with replacement on the underlying experimental data. These techniques are nonparametric, and more discriminatory than standard, formula-based nonparametric analysis.
RESULTS Cefoperazone concentrations at the infection site for each isolate w h e n treated with cefoperazone alone
150
C.E. Fasching et al.
and with the c o m b i n a t i o n of c e f o p e r a z o n e plus sulb a c t a m are listed in Table 1. The values g i v e n are arithmetic m e a n s a n d reflect an a v e r a g e of the 92hr (peak) a n d the 96-hr (trough) c h a m b e r levels. The p e a k a n d t r o u g h m e a s u r e m e n t s w e r e a v e r a g e d to p r o v i d e a basis for e a s y c o m p a r i s o n of antibiotic concentrations at the infection sites, as considerable variability w a s occasionally s e e n b e t w e e n isolates, infection g r o u p s , a n d t r e a t m e n t r e g i m e n . This variability is m o s t p r o n o u n c e d in the n o n - s u l b a c t a m containing r e g i m e n . C h a m b e r s in w h i c h cefoperazone concentrations w e r e low (128 >128 NT 8 32-64
NT NT 32 NT NT 32-64
0.5 2.0 0.25 0.25 0.25 0.25
NT NT NT NT NT NT
NT NT NT NT NT NT
0.5 0.25 0.5 ~ 128 2-32 > 128 > 128
4 NT ~>128 0.5 128 >128
>128 32 32-64 64 >128 64
>128 32 32 32-64 >128 64
CP
CP + SL
STA1295 STA1506 StAl115 STE2841 STE1594 STE3017
5.9 18.8 5.0 20.2 18.1 20.7
20.6 21.6 21.1 20.3 18.8 22.2
SPn5 SPn17 SPn20 SPn52 SPn61 SPn131
35.1 31.4 34.0 40.8 36.4 43.6
18.6 21.2 20.5 20.8 20.7 23.0
0.5 2.0 0.25 0.25 0.25 0.25
Ecol CiD10 KPn85 KPn95 SEM157 SeM230
0.4 15.2 0 0 0 0
15.1 22.3 23.2 10.9 20.5 14.8
1.0 0.25 0.5 ~128 32 64 64 >128 64
>128 32 64 32 >128 64
PsA833 PsA845 PsA864 PsA876 PsA913 PsA915
4.8 5.7 14.1 1.5 25.6 4.9
13.9 14.1 11.5 13.2 10.2 11.2
8 8 64 32 16 16
8 8 32 16 8 8
8 32 >128 1 8 32
CP + SL 4 32 128 1 8 32
CP, cefoperazone; SL, sulbactam; and NT, not tested due to organism elimination.
8-64 8->128 64-> 128 64-> 128 >128 16->128
8 8->128 16-128 32-64 8-64 16
Addition of Sulbactam to Cefoperazone
TABLE 2
151
Pharmacokinetic Data: Cefoperazone AUCs given in ixg • hr/ml Treatment Regimen
Infection Group
Cefoperazone
Cefoperazone plus Sulbactam
Staphylococci Pneumococci Enterobacteriaceae Enterococci Pseudomonads
18,608 28, 610 30,850 19,643 22,080
11,686 20,270 24,011 22,016 25,653
Average AUC
23,958
20,727
the addition of sulbactam. MBC testing demonstrated values that were within two wells of the MIC on all isolates except CiD10 (data not shown). High inoculum microdilution broth testing demonstrated inoculum effects (a ratio of high-conventional MIC >4) were present in some isolates of both the Enterobacteriaceae and the P. aeruginosa for cefoperazone (eight of 12 isolates) and cefoperazone plus sulbactam (eight of 12 isolates, data not shown). Agar dilution testing done on the staphylococci, enterococci, Enterobacteriaceae, and P. aeruginosa isolates indicated partial synergy between cefoperazone and sulbactam in isolate StAl115 at conventional inocula, and in isolates SEM157, SeM230, PsA876, and PsA915 at high inocula. Inoculum effects using agar dilution were found with cefoperazone and cefoperazone plus sulbactam for all isolates of Enterobacteriaceae, with cefoperazone in three of six and with cefoperazone plus sulbactam in one of six of the P. aeruginosa, but not in isolates of staphylococci or enterococci (data not shown). Microdilution susceptibility testing results at conventional inoculum done on isolates that survived in vivo treatment are also in Table 1. The incidence of resistance development as evidenced by an MIC increase of greater than two doubling dilutions was seen in nine of 30 isolates with the cefoperazone regimen (seven of these nine changed from susceptible to resistant) and five of 30 with the cefoperazone plus sulbactam regimen (four of these five changed from susceptible to resistant). Table 3 shows results of bacterial quantitation performed on infected chambers at 92 hr. Chambers were initially inoculated with 5.0 log10 CFU/ml, after 4 hr (when therapy was begun) the isolate density had increased from 1 to 3 logs. Control animals (no antibiotics) demonstrated growth of isolates that reached 8.0-9.0 log~0 CFU/ml at 24 hr after infection, and maintained or increased this number of bacterial counts by the end of the study (92 hr). To facilitate comparison of treatment regimens,
we summed mean log reductions for each infection group (n = 6 organisms per group). This represents a "total" reduction of the infection group by the treatment regimen. Confidence levels (n = 18, each organism studied three times) were calculated and are represented in Figure 1 along with reduction totals. The p values listed in Figure 1 were determined by comparing cefoperazone reductions to cefoperazone plus sulbactam reductions for each infection group. Statistically better (p ~< 0.05) bacterial reductions were seen u p o n the addition of sulbactam to cefoperazone with the staphylococci, Enterobacteriaceae, and P. aeruginosa infection groups. Among the staphylococci, a ~>5.0 log10 CFU/ml reduction with cefoperazone alone was seen in one of the six isolates. Four isolates (STA1295, StAl115, STE1594, and STE3017) were ~-lactamase-producing staphylococci as determined by nitrocefin testing. isolates STA1506 and STE2841 did not produce 13-1actamase even after oxacillin induction on agar. The addition of sulbactam to cefoperazone increased the treatment efficacy from one to five staphylococci. Three of the four methicillin-resistant staphylococcal isolates were reduced />5..0 log10 CFU/ml with the cefoperazone plus sulbactam therapy. Vancomycin therapy had previously been demonstrated to reduce four out of six isolates i>5.0 loga0 CFU/ml, despite all six isolates testing susceptible in vitro to vancomycin. OxaciUin treatment also previously gave efficacious reductions in four of the six staphylococci, with only two of the six staphylococci testing susceptible in vitro. The six pneumococci tested in this study demonstrated susceptible cefoperazone MICs and all showed in vivo bacterial reductions of 1>5.0 loglo CFU/ml. There was no antagonism demonstrated by the addition of sulbactam. Among the Enterobacteriaceae, only one isolate (CiD10) demonstrated a difference of ~5.0 log10 in bacterial growth from untreated control with cefoperazone treatment, despite four of six isolates testing susceptible to cefoperazone alone in vitro. CiD10 had a cefoperazone chamber concentration which was much greater than the other five Enterobacteriaceae. Cefoperazone concentrations at infection sites were increased and the number of isolates (five of six) which showed efficacy in vivo was increased when sulbactam was added to cefoperazone therapy. In vivo efficacy was not demonstrated with cefoperazone or cefoperazone plus sulbactam treatment regimens against the enterococci that we tested. These isolates were 13-1actamase negative by nitrocefin testing. The addition of amikacin to cefoperazone plus sulbactam was investigated as in vitro susceptibility testing indicated synergy (data not show) in two of the six enterococcal isolates (EnF21 and
152
TABLE 3
C.E. Fasching et al.
Bacterial Quantitation in Log~0 CFU/ml at End of Study a n d Reductions ( Growth Control
Isolate
GC
CP
CP + SL
STA1295 STA1506 StAl115 STE2841 STE1594 STE3017
7.4 9.3 10.2 9.4 8.9 9.1
6.8 6.0 7.7 2.5 5.2 6.6
(0.6) (3.3) (2.5) (6.9) (3.7) (2.5)
~2.0 3.8 4.8 ~2.0 ~2.0 5.8
(5.4) (5.5) (5.4) (7.4) (6.9) (3.3)
SPn5 SPn17 SPn20 SPn25 SPn61 SPn131
8.7 8.8 9.0 9.1 9.1 9.1
~2.0 ~2.0 ~2.0 ~2.0 ~2.0 ~2.0
(6.7) (6.8) (7.0) (7.1) (7.1) (7.1)
~2.0 ~2.0 ~2.0 ~2.0 ~2.0 ~2.0
(6.7) (6.8) (7.0) (7.1) (7.1) (7.1)
ECol CiD10 KPn85 KPn95 SEM157 SeM230
10.6 10.3 10.3 9.6 9.9 10.1
6.9 ~3.0 8.0 8.9 9.7 9.4
(3.7) (7.3) (2.3) (0.7) (0.2) (0.7)
2.9 ~3.0 ~4.1 ~3.0 ~3.8 ~5.3
(7.7) (7.3) (6.2) (6.6) (6.1) (4.8)
) from
OX ~2.0 2.8 7.0 2.0 2.9 8.4
(5.4) (6.5) (3.2) (7.4) (6.0) (0.7)
VN 2.1 3.3 4.5 6.3 2.0 6.7
(7.8) (6.0) (5.7) (3.1) (6.9) (2.4)
CP + SL + AK SAvl EnF10 EnF20 EnF21 SAv25 EnF30
9.3 9.7 9.8 9.8 8.8 10.1
8.0 8.4 6.9 8.7 8.8 9.0
(1.3) (1.3) (2.9) (1.1) (0.0) (1.1)
8.1 8.2 7.6 8.6 9.0 8.5
(1.2) (1.5) (2.1) (1.2) (--) (1.6)
6.5 4.8 6.1 7.0 7.0 5.6
(2.8) (4.9) (3.7) (2.8) (1.8) (4.5)
~2.0 ~2.0 2.3 5.5 2.3 ~2.0
(6.3) (7.0) (6.7) (2.8) (6.1) (7.3)
CP + AK PsA833 PsA845 PsA864 PsA876 PsA913 PsA915
8.3 9.0 9.0 8.3 8.4 9.3
7.5 8.3 8.4 8.9 7.2 8.1
(0.8) (0.7) (0.6) (--) (1.2) (1.2)
5.6 5.7 6.5 7.7 6.6 8.1
(2.7) (3.3) (2.5) (0.6) (1.8) (1.2)
~2.0 6.2 3.4 8.8 ~2.0 ~2.0
(6.3) (2.8) (5.6) (--) (6.4) (7.3)
GC, growth control; OX, oxacillin; VN, vancomycin; and AK, amikacin. Other abbreviations as in Table 1.
SAv25). One isolate, EnF20, r e s p o n d e d in vivo to the addition of amikacin with a reduction of I>5.0 log10 CFU/ml. A m o n g the six P. aeruginosa tested, cefoperazone and cefoperazone plus sulbactam failed to achieve a />5.0 log10 CFU/ml reduction in bacterial growth in vivo. The addition of sulbactam increased the number of susceptible isolates in vitro from four to five, and increased the average cefoperazone concentrations by 2.3- to 8.8-fold in four of the six chambers, however, it did not increase the in vivo efficacy according to the I>5.0 loglo CFU/ml reduction criteria. High inoculum susceptibility testing (microdilution) for these P. aeruginosa indicated cefoperazone resistance in five of six, and indicated
cefoperazone plus sulbactam resistance in three of six. All the p s e u d o m o n a l isolates h a d sensitive amikacin MICs, and synergy testing b e t w e e n amikacin versus cefoperazone and, amikacin versus cefoperazone plus sulbactam, d e m o n s t r a t e d synergism in two of six and five of six isolates, respectively (data not show). Five of six isolates were reduced >5.0 log10 CFU/ml w h e n cefoperazone plus sulbactam plus amikacin was tested against P. aeruginosa (Table 3).
DISCUSSION Cefoperazone acts u p o n penicillin-binding proteins to inhibit peptidoglycan synthesis in Gram-negative
153
Addition of Sulbactam to Cefoperazone 45 -
>
40
c-
0 ¢..
,.,..
c 0 o
35 30
c-
•~
25
cO 0
-~
20
"I3
m o &
15
0 -..1
E -"
5
0 Spn p=0.50
Enc p=0.47
Stc p=O.01
Ent p
147
ANTIMICROBIAL CLINICAL STUDIES
Antibacterial Activity of Cefoperazone and Cefoperazone Plus Sulbactam in a Neutropenic Site Model Claudine E. Fasching, Julia A. Moody, Leann M. Sinn, Jane Tenquist, Dale N. Gerding, and Lance R. Peterson
Efficacy of cefoperazone versus cefoperazone plus sulbactam was studied in a rabbit neutropenic site infection model against a broad range of clinical isolates including six isolates each of staphylococci, enterococci, pneumococci, Enterobacteriaceae, and Pseudomonas aeruginosa. Therapy of cefoperazone plus sulbactam demonstrated enhanced efficacy against the staphylococci, pseudomonads, and Enterobacteriaceae. The activity of cefoperazone against enterococci and pneumococci
was not enhanced or inhibited by the addition of sulbactam. Increased concentrations of cefoperazone found at the infection sites when sulbactam was added to the therapeutic regimen indicates that sulbactam provided a protection to cefoperazone from [3-1actamases produced by staphylococci and Enterobacteriaceae. The combination improved the efficacy of cefoperazone in this animal model.
INTRODUCTION
Sulbactam inhibits B-lactamases from Staphylococcus aureus and plasmid-mediated [3-1actamases from Enterobacteriaceae (Vu and Nikaido, 1985), and sulbactam has not been shown to routinely induce J3-1actamase production in strains of Enterobacteriaceae (Moosdeen et al., 1986). The combination of cefoperazone plus sulbactam has shown a synergistic action in vitro by reducing minimum inhibitory concentration (MIC) of cefoperazone 2- to 32-fold in strains susceptible to cefoperazone (Jones et al., 1985), and by extending the antimicrobial spectrum of cefoperazone (Jones et al., 1987). We evaluated the activity of cefoperazone and cefoperazone plus sulbactam against organisms that have previously been studied in a rabbit model, developed in our laboratory, which simulates a closed-space, neutropenic site of infection. The purpose of this study was (a) to compare the efficacy of cefoperazone to the combination of cefoperazone plus sulbactam in an in vivo model simulating a neutropenic infection site, (b) to compare results from in vitro susceptibility testing to in vivo quantitative bacterial reductions achieved through antimicrobial therapy, and (c) to investigate the occurrence of any resistance development during drug therapy.
Cefoperazone is a third-generation, extended spectrum cephalosporin that has broad antimicrobial activity including Pseudomonas aeruginosa and anaerobes (Jones and Barry, 1983). Hydrolysis of cefoperazone by most Gram-negative f3-1actamases is relatively slow (Matsubara et al., 1979). Reports that demonstrate hydrolysis of cefoperazone by TEM enzymes (Jones et al., 1987) and B-lactamase produced by Enterobacter cloacae (Fass, 1981) reveal that resistance to cefoperazone may be negated by the addition of an appropriate [3-1actamase inhibitor. From the Infectious Disease Section, Medical Service (C.E.F., J.A.M., L.M.S., J.T., D.N.G., L.R.P.), and the Microbiology Section, Laboratory Service (D.N.G., L.R.P.), Department of Veterans Affairs Medical Center, Minneapolis, Minnesota, USA. Present address of J.A. Moody: St. Paul-RamseyMedical Center, St. Paul, Minnesota. Address reprint requests to C. E. Fasching, VA Medical Center, One Veterans Drive, Infectious Disease Section (111F), Minneapolis, MN 55417, USA. Received 29 March 1990; revised and accepted 22 August 1990. © 1991 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010 0732-8893/91/$3.50
148
METHODS Bacteria The microorganisms tested comprised five different “infection groups” that were used in the subsequent data analysis. Enferococcus fuecuZis (EnFlO, EnF20, EnF21, and EnF30), Streptococcus avium (SAvl and SAv25), the pneumococci group of Pneumococcus (SPn5, SPnl7, SPn20, SPn25, SPn61, and SPnl31), the Enterobacteriaceae group of Escherichia coli (ECol), Cifrobucfer diversus (CiDlO), KZebsieZZupneumoniue (KPn85 and KPn95), Serrufiu murcescens (SeM157 and SeM230), the pseudomonads group of Pseudomonas
ueruginosu (PsA833, PsA845, PsA864, PsA876, PsA913, and PsA915), and the staphylococci group of SfuphyZococcus uureus (StA1295, StA1506, and StA1115), and SfuphyZococcus epidermidis (StE2841, StE1594, and StE3017). The staphylococci were selected on the basis of oxacillin (OX) and cephalothin (CE) susceptibility which were StA1295, OX MIC = 2 kg/ml and CE MIC = 2 pg/ml; StA1506, OX MIC = 16 pg/ml and CE MIC = 8 kg/ml; StA1115, OX MIC 2 128 pg/ml and CE MIC = 128 kg/ml; StE2841, OX MIC = 0.25 pg/ml and CE MIC = 0.25 kg/ml; StE1594, OX MIC 2 128 pg/ml and CE MIC = 4 Fgiml; and StE3017, OX MIC = 64 kg/ml and CE MIC = 64 @nl. Isolates tested were recovered from clinical specimens at the VA Medical Center, Minneapolis, and stored at - 70°C until used.
Animal Model The animal model was one previously described for the study to extravascular penetration of antibiotics (Peterson and Gerding 1978) and has been used to study the interaction of antimicrobials and bacteria in an in vivo model that is meant to simulate host neutropenia (Bamberger et al., 1986; Gerding et al., 1985; Moody et al., 1987; Peterson et al. 1984 and 1987). Extravascular spaces consisted of regenerated cellulose tubing (Union Carbide, Chicago, IL) that were tied at one end and occluded at the other with a cork through which tubing from a 21-gauge butterfly intermittent infusion set (Abbott Hospital Products, North Chicago, IL) was passed. These chambers allow for the free diffusion of molecules with molecular weights of 5.0 log10 CFU/ml from the growth control observed in the animal model correlated to a bactericidal action of the drug based on the number of organisms present in the original inoculum. Reductions between 5.0 and 3.0 log10 CFU/ml correspond to a bacteriostatic action of the therapy, and reductions of 43.0 log10 CFU/ml indicated growth at the infection site over the treatment period as compared to the original inoculum. We defined reductions of/>5.0 log~0 to indicate efficacy of the antimicrobial regimen.
Statistical Methods Statistical analysis was performed to determine confidence levels and significance values of the lOgl0 CFU/ml reductions seen in each infection group for the cefoperazone versus cefoperazone plus sulbactam treatment outcomes. These statistics were generated by a computer bootstrap analysis method (Diaconis and Efron, 1983) in which the distribution of outcome measurements was generated by repeated sampling with replacement on the underlying experimental data. These techniques are nonparametric, and more discriminatory than standard, formula-based nonparametric analysis.
RESULTS Cefoperazone concentrations at the infection site for each isolate w h e n treated with cefoperazone alone
150
C.E. Fasching et al.
and with the c o m b i n a t i o n of c e f o p e r a z o n e plus sulb a c t a m are listed in Table 1. The values g i v e n are arithmetic m e a n s a n d reflect an a v e r a g e of the 92hr (peak) a n d the 96-hr (trough) c h a m b e r levels. The p e a k a n d t r o u g h m e a s u r e m e n t s w e r e a v e r a g e d to p r o v i d e a basis for e a s y c o m p a r i s o n of antibiotic concentrations at the infection sites, as considerable variability w a s occasionally s e e n b e t w e e n isolates, infection g r o u p s , a n d t r e a t m e n t r e g i m e n . This variability is m o s t p r o n o u n c e d in the n o n - s u l b a c t a m containing r e g i m e n . C h a m b e r s in w h i c h cefoperazone concentrations w e r e low (128 >128 NT 8 32-64
NT NT 32 NT NT 32-64
0.5 2.0 0.25 0.25 0.25 0.25
NT NT NT NT NT NT
NT NT NT NT NT NT
0.5 0.25 0.5 ~ 128 2-32 > 128 > 128
4 NT ~>128 0.5 128 >128
>128 32 32-64 64 >128 64
>128 32 32 32-64 >128 64
CP
CP + SL
STA1295 STA1506 StAl115 STE2841 STE1594 STE3017
5.9 18.8 5.0 20.2 18.1 20.7
20.6 21.6 21.1 20.3 18.8 22.2
SPn5 SPn17 SPn20 SPn52 SPn61 SPn131
35.1 31.4 34.0 40.8 36.4 43.6
18.6 21.2 20.5 20.8 20.7 23.0
0.5 2.0 0.25 0.25 0.25 0.25
Ecol CiD10 KPn85 KPn95 SEM157 SeM230
0.4 15.2 0 0 0 0
15.1 22.3 23.2 10.9 20.5 14.8
1.0 0.25 0.5 ~128 32 64 64 >128 64
>128 32 64 32 >128 64
PsA833 PsA845 PsA864 PsA876 PsA913 PsA915
4.8 5.7 14.1 1.5 25.6 4.9
13.9 14.1 11.5 13.2 10.2 11.2
8 8 64 32 16 16
8 8 32 16 8 8
8 32 >128 1 8 32
CP + SL 4 32 128 1 8 32
CP, cefoperazone; SL, sulbactam; and NT, not tested due to organism elimination.
8-64 8->128 64-> 128 64-> 128 >128 16->128
8 8->128 16-128 32-64 8-64 16
Addition of Sulbactam to Cefoperazone
TABLE 2
151
Pharmacokinetic Data: Cefoperazone AUCs given in ixg • hr/ml Treatment Regimen
Infection Group
Cefoperazone
Cefoperazone plus Sulbactam
Staphylococci Pneumococci Enterobacteriaceae Enterococci Pseudomonads
18,608 28, 610 30,850 19,643 22,080
11,686 20,270 24,011 22,016 25,653
Average AUC
23,958
20,727
the addition of sulbactam. MBC testing demonstrated values that were within two wells of the MIC on all isolates except CiD10 (data not shown). High inoculum microdilution broth testing demonstrated inoculum effects (a ratio of high-conventional MIC >4) were present in some isolates of both the Enterobacteriaceae and the P. aeruginosa for cefoperazone (eight of 12 isolates) and cefoperazone plus sulbactam (eight of 12 isolates, data not shown). Agar dilution testing done on the staphylococci, enterococci, Enterobacteriaceae, and P. aeruginosa isolates indicated partial synergy between cefoperazone and sulbactam in isolate StAl115 at conventional inocula, and in isolates SEM157, SeM230, PsA876, and PsA915 at high inocula. Inoculum effects using agar dilution were found with cefoperazone and cefoperazone plus sulbactam for all isolates of Enterobacteriaceae, with cefoperazone in three of six and with cefoperazone plus sulbactam in one of six of the P. aeruginosa, but not in isolates of staphylococci or enterococci (data not shown). Microdilution susceptibility testing results at conventional inoculum done on isolates that survived in vivo treatment are also in Table 1. The incidence of resistance development as evidenced by an MIC increase of greater than two doubling dilutions was seen in nine of 30 isolates with the cefoperazone regimen (seven of these nine changed from susceptible to resistant) and five of 30 with the cefoperazone plus sulbactam regimen (four of these five changed from susceptible to resistant). Table 3 shows results of bacterial quantitation performed on infected chambers at 92 hr. Chambers were initially inoculated with 5.0 log10 CFU/ml, after 4 hr (when therapy was begun) the isolate density had increased from 1 to 3 logs. Control animals (no antibiotics) demonstrated growth of isolates that reached 8.0-9.0 log~0 CFU/ml at 24 hr after infection, and maintained or increased this number of bacterial counts by the end of the study (92 hr). To facilitate comparison of treatment regimens,
we summed mean log reductions for each infection group (n = 6 organisms per group). This represents a "total" reduction of the infection group by the treatment regimen. Confidence levels (n = 18, each organism studied three times) were calculated and are represented in Figure 1 along with reduction totals. The p values listed in Figure 1 were determined by comparing cefoperazone reductions to cefoperazone plus sulbactam reductions for each infection group. Statistically better (p ~< 0.05) bacterial reductions were seen u p o n the addition of sulbactam to cefoperazone with the staphylococci, Enterobacteriaceae, and P. aeruginosa infection groups. Among the staphylococci, a ~>5.0 log10 CFU/ml reduction with cefoperazone alone was seen in one of the six isolates. Four isolates (STA1295, StAl115, STE1594, and STE3017) were ~-lactamase-producing staphylococci as determined by nitrocefin testing. isolates STA1506 and STE2841 did not produce 13-1actamase even after oxacillin induction on agar. The addition of sulbactam to cefoperazone increased the treatment efficacy from one to five staphylococci. Three of the four methicillin-resistant staphylococcal isolates were reduced />5..0 log10 CFU/ml with the cefoperazone plus sulbactam therapy. Vancomycin therapy had previously been demonstrated to reduce four out of six isolates i>5.0 loga0 CFU/ml, despite all six isolates testing susceptible in vitro to vancomycin. OxaciUin treatment also previously gave efficacious reductions in four of the six staphylococci, with only two of the six staphylococci testing susceptible in vitro. The six pneumococci tested in this study demonstrated susceptible cefoperazone MICs and all showed in vivo bacterial reductions of 1>5.0 loglo CFU/ml. There was no antagonism demonstrated by the addition of sulbactam. Among the Enterobacteriaceae, only one isolate (CiD10) demonstrated a difference of ~5.0 log10 in bacterial growth from untreated control with cefoperazone treatment, despite four of six isolates testing susceptible to cefoperazone alone in vitro. CiD10 had a cefoperazone chamber concentration which was much greater than the other five Enterobacteriaceae. Cefoperazone concentrations at infection sites were increased and the number of isolates (five of six) which showed efficacy in vivo was increased when sulbactam was added to cefoperazone therapy. In vivo efficacy was not demonstrated with cefoperazone or cefoperazone plus sulbactam treatment regimens against the enterococci that we tested. These isolates were 13-1actamase negative by nitrocefin testing. The addition of amikacin to cefoperazone plus sulbactam was investigated as in vitro susceptibility testing indicated synergy (data not show) in two of the six enterococcal isolates (EnF21 and
152
TABLE 3
C.E. Fasching et al.
Bacterial Quantitation in Log~0 CFU/ml at End of Study a n d Reductions ( Growth Control
Isolate
GC
CP
CP + SL
STA1295 STA1506 StAl115 STE2841 STE1594 STE3017
7.4 9.3 10.2 9.4 8.9 9.1
6.8 6.0 7.7 2.5 5.2 6.6
(0.6) (3.3) (2.5) (6.9) (3.7) (2.5)
~2.0 3.8 4.8 ~2.0 ~2.0 5.8
(5.4) (5.5) (5.4) (7.4) (6.9) (3.3)
SPn5 SPn17 SPn20 SPn25 SPn61 SPn131
8.7 8.8 9.0 9.1 9.1 9.1
~2.0 ~2.0 ~2.0 ~2.0 ~2.0 ~2.0
(6.7) (6.8) (7.0) (7.1) (7.1) (7.1)
~2.0 ~2.0 ~2.0 ~2.0 ~2.0 ~2.0
(6.7) (6.8) (7.0) (7.1) (7.1) (7.1)
ECol CiD10 KPn85 KPn95 SEM157 SeM230
10.6 10.3 10.3 9.6 9.9 10.1
6.9 ~3.0 8.0 8.9 9.7 9.4
(3.7) (7.3) (2.3) (0.7) (0.2) (0.7)
2.9 ~3.0 ~4.1 ~3.0 ~3.8 ~5.3
(7.7) (7.3) (6.2) (6.6) (6.1) (4.8)
) from
OX ~2.0 2.8 7.0 2.0 2.9 8.4
(5.4) (6.5) (3.2) (7.4) (6.0) (0.7)
VN 2.1 3.3 4.5 6.3 2.0 6.7
(7.8) (6.0) (5.7) (3.1) (6.9) (2.4)
CP + SL + AK SAvl EnF10 EnF20 EnF21 SAv25 EnF30
9.3 9.7 9.8 9.8 8.8 10.1
8.0 8.4 6.9 8.7 8.8 9.0
(1.3) (1.3) (2.9) (1.1) (0.0) (1.1)
8.1 8.2 7.6 8.6 9.0 8.5
(1.2) (1.5) (2.1) (1.2) (--) (1.6)
6.5 4.8 6.1 7.0 7.0 5.6
(2.8) (4.9) (3.7) (2.8) (1.8) (4.5)
~2.0 ~2.0 2.3 5.5 2.3 ~2.0
(6.3) (7.0) (6.7) (2.8) (6.1) (7.3)
CP + AK PsA833 PsA845 PsA864 PsA876 PsA913 PsA915
8.3 9.0 9.0 8.3 8.4 9.3
7.5 8.3 8.4 8.9 7.2 8.1
(0.8) (0.7) (0.6) (--) (1.2) (1.2)
5.6 5.7 6.5 7.7 6.6 8.1
(2.7) (3.3) (2.5) (0.6) (1.8) (1.2)
~2.0 6.2 3.4 8.8 ~2.0 ~2.0
(6.3) (2.8) (5.6) (--) (6.4) (7.3)
GC, growth control; OX, oxacillin; VN, vancomycin; and AK, amikacin. Other abbreviations as in Table 1.
SAv25). One isolate, EnF20, r e s p o n d e d in vivo to the addition of amikacin with a reduction of I>5.0 log10 CFU/ml. A m o n g the six P. aeruginosa tested, cefoperazone and cefoperazone plus sulbactam failed to achieve a />5.0 log10 CFU/ml reduction in bacterial growth in vivo. The addition of sulbactam increased the number of susceptible isolates in vitro from four to five, and increased the average cefoperazone concentrations by 2.3- to 8.8-fold in four of the six chambers, however, it did not increase the in vivo efficacy according to the I>5.0 loglo CFU/ml reduction criteria. High inoculum susceptibility testing (microdilution) for these P. aeruginosa indicated cefoperazone resistance in five of six, and indicated
cefoperazone plus sulbactam resistance in three of six. All the p s e u d o m o n a l isolates h a d sensitive amikacin MICs, and synergy testing b e t w e e n amikacin versus cefoperazone and, amikacin versus cefoperazone plus sulbactam, d e m o n s t r a t e d synergism in two of six and five of six isolates, respectively (data not show). Five of six isolates were reduced >5.0 log10 CFU/ml w h e n cefoperazone plus sulbactam plus amikacin was tested against P. aeruginosa (Table 3).
DISCUSSION Cefoperazone acts u p o n penicillin-binding proteins to inhibit peptidoglycan synthesis in Gram-negative
153
Addition of Sulbactam to Cefoperazone 45 -
>
40
c-
0 ¢..
,.,..
c 0 o
35 30
c-
•~
25
cO 0
-~
20
"I3
m o &
15
0 -..1
E -"
5
0 Spn p=0.50
Enc p=0.47
Stc p=O.01
Ent p
