Skip to main content
NCBI home page
Antimicrobial Agents and Chemotherapy logoLink to Antimicrobial Agents and Chemotherapy
. 2008 Nov 10;53(2):370–384. doi: 10.1128/AAC.01047-08

Establishment of In Vitro Susceptibility Testing Methodologies and Comparative Activities of Piperacillin in Combination with the Penem β-Lactamase Inhibitor BLI-489 ▿

Peter J Petersen 1,*, C Hal Jones 1, Aranapakam M Venkatesan 2, Tarek S Mansour 2, Steven J Projan 3,†, Patricia A Bradford 1
PMCID: PMC2630610  PMID: 19001109

Abstract

The novel bicyclic penem inhibitor BLI-489 has demonstrated activity as an inhibitor of class A, C, and D β-lactamases. To determine the combination of piperacillin and BLI-489 to be used in susceptibility testing that would most accurately identify susceptible and resistant isolates, a predictor panel of β-lactamase-producing bacteria was utilized to determine the reliability of the combination of piperacillin-BLI-489 at a constant inhibitor concentration of 2 or 4 μg/ml and at ratios of 1:1, 2:1, 4:1, and 8:1. There were a number of strains that would be falsely reported as susceptible or intermediate if tested with the ratios of 1:1 and 2:1, whereas the constant concentration of 2 μg/ml of BLI-489 and the ratio of 8:1 had a tendency to overpredict resistance. Similar MICs were obtained with piperacillin-BLI-489 in a 4:1 ratio and when BLI-489 was held constant at 4 μg/ml. Based on these results, an in vitro testing methodology employing a constant concentration of 4 μg/ml BLI-489 was used to evaluate the combination of piperacillin-BLI-489 against a larger panel of recently identified clinical isolates. Approximately 55% of all of the enteric bacilli tested were nonsusceptible to piperacillin alone (MIC ≥ 32 μg/ml). However, 92% of these piperacillin nonsusceptible strains were inhibited by ≤16 μg/ml piperacillin-BLI-489; in contrast, only 66% were inhibited by ≤16 μg/ml piperacillin-tazobactam. The combination of piperacillin-BLI-489 also demonstrated improved activity compared to that of piperacillin-tazobactam against the problematic extended-spectrum β-lactamase- and AmpC-expressing strains.

The continued emergence of resistance to β-lactam antibiotics due to the increasing variety of β-lactamase enzymes, including extended-spectrum β-lactamases (ESBL) and AmpC, is recognized as a global medical problem (6, 23, 47). One successful strategy that has been employed to circumvent this resistance has been the coadministration of a β-lactamase inhibitor along with a β-lactam antibiotic. The current commercially available β-lactam-β-lactamase inhibitor combinations include the coadministration of a penicillin such as piperacillin, amoxicillin (amoxicilline), or ticarcillin, and ampicillin with a β-lactamase inhibitor such as tazobactam, clavulanic acid, or sulbactam (31). Although these β-lactamase inhibitors effectively target the molecular class A enzymes, which historically were the predominant β-lactamase produced by bacteria, they lack significant activity against class C (AmpC) β-lactamase-producing organisms and ESBL-producing (classes A and D) organisms, which are now much more prevalent among clinical pathogens (17, 31, 33).

The class C (AmpC) β-lactamase enzymes are naturally occurring inducible enzymes in gram-negative bacteria such as Citrobacter spp., Enterobacter spp., Pseudomonas aeruginosa, Morganella morganii, Providencia spp., and Serratia spp. However, there now have been reports of infections caused by strains that are stably derepressed, causing the overexpression of the class C enzymes and thereby conferring resistance to current inhibitor combinations and expanded-spectrum cephalosporins, including cefepime (1, 2, 17, 41). The prevalence of class A ESBL-producing strains among Escherichia coli and Klebsiella pneumoniae has exposed the limited treatment options for these serious infections (22). Case reports of patient treatment failures with cefepime for pathogens for which the MICs were susceptible further demonstrates the need for novel antibacterial agents (4, 28). Tigecycline, carbapenems, and the polymyxins remain the only existing therapeutic alternative for many problematic gram-negative pathogens (26, 30, 39). However, the recent increase and spread of carbapenemases such as KPC-, GES- and VIM-type enzymes already are starting to limit the clinical use of carbapenems (32, 46).

It was reported that bicyclic and tricyclic substitution via a methylidene linkage to the 6 position of the penem molecule conferred activity as an inhibitor of class A, C, and D β-lactamase enzymes (44, 45, 48). The enhancement of the activity and efficacy of piperacillin when administered in combination both in vitro and in vivo with the 6-substituted penems was established (48). These new β-lactamase inhibitors offer a broader spectrum of activity compared to those of the current commercially available inhibitors. From these compounds, the bicyclic-6-methylidene penem molecule BLI-489 was chosen as the lead compound to be combined with piperacillin (Fig. 1). In this study, a predictor panel approach was employed to determine the optimal concentration of BLI-489 to use in combination with piperacillin for in vitro testing. After establishing the precise testing methodology, the in vitro activity of piperacillin-BLI-489 and comparative agents were determined against a larger and more diverse collection of recently identified clinical isolates.

FIG. 1.

FIG. 1.

Open in a new tab

Chemical structure of the penem β-lactamase inhibitor BLI-489.

MATERIALS AND METHODS

Organisms.

Organisms used in this study were either isogenic laboratory strains expressing various β-lactamases or clinical isolates taken from the Wyeth Research culture collection and were collected from various worldwide medical centers. The predictor panel (Table 1) contained 113 strains producing β-lactamase enzymes from molecular classes A, B, C, and D (18). Quality control strains were from the American Type Culture Collection (Rockville, MD) as recommended by the Clinical Laboratory Standards Institute (CLSI) (20, 21).

TABLE 1.

Strains of the predictor panel

Organism Reference no. β-Lactamaseb Molecular class Strain type Source or reference
A. baumannii GC 7684 AmpC C Clinical Wyeth strain collectiona
A. baumannii GC 7685 TEM-1, AmpC A, C Clinical Wyeth strain collectiona
A. baumannii GC 7692 TEM-1, AmpC A, C Clinical Wyeth strain collectiona
C. freundii GC 4164 AmpC(i) C Clinical Wyeth strain collection
C. freundii GC 4171 TEM-1, AmpC(i) A, C Clinical Wyeth strain collection
C. freundii GC 4187 AmpC(c) C Clinical Wyeth strain collection
C. freundii PT 1499 AmpC(c) C Clinical 29
E. aerogenes GC 7845 AmpC C Clinical Wyeth strain collection
E. aerogenes GC 7036 TEM-24, AmpC(i) A (ESBL), C Clinical 5
E. aerogenes GC 7039 TEM-4, AmpC(i) A (ESBL), C Clinical 5
E. cloacae GC 1475 P99 C Clinical Wyeth strain collection
E. cloacae GC 1477 AmpC C Laboratory Wyeth strain collection
E. cloacae GC 1713 AmpC(c) C Laboratory Wyeth strain collection
E. cloacae GC 4142 AmpC(c) C Clinical Wyeth strain collection
E. cloacae GC 6991 AmpC C Clinical Wyeth strain collection
E. cloacae GC 7052 SHV-5, AmpC(i) A (ESBL), C Clinical 5
E. cloacae GC 7055 TEM-1, SHV-12, AmpC(i) A, A (ESBL), C Clinical 5
E. cloacae GC 7065 TEM-26, SHV-12, AmpC(i) A, A (ESBL), C Clinical 5
E. cloacae PT 1494 AmpC(i) C Clinical 29
E. cloacae PT 197 AmpC(c) C Clinical 29
E. coli GAR 5929 TEM-1, CTX-M-15, OXA-1, AmpC A, A (ESBL), D, C Clinical Wyeth strain collection
E. coli GAR 6649 TEM-1, CTX-M-5, OXA-1 A, A (ESBL), D Clinical Wyeth strain collection
E. coli GC 1480 OXA-1 D Clinical Wyeth strain collection
E. coli GC 1807 OXA-7 D Clinical Wyeth strain collection
E. coli GC 2253 IRT-2 A (IRT)i Clinical Wyeth strain collectionc
E. coli GC 2295 TEM-1, AmpC A, C Clinical 9
E. coli GC 2805 CcrA B Laboratory 38
E. coli GC 2847 TEM-1 (in C600) A Laboratory 11
E. coli GC 2851 TEM-2 (in C600) A Laboratory 11
E. coli GC 2852 TEM-3 (in C600) A (ESBL) Laboratory 11
E. coli GC 2853 TEM-4 (in C600) A (ESBL) Laboratory 11
E. coli GC 2856 TEM-5 (in C600) A (ESBL) Laboratory 11
E. coli GC 2857 TEM-7 (in C600) A (ESBL) Laboratory 11
E. coli GC 2858 TEM-8 (in C600) A (ESBL) Laboratory 11
E. coli GC 2859 TEM-9 (in C600) A (ESBL) Laboratory 11
E. coli GC 2863 TEM-10 (in C600) A (ESBL) Laboratory 11
E. coli GC 2864 TEM-12 (in C600) A (ESBL) Laboratory 11
E. coli GC 2865 SHV-1 (in C600) A Laboratory 11
E. coli GC 2869 SHV-2 (in C600) A (ESBL) Laboratory 11
E. coli GC 2870 SHV-3 (in C600) A (ESBL) Laboratory 11
E. coli GC 2871 SHV-4 (in C600) A (ESBL) Laboratory 11
E. coli GC 2874 SHV-5 (in C600) A (ESBL) Laboratory 11
E. coli GC 2875 OXA-1 (in C600) D Laboratory 11
E. coli GC 2876 OXA-2 (in C600) D Laboratory 11
E. coli GC 2877 OXA-3 (in C600) D Laboratory 11
E. coli GC 2878 OXA-4 (in C600) D Laboratory 11
E. coli GC 2879 OXA-5 (in C600) D Laboratory 11
E. coli GC 2880 OXA-6 (in C600) D Laboratory 11
E. coli GC 2881 OXA-7 (in C600) D Laboratory 11
E. coli GC 2882 PSE-1 (in C600) A Laboratory 11
E. coli GC 2883 OXA-10/PSE-2 (in C600) D Laboratory 11
E. coli GC 2884 PSE-3 (in C600) A Laboratory 11
E. coli GC 2885 PSE-4 (in C600) A Laboratory 11
E. coli GC 2886 TEM-8 (in C600) A Laboratory 11
E. coli GC 2891 MIR-1 (in C600) C Laboratory 11
E. coli GC 2894 AmpC(i) (in C600) C Laboratory 11
E. coli GC 2904 TEM-6 (in C600) A Laboratory 11
E. coli GC 2905 P99 (in C600) C Laboratory 11
E. coli GC 2906 IMI-1 (in C600) A (cpase)j Laboratory 11
E. coli GC 4972 TEM-1, OXA-1 A, D Clinical Wyeth strain collectiond
E. coli GC 6539 CMY-2 C Bovine 10
E. coli GC 6265 TEM-1 A Clinical Wyeth strain collection
E. coli GC 1499 TEM-1, TEM-4 A, A (ESBL) Clinical Wyeth strain collection
E. coli GC 1684 TEM-10 A (ESBL) Clinical 37
E. coli GC 1695 TEM-1, TEM-10 A, A (ESBL) Clinical 8
E. coli GC 1995 TEM-10 A (ESBL) Clinical 8
E. coli GC 2009 TEM-10 A (ESBL) Clinical 8
E. coli GC 2015 SHV-1, TEM-10 A, A (ESBL) Clinical 8
E. coli GC 2146 TEM-1, SHV-7 A, A (ESBL) Clinical 15
E. coli GC 2300 TEM-28 A (ESBL) Clinical 9
E. coli GC 2400 TEM-43 A (ESBL) Clinical 51
E. coli GC 4971 TEM-29, OXA-1 A (ESBL), D Clinical Wyeth strain collectiond
E. coli GC 5901 TEM-1, SHV-8 A, A (ESBL) Clinical 36
E. coli GC 6197 TEM-1, SHV-7, CMY-2 A, A (ESBL), C Clinical Wyeth strain collectione
E. coli GC 6260 TEM-1, SHV-5 A, A (ESBL) Clinical Wyeth strain collectione
E. coli GC 6368 SHV-7 A Laboratory Wyeth strain collectionf
K. oxytoca GC 7627 TEM-1, K1, KPC-2 A, C, A (cpase) Clinical 7
K. pneumoniae GC 7632 TEM-1 KPC-2, SHV-7, SHV-12 A, A (cpase), A (ESBL), A (ESBL) Clinical 7
K. pneumoniae GC 7635 SHV-1, KPC-2 A, A (cpase) Clinical 7
K. pneumoniae GC 7636 TEM-1, KPC-2, SHV-12 A, A (cpase), A (ESBL) Clinical 7
K. pneumoniae GC 7645 TEM-1, TEM-30, KPC-2, SHV-12 A, A (IRT), A (cpase), A (ESBL) Clinical 7
K. pneumoniae GC 7820 ACT-1 C Clinical Wyeth strain collectiong
K. pneumoniae GC 7821 DHA-1 C Clinical Wyeth strain collectiong
K. pneumoniae GC 7822 ACT-1 C Clinical Wyeth strain collectiong
K. pneumoniae GC 7823 FOX-5 C Clinical Wyeth strain collectiong
K. pneumoniae GC 7824 FOX-5 C Clinical Wyeth strain collectiong
K. pneumoniae GC 1507 TEM-9, SHV-1 A (ESBL), A Clinical Wyeth strain collection
K. pneumoniae GC 1510 TEM-10 A (ESBL) Clinical 37
K. pneumoniae GC 1516 TEM-26, SHV-12, AmpC(i) A (ESBL), A (ESBL), C Clinical 37
K. pneumoniae GC 1554 TEM-1, TEM-26, SHV-1 A, A (ESBL), A Clinical 43
K. pneumoniae GC 1827 TEM-3 A (ESBL) Clinical Wyeth strain collection
K. pneumoniae GC 1830 SHV-2 A (ESBL) Clinical Wyeth strain collection
K. pneumoniae GC 1832 SHV-4 A (ESBL) Clinical Wyeth strain collection
K. pneumoniae GC 2006 SHV-1, TEM-10 A, A (ESBL) Clinical 8
K. pneumoniae GC 3104 TEM-1, MIR-1 A, C Clinical 34
K. pneumoniae GC 6488 TEM-1, SHV-5, SHV-7 A, A (ESBL), A (ESBL) Clinical 7
K. pneumoniae GC 6494 TEM-1, SHV-5 A, A (ESBL) Clinical 7
K. pneumoniae GC 6639 SHV-13 A (ESBL) Clinical Wyeth strain collectionh
K. pneumoniae GC 6651 TEM-1, SHV-1, SHV-5 A, A, A (ESBL) Clinical 7
K. pneumoniae GC 6655 TEM-1, ACT-1 A, C Clinical 7
K. pneumoniae GC 6657 TEM-1, SHV-27 A, A (ESBL) Clinical 7
K. pneumoniae PT 9618 SHV-48 A Clinical Wyeth strain collection
M. morganii GC 1617 TEM-10, AmpC(i) A, C Clinical 37
P. mirabilis GAR 10941 TEM-155 A (ESBL) Clinical Wyeth strain collection
S. maltophilia GC 1712 L1 B Clinical Wyeth strain collection
S. marcescens GC 4132 AmpC(c) C Clinical Wyeth strain collection
S. marcescens GC 4145 AmpC(i) C Clinical Wyeth strain collection
S. marcescens GC 4150 AmpC(c) C Clinical Wyeth strain collection
S. marcescens PT 488 AmpC(c) C Clinical 29
S. marcescens PT 6003 AmpC(c) C Clinical 29
S. marcescens PT 696 AmpC(i) C Clinical 29
Salmonella enterica serovar Typhimurium GC 4197 CTX-M-5 A (ESBL) Clinical 16
Salmonella enterica serovar Typhimurium GC 4198 SHV-1, CTX-M-5 A, A (ESBL) Clinical 16
Open in a new tab
a

A gift from Jana Swensen.

b

(i), inducible; (c), constitutive.

c

A gift from Alain Philippon.

d

A gift from Philip Coudron.

e

A gift from Gregory Storch.

f

A gift from Mary Hayden.

g

A gift from George Jacoby.

h

A gift from David Livermore.

i

IRT, inhibitor-resistant TEM.

j

cpase, carbapenemase.

Antibiotics.

Standard powders of all drugs were used. The 6-methylidene bicyclic penem BLI-489 was synthesized by Wyeth Research, Pearl River, NY, and Wyeth-Lederle Japan, Saitama, Japan; piperacillin and tazobactam powder were obtained from Wyeth Research, Pearl River, NY; cefepime, ceftriaxone, and imipenem were obtained from US Pharmacopeia, Rockville, MD; cefoxitin, levofloxacin, and vancomycin were from Sigma-Aldrich, St. Louis, MO; and linezolid was from American Custom Chemical Corp., San Diego, CA.

Antimicrobial susceptibility testing.

The in vitro activities of the antibiotics against aerobic bacteria were determined by the broth microdilution method as recommended by the CLSI (20) using Mueller-Hinton II broth (MHB II; BBL, Cockeysville, MD). The MIC testing of Streptococcus spp. was performed using MHB II supplemented with 5% lysed horse blood, and Haemophilus test medium (HTM) was employed when testing Haemophilus spp. (20). Microtiter plates containing serial dilutions of each antimicrobial agent were inoculated with each organism to yield the appropriate density (105 CFU/ml) in a 100-μl final volume. The plates were incubated for 18 to 22 h at 35°C in ambient air. The in vitro activities of the antibiotics against anaerobic bacteria were determined by the agar dilution method as recommended by the CLSI (19) using Brucella agar (BBL, Sparks, MD) supplemented with hemin, vitamin K1, and laked sheep blood. Growth from a 48-h Brucella blood agar plate (Remel, Lexan, KS) was used to inoculate supplemented brain heart infusion broth and incubated for 6 h at 35°C in an anaerobic chamber. The turbidity was adjusted to match that of a McFarland 0.5 standard (108 CFU/ml) and applied to the surface of the agar plates with a Steers' replicator. Test plates were incubated at 35°C for 48 h in an anaerobic chamber. The MIC for all isolates was defined as the lowest concentration of antimicrobial agent that completely inhibited the growth of the organism as detected by the unaided eye.

RESULTS

In vitro activity against a predictor panel.

A predictor panel of gram-negative pathogens expressing well-characterized β-lactamases was used to assess the testing conditions that would most accurately separate susceptible from resistant strains in broth microdilution tests. The results for the predictor panel evaluation of piperacillin combined with BLI-489 at a constant concentration of 2 or 4 μg/ml as well as when combined with piperacillin in ratios of 1:1, 2:1, 4:1, and 8:1 (piperacillin-BLI-489) are displayed in Fig. 2A to E. For all analyses with piperacillin-BLI-489, the interpretive criteria utilized were those for piperacillin-tazobactam. For testing performed with a constant concentration of 2 or 4 μg/ml, approximately 41% of the gram-negative pathogens had lower MICs when tested with a piperacillin-BLI-489 combination using 4 μg/ml of BLI-489 than when tested using 2 μg/ml of BLI-489 (Fig. 2A). There were several minor errors: three strains (K. pneumoniae GC 6488, K. oxytoca GC 7627, and K. pneumoniae GC 7822) changed their interpretive category from resistant to intermediate, and four strains (E. coli GC 2015, K. pneumoniae GC 2006, K. pneumoniae GC 6651, and K. pneumoniae PT 9618) changed from intermediate to susceptible when the concentration of BLI-489 tested was analyzed at a constant concentration of 2 or 4 μg/ml, respectively. The strains involved were predominately Klebsiella pneumoniae strains that possessed two or more class A enzymes, including ESBLs.

FIG. 2.

FIG. 2.

FIG. 2.

Open in a new tab

Error rate-bound analysis comparing piperacillin MICs to those of various combinations of BLI-489. In each graph, the MICs of piperacillin tested with one concentration or ratio of BLI-489 is plotted against a different testing condition. (A) A constant concentration of 4 μg/ml BLI-489 versus a constant concentration of 2 μg/ml BLI-489; (B) a constant concentration of 4 μg/ml BLI-489 versus a 1:1 ratio of piperacillin to BLI-489; (C) a constant concentration of 4 μg/ml BLI-489 versus a 2:1 ratio of piperacillin to BLI-489; (D) a constant concentration of 4 μg/ml BLI-489 versus a 4:1 ratio of piperacillin to BLI-489; (E) a constant concentration of 4 μg/ml BLI-489 versus an 8:1 ratio of piperacillin to BLI-489; and (F) a constant concentration of 4 μg/ml BLI-489 versus a constant concentration of 4 μg/ml tazobactam. The data points that fall along the diagonal line of the scattergram represent essential agreement between the different concentrations of inhibitor. Horizontal and vertical lines demarcate the susceptible (≤16 μg/ml), intermediate (32 to 64 μg/ml), and resistant (≥64 μg/ml) categories (based on current interpretative categories for piperacillin-tazobactam), with category discrepancies highlighted in gray. The numbers in the figures indicate the number of strains at the indicated MIC.

The comparison of a constant concentration of 4 μg/ml BLI-489 to a ratio (piperacillin-BLI-489) of 1:1, 2:1, 4:1, or 8:1 is displayed in Fig. 2B to E. Overall, when the MICs were determined in tests with piperacillin-BLI-489 at a ratio of 1:1 or 2:1, a few strains appeared more susceptible than those determined at a constant 4 μg/ml of BLI-489. There were seven strains (Acinetobacter baumannii GC 7684, E. coli GC 2885, K. pneumoniae GC 7645, K. pneumoniae GC 7632, K. pneumoniae GC 7635, K. pneumoniae GC 7636, and A. baumannii GC 7685) that changed their interpretive category from resistant to intermediate, and four strains (K. oxytoca GC 7627, E. cloacae GC 1477, E. coli GC 5901, and K. pneumoniae GC 7822) changed from intermediate to susceptible when the 1:1 ratio (piperacillin-BLI-489) was compared to the constant concentration of 4 μg/ml BLI-489 (Fig. 2B). Similarly, six strains (A. baumannii GC 7684, E. coli GC 2885, K. pneumoniae GC 7632, K. pneumoniae GC 7635, K. pneumoniae GC 7636, and A. baumannii GC 7685) had changes in their interpretive category from resistant to intermediate, and three (E. cloacae GC 1477, E. coli GC 5901, and K. pneumoniae GC 7822) changed from intermediate to susceptible when the results of the 2:1 ratio (piperacillin-BLI-489) were compared to those of the constant BLI-489 concentration of 4 μg/ml (Fig. 2C). These results most likely were due to the higher concentration of BLI-489 in the 1:1 and 2:1 ratios at MICs above 4 and 8 μg/ml, respectively. The same subset of organisms was involved in these discrepancies and produced either a class C enzyme or multiple class A enzymes. There was a trend in which more than half of the organisms were more susceptible to piperacillin plus 4 μg/ml constant BLI-489 than to piperacillin-BLI-489 tested at a 4:1 ratio (Fig. 2D). Although the individual MICs were lower with BLI-489 at a constant 4 μg/ml, there were very few discrepancies. Two strains (E. cloacae GC 1477 and K. pneumoniae PT 9618) were categorized as susceptible with the constant concentration of 4 μg/ml of BLI-489 and intermediate with the 4:1 ratio (piperacillin-BLI-489), and one strain (K. pneumoniae GC 1516) was susceptible to the 4:1 ratio but was categorized as intermediate to piperacillin when tested with BLI-489 at a 4-μg/ml constant concentration. MICs determined in tests performed with the 8:1 ratio (piperacillin-BLI-489) generally were higher than those obtained with the constant concentration of 4 μg/ml BLI-489 (Fig. 2E). There were five strains (E. coli GC 2015, K. pneumoniae GC 2006, K. pneumoniae PT 9618, K. pneumoniae GC 1516, and K. pneumoniae GC 1554) that were categorized as intermediate when tested with the 8:1 ratio but susceptible with the constant concentration of 4 μg/ml of BLI-489. Based on the ability of piperacillin combined with a constant 4 μg/ml of BLI-489 to effectively separate resistant and susceptible strains, this appears to be the most appropriate methodology for in vitro testing.

To compare the activities of BLI-489 and tazobactam in inhibiting the β-lactamases produced by the predictor panel of isolates, the in vitro activity of piperacillin combined with either BLI-489 or tazobactam at a constant concentration of 4 μg/ml is displayed in Fig. 2F. There were a significant number of strains in the panel (37%) that would be classified as nonsusceptible (resistant or intermediate) to the piperacillin-tazobactam combination but susceptible to piperacillin combined with BLI-489. The majority of these strains produced either class C enzymes or ESBLs. Only one strain of Escherichia coli, which produced a PSE-4 β-lactamase, was susceptible to piperacillin-tazobactam but resistant to piperacillin-BLI-489.

In vitro activity against recent clinical isolates.

Based on the predictor panel results, the in vitro antibacterial activities of piperacillin in combination with BLI-489 (4 μg/ml constant concentration), piperacillin-tazobactam, and comparative antibacterial agents was assessed against 1,163 gram-negative and 712 gram-positive recently identified clinical isolates (Tables 2 and 3). In general, based on the MICs at which 90% of the tested isolates are inhibited (MIC90s), the activity demonstrated by the piperacillin-BLI-489 combination equaled or exceeded the activity of piperacillin-tazobactam for all isolates tested.

TABLE 2.

In vitro activities of piperacillin in combination with BLI-489 and comparative antibacterial agents against recently identified gram-negative clinical isolates

Organism (no. of isolates) Antibiotic MIC (μg/ml)
Range 50% 90%
Escherichia coli (52) Piperacillin 0.5->128 2 >128
Piperacillin-tazobactam 0.5-128 2 2
Piperacillin-BLI-489 0.25-64 1 2
Levofloxacin ≤0.015->16 0.03 8
Cefepime ≤0.015-16 0.03 0.06
Ceftriaxone ≤0.03->32 0.06 0.06
Imipenem 0.06-0.25 0.12 0.12
Escherichia coli class A ESBL (31) Piperacillin >128 >128 >128
Piperacillin-tazobactam 1->128 8 >128
Piperacillin-BLI-489 1-32 4 16
Levofloxacin ≤0.015->16 8 >16
Cefepime 0.5->16 >16 >16
Ceftriaxone 8->32 >32 >32
Imipenem 0.12-1 0.12 0.5
Escherichia coli class A ESBL plus class D OXA (17) Piperacillin >128 >128 >128
Piperacillin-tazobactam 1->128 16 32
Piperacillin-BLI-489 2-16 4 8
Levofloxacin 0.03->16 16 >16
Cefepime 8->16 >16 >16
Ceftriaxone >32 >32 >32
Imipenem 0.12-1 0.12 0.25
Escherichia coli AmpC (17) Piperacillin 16->128 >128 >128
Piperacillin-tazobactam 2-64 8 32
Piperacillin-BLI-489 1-16 8 16
Levofloxacin 0.03->16 16 >16
Cefepime 0.25->16 >16 >16
Ceftriaxone 4->32 >32 >32
Imipenem 0.12-0.5 0.12 0.25
Miscellaneous Enterobacteriaceaea (AmpC) (17) Piperacillin 32->128 >128 >128
Piperacillin-tazobactam 8->128 64 128
Piperacillin-BLI-489 0.5-128 2 16
Levofloxacin ≤0.015-16 0.12 8
Cefepime 0.25->16 1 4
Ceftriaxone 4->32 32 >32
Imipenem 0.5-2 0.5 2
Enterobacter aerogenes (50) Piperacillin 0.5->128 4 128
Piperacillin-tazobactam 0.25->128 4 64
Piperacillin-BLI-489 0.5-32 2 8
Levofloxacin 0.03->16 0.06 0.5
Cefepime ≤0.015->16 0.03 0.5
Ceftriaxone ≤0.03->32 0.12 >32
Imipenem 0.5->32 1 2
Enterobacter cloacae (52) Piperacillin 1->128 4 >128
Piperacillin-tazobactam 0.5->128 2 >128
Piperacillin-BLI-489 0.5-16 2 16
Levofloxacin ≤0.015->16 0.06 >16
Cefepime ≤0.015->16 0.12 >16
Ceftriaxone ≤0.03->32 0.5 >32
Imipenem 0.12-2 0.5 1
Citrobacter koseri (51) Piperacillin 4->128 32 >128
Piperacillin-tazobactam 1->128 2 8
Piperacillin-BLI-489 1-64 2 8
Levofloxacin ≤0.015-0.25 0.03 0.06
Cefepime ≤0.015->16 0.03 0.12
Ceftriaxone ≤0.03->32 0.06 0.25
Imipenem 0.06-0.5 0.12 0.12
Citrobacter freundii complex (51) Piperacillin 1->128 4 >128
Piperacillin-tazobactam 0.5->128 2 64
Piperacillin-BLI-489 1->128 2 8
Levofloxacin ≤0.015->16 0.12 2
Cefepime ≤0.015->16 0.03 8
Ceftriaxone 0.12->32 0.5 >32
Imipenem 0.5-2 1 1
Klebsiella pneumoniae (54) Piperacillin 2->128 8 128
Piperacillin-tazobactam 1->128 2 16
Piperacillin-BLI-489 1-16 2 8
Levofloxacin 0.03->16 0.06 0.5
Cefepime ≤0.015-0.25 0.03 0.12
Ceftriaxone ≤0.03-0.25 0.06 0.12
Imipenem 0.12-1 0.25 0.5
Klebsiella pneumoniae class A ESBL (36) Piperacillin 128->128 >128 >128
Piperacillin-tazobactam 2->128 32 >128
Piperacillin-BLI-489 2-128 16 32
Levofloxacin 0.03->16 0.5 8
Cefepime 0.5->16 8 >16
Ceftriaxone 0.12->32 >32 >32
Imipenem 0.06-2 0.25 0.5
Klebsiella pneumoniae class A ESBL plus class D OXA (20) Piperacillin 64->128 >128 >128
Piperacillin-tazobactam 4->128 16 >128
Piperacillin-BLI-489 4-128 8 64
Levofloxacin 0.06->16 1 >16
Cefepime 2->16 >16 >16
Ceftriaxone 16->32 >32 >32
Imipenem 0.12-1 0.25 0.5
Klebsiella pneumoniae (AmpC) (30) Piperacillin 32->128 >128 >128
Piperacillin-tazobactam 8->128 32 >128
Piperacillin-BLI-489 4->128 16 >128
Levofloxacin 0.06->16 2 >16
Cefepime 0.12->16 2 >16
Ceftriaxone 8->32 32 >32
Imipenem 0.12-32 1 16
Klebsiella oxytoca (53) Piperacillin 0.5->128 8 64
Piperacillin-tazobactam 0.25->128 2 8
Piperacillin-BLI-489 0.25-16 2 8
Levofloxacin ≤0.015-8 0.03 0.25
Cefepime ≤0.015->16 0.03 0.06
Ceftriaxone ≤0.03->32 0.06 0.12
Imipenem 0.12-0.5 0.25 0.5
Serratia marcescens (52) Piperacillin 1->128 4 >128
Piperacillin-tazobactam 0.5-64 2 64
Piperacillin-BLI-489 0.5-16 1 4
Levofloxacin 0.06-8 0.12 4
Cefepime 0.03->16 0.06 8
Ceftriaxone 0.06->32 0.5 >32
Imipenem 0.25-4 1 2
Morganella morganii (54) Piperacillin 0.25->128 8 >128
Piperacillin-tazobactam ≤0.12-16 0.5 1
Piperacillin-BLI-489 ≤0.12-8 0.25 2
Levofloxacin ≤0.015->16 0.03 >16
Cefepime ≤0.015->16 0.03 0.25
Ceftriaxone ≤0.03->32 0.06 8
Imipenem 0.5-4 4 4
Proteus mirabilis (50) Piperacillin ≤0.12->128 >128 >128
Piperacillin-tazobactam ≤0.12-64 0.25 2
Piperacillin-BLI-489 ≤0.12-2 0.25 0.5
Levofloxacin 0.03->16 0.5 16
Cefepime 0.03->16 0.06 >16
Ceftriaxone ≤0.03->32 ≤0.03 >32
Imipenem 1-16 2 4
Proteus vulgaris (32) Piperacillin 0.25->128 64 >128
Piperacillin-tazobactam 0.25-8 0.5 2
Piperacillin-BLI-489 0.25-16 0.5 1
Levofloxacin 0.03-1 0.03 0.12
Cefepime 0.03-2 0.12 0.5
Ceftriaxone ≤0.03->32 32 >32
Imipenem 0.5-4 2 4
Providencia spp.b (31) Piperacillin 0.25->128 2 >128
Piperacillin-tazobactam 0.25->128 2 16
Piperacillin-BLI-489 0.25-64 1 8
Levofloxacin 0.06->16 1 16
Cefepime ≤0.015->16 0.12 >16
Ceftriaxone ≤0.03->32 0.06 >32
Imipenem 0.5-2 2 2
Acinetobacter calcoaceticus/A. baumannii complex (54) Piperacillin 8->128 >128 >128
Piperacillin-tazobactam ≤0.12->128 >128 >128
Piperacillin-BLI-489 2->128 64 >128
Levofloxacin 0.06-16 8 16
Cefepime 1->16 >16 >16
Ceftriaxone 4->32 >32 >32
Imipenem 0.12->32 1 32
Acinetobacter spp.c (30) Piperacillin 0.5->128 4 128
Piperacillin-tazobactam ≤0.12->128 ≤0.12 32
Piperacillin-BLI-489 0.5-32 4 16
Levofloxacin 0.03-2 0.12 2
Cefepime 0.06->16 0.5 4
Ceftriaxone 0.5->32 2 8
Imipenem ≤0.03-4 0.06 0.5
Burkholderia cepacia (31) Piperacillin 2->128 32 128
Piperacillin-tazobactam 0.25-128 4 64
Piperacillin-BLI-489 0.25-16 1 2
Levofloxacin 0.12-8 2 4
Cefepime 0.25->16 16 >16
Ceftriaxone 2->32 32 >32
Imipenem 0.25->32 8 16
Stenotrophomonas maltophilia (50) Piperacillin >128 >128 >128
Piperacillin-tazobactam 128->128 >128 >128
Piperacillin-BLI-489 32->128 >128 >128
Levofloxacin 0.25-8 1 4
Cefepime 1->16 >16 >16
Ceftriaxone >32 >32 >32
Imipenem >32 >32 >32
Pseudomonas aeruginosa (54) Piperacillin 4->128 16 >128
Piperacillin-tazobactam 4->128 16 >128
Piperacillin-BLI-489 4->128 8 64
Levofloxacin 0.12->16 1 >16
Cefepime 0.5->16 4 16
Ceftriaxone 8->32 >32 >32
Imipenem 0.25->32 2 32
Haemophilus influenzae β-lactamase positive (37) Piperacillin 0.5-128 8 32
Piperacillin-tazobactam ≤0.004-0.06 0.008 0.03
Piperacillin-BLI-489 ≤0.004-0.015 ≤0.004 ≤0.004
Levofloxacin ≤0.015-4 ≤0.015 ≤0.015
Cefepime 0.03-0.25 0.06 0.12
Ceftriaxone ≤0.03 ≤0.03 ≤0.03
Imipenem 0.06-2 0.5 0.5
Haemophilus influenzae β-lactamase negative (51) Piperacillin ≤0.004-0.12 0.015 0.03
Piperacillin-tazobactam ≤0.004-0.12 0.008 0.03
Piperacillin-BLI-489 ≤0.004-0.06 ≤0.004 0.015
Levofloxacin ≤0.015-8 ≤0.015 ≤0.015
Cefepime 0.03-0.12 0.06 0.12
Ceftriaxone ≤0.03 ≤0.03 ≤0.03
Imipenem 0.12-2 0.05 1
Haemophilus parainfluenzae (47) Piperacillin ≤0.004-128 0.06 0.5
Piperacillin-tazobactam ≤0.004-2 0.06 0.25
Piperacillin-BLI-489 ≤0.004-0.12 ≤0.004 ≤0.004
Levofloxacin ≤0.015-4 ≤0.015 0.06
Cefepime ≤0.015-4 0.03 0.25
Ceftriaxone ≤0.03-0.25 ≤0.03 ≤0.03
Imipenem ≤0.03-1 0.25 0.5
Moraxella catarrhalis (29) Piperacillin ≤0.12-32 0.25 8
Piperacillin-tazobactam ≤0.004-0.015 ≤0.004 0.008
Piperacillin-BLI-489 ≤0.004-0.03 ≤0.004 0.015
Levofloxacin ≤0.015-0.06 0.03 0.06
Cefepime 0.06-8 0.25 4
Ceftriaxone ≤0.03-1 ≤0.03 0.5
Imipenem ≤0.03-0.12 ≤0.03 0.06
Open in a new tab
a

Species (no. of isolates) included C. freundii (3), E. aerogenes (1), E. cloacae (7), and S. marcescens (6).

b

Species (no. of isolates) included P. rettgeri (11) and P. stuartii (20).

c

Species (no. of isolates) included A. haemolyticus (5), A. johnsonii/A. junii (7), and A. lwoffi (18).

TABLE 3.

In vitro activities of piperacillin in combination with BLI-489 and comparative antibacterial agents against recently identified gram-positive clinical isolates

Organism (no. of isolates) Antibiotic MIC (μg/ml)
Range 50% 90%
Staphylococcus aureus, methicillin resistant (50) Piperacillin 16->128 >128 >128
Piperacillin-tazobactam 2->128 64 >128
Piperacillin-BLI-489 0.5->128 32 >128
Levofloxacin 0.12->16 4 >16
Cefepime 4->16 >16 >16
Vancomycin 0.5-2 1 1
Linezolid 2-4 2 4
Staphylococcus aureus, methicillin susceptible (50) Piperacillin 0.5->128 64 >128
Piperacillin-tazobactam 0.5-2 1 2
Piperacillin-BLI-489 ≤0.12-0.5 0.25 0.5
Levofloxacin 0.12-8 0.25 0.5
Cefepime 2-4 2 4
Vancomycin 0.5-1 0.5 1
Linezolid 2-4 4 4
Staphylococcus epidermidis, methicillin resistant (50) Piperacillin 0.25-128 8 64
Piperacillin-tazobactam ≤0.12-32 1 8
Piperacillin-BLI-489 ≤0.12-32 0.5 8
Levofloxacin 0.12->16 2 >16
Cefepime 0.25->16 4 >16
Vancomycin 0.5-4 2 2
Linezolid 1-2 1 2
Staphylococcus epidermidis, methicillin susceptible (50) Piperacillin ≤0.12-8 2 8
Piperacillin-tazobactam ≤0.12-1 0.25 0.5
Piperacillin-BLI-489 ≤0.004-0.5 ≤0.004 ≤0.004
Levofloxacin 0.06-8 0.12 0.25
Cefepime 0.25-2 0.5 1
Vancomycin 0.5-1 1 1
Linezolid 1-2 1 2
Staphylococcus haemolyticus (52) Piperacillin 2->128 >128 >128
Piperacillin-tazobactam 0.5->128 32 >128
Piperacillin-BLI-489 0.25->128 32 >128
Levofloxacin 0.12->16 8 16
Cefepime 1->16 >16 >16
Vancomycin 0.25-2 1 1
Linezolid 1-2 1 2
Enterococcus faecalis (52) Piperacillin 2-32 2 4
Piperacillin-tazobactam 2-32 2 4
Piperacillin-BLI-489 1-16 2 4
Levofloxacin 0.5->16 1 >16
Cefepime 1->16 >16 >16
Vancomycin 0.5-2 1 2
Linezolid 1-2 2 2
Enterococcus faecium (54) Piperacillin 1->128 32 >128
Piperacillin-tazobactam 1->128 32 >128
Piperacillin-BLI-489 0.25->128 4 >128
Levofloxacin 0.5->16 4 >16
Cefepime 0.12->16 >16 >16
Vancomycin 0.5-2 0.5 1
Linezolid 0.5-4 2 2
Enterococcus faecalis, vancomycin resistant (16) Piperacillin 2->128 2 >128
Piperacillin-tazobactam 2->128 2 >128
Piperacillin-BLI-489 2-128 2 64
Levofloxacin 1->16 2 >16
Cefepime 16->16 >16 >16
Vancomycin 16->32 >32 >32
Linezolid 1-2 2 2
Enterococcus faecium, vancomycin resistant (34) Piperacillin 128->128 >128 >128
Piperacillin-tazobactam 128->128 >128 >128
Piperacillin-BLI-489 32->128 >128 >128
Levofloxacin >16 >16 >16
Cefepime >16 >16 >16
Vancomycin 32->32 >32 >32
Linezolid 0.5-8 2 2
Enterococcus avium (51) Piperacillin 4->128 16 64
Piperacillin-tazobactam 4->128 16 64
Piperacillin-BLI-489 1-128 8 32
Levofloxacin 0.5->16 2 2
Cefepime 2->16 4 8
Vancomycin 0.25-0.5 0.25 0.5
Linezolid 1-2 2 2
Enterococcus spp.a (41) Piperacillin 0.25->128 4 16
Piperacillin-tazobactam 0.25->128 4 16
Piperacillin-BLI-489 ≤0.12->128 2 8
Levofloxacin 0.25->16 1 8
Cefepime 0.25->16 >16 >16
Vancomycin 0.25-8 0.5 4
Linezolid 0.5-2 2 2
Streptococcus pneumoniae, penicillin susceptibleb (42) Piperacillin ≤0.004-0.5 0.015 0.03
Piperacillin-tazobactam ≤0.004-0.12 0.008 0.015
Piperacillin-BLI-489 ≤0.004 ≤0.004 ≤0.004
Levofloxacin 0.25-2 1 1
Cefepime ≤0.015-0.06 ≤0.015 ≤0.015
Vancomycin ≤0.03-0.5 0.12 0.25
Linezolid 0.12-1 0.5 1
Streptococcus pneumoniae, penicillin intermediatec (20) Piperacillin ≤0.12-2 0.25 1
Piperacillin-tazobactam ≤0.12-2 0.5 1
Piperacillin-BLI-489 ≤0.12-2 0.25 1
Levofloxacin 0.5-1 0.5 1
Cefepime ≤0.015-0.5 0.06 0.25
Vancomycin 0.12-0.25 0.25 0.25
Linezolid 0.25-1 0.5 1
Streptococcus pneumoniae, penicillin resistantd (27) Piperacillin 1-16 2 4
Piperacillin-tazobactam 0.5-16 2 4
Piperacillin-BLI-489 0.25-16 2 4
Levofloxacin 0.5-16 1 1
Cefepime 0.12-8 0.5 1
Vancomycin 0.12-0.25 0.25 0.25
Linezolid 0.25-1 1 1
Streptococcus pyogenes (52) Piperacillin 0.015-0.06 0.03 0.06
Piperacillin-tazobactam 0.015-0.06 0.03 0.06
Piperacillin-BLI-489 ≤0.004 ≤0.004 ≤0.004
Levofloxacin 0.12-1 0.5 0.5
Cefepime ≤0.015 ≤0.015 ≤0.015
Vancomycin 0.12-0.5 0.25 0.25
Linezolid 0.5-1 1 1
Streptococcus agalactiae (41) Piperacillin 0.06-0.25 0.12 0.25
Piperacillin-tazobactam 0.06-0.25 0.12 0.25
Piperacillin-BLI-489 ≤0.004-0.06 ≤0.004 0.03
Levofloxacin 0.5-2 1 1
Cefepime 0.03-0.12 0.06 0.06
Vancomycin 0.25-0.5 0.25 0.5
Linezolid 0.5-2 1 2
Open in a new tab
a

Species (no. of isolates) included E. casseliflavus (10), E. durans (10), E. gallinarum (10), and E. hirae (11).

b

Penicillin MIC, ≤0.06 μg/ml.

c

Penicillin MIC, 0.12 to 1 μg/ml.

d

Penicillin MIC, ≥2 μg/ml.

Overall, against most enteric bacilli (Table 2), the activity of piperacillin-BLI-489 resulted in MIC90s of 0.5 to 16 μg/ml. The exceptions were K. pneumoniae strains producing class A ESBL, class A ESBL plus class D OXA, and AmpC, for which approximately 78, 65, and 67% were inhibited by the combination of piperacillin-BLI-489 at a concentration of ≤16 μg/ml, respectively. In contrast, only 47% of the K. pneumoniae strains producing class A ESBL, 60% producing class A ESBL plus class D OXA, and 23% producing AmpC were inhibited by a ≤16-μg/ml concentration of the piperacillin-tazobactam combination. In addition, the combination of piperacillin-tazobactam demonstrated poor activity against Enterobacter aerogenes, Enterobacter cloacae, Citrobacter freundii complex, Serratia marcescens, class A ESBL-producing, class A ESBL plus class D OXA-producing, or AmpC-producing Escherichia coli and AmpC-producing Enterobacteriaceae (MIC90s, 32 to >128 μg/ml). It should be noted that approximately 55% of all of the enteric bacilli tested were nonsusceptible to piperacillin alone (MICs, ≥32 μg/ml). However, 92% of the piperacillin nonsusceptible strains were inhibited by ≤16 μg/ml piperacillin-BLI-489; in contrast, only 66% were inhibited by ≤16 μg/ml piperacillin-tazobactam. When the MIC90s were compared to those for the other antimicrobial agents, the combination of piperacillin-BLI-489 demonstrated better activity than cefepime, ceftriaxone, and levofloxacin against ESBL- and AmpC-producing E. coli, Enterobacter cloacae, Proteus mirabilis, and Providencia spp. strains. With the exception of the AmpC-producing K. pneumoniae (MIC90, 16 μg/ml), a high level of activity for imipenem was observed against all of the gram-negative bacilli (MIC90s, 0.12 to 4 μg/ml).

The combination of piperacillin-BLI-489 demonstrated good activity against the nonfermentative bacteria: Burkholderia cepacia, Acinetobacter spp., and Pseudomonas aeruginosa, with MIC90s of 2, 16, and 64 μg/ml, respectively. However, piperacillin-BLI-489 was less active against Acinetobacter calcoaceticus-A. baumannii complex and Stenotrophomonas maltophilia (MIC90s, >128 μg/ml). In contrast, piperacillin-tazobactam had poor activity against all nonfermentative isolates tested (MIC90s, 32 to >128 μg/ml). The remaining comparative antimicrobial agents demonstrated moderate to poor overall activity against the majority of the nonfermentative isolates tested, with the exception of Acinetobacter spp.

The fastidious gram-negative organisms Haemophilus influenzae, Haemophilus parainfluenzae, and Moraxella catarrhalis were inhibited equally by both piperacillin-BLI-489 and piperacillin-tazobactam (MICs, ≤0.004 to 2 μg/ml). Both combinations demonstrated a marked improvement in activity compared to that of piperacillin tested as a single agent (MICs, ≤0.004 to 128 μg/ml). Most notable was the improvement in the activity of piperacillin tested alone (MIC90, 32 μg/ml) compared to that of the piperacillin-BLI-489 combination against β-lactamase-positive H. influenzae (MIC90 ≤ 0.004 μg/ml). The comparator agents also were highly active against these fastidious isolates (MIC90s, ≤0.015 to 4 μg/ml).

As shown in Table 3, the in vitro activity of piperacillin (MIC90, 8 to >128 μg/ml) against methicillin (meticillin)-susceptible Staphylococcus aureus, methicillin-resistant Staphylococcus epidermidis, and methicillin-susceptible S. epidermidis was significantly enhanced by the addition of BLI-489 or tazobactam (MIC90s, ≤0.004 to 8 μg/ml). Compared to the activity of piperacillin alone, there was no improvement in activity shown by either the piperacillin-BLI-489 or piperacillin-tazobactam combination against methicillin-resistant S. aureus or Staphylococcus haemolyticus (MIC90 > 128 μg/ml). Vancomycin and linezolid, with MIC90s of 1 to 2 and 2 to 4 μg/ml, respectively, demonstrated potent activity against all staphylococcal isolates tested. The addition of BLI-489 or tazobactam also failed to demonstrate improvement in the activity of piperacillin alone (MIC90s, 4 to >128 μ/ml) against any of the enterococci tested. Linezolid (MIC90s, 2 μg/ml) demonstrated the best overall activity against both vancomycin-susceptible and -resistant enterococcal strains.

Piperacillin alone or in combination with BLI-489 showed increasing MIC90s (0.03 to ≤0.004, 1, and 4 μg/ml), corresponding to increasing penicillin resistance against the Streptococcus pneumoniae isolates tested. The combination of neither piperacillin-BLI-489 nor piperacillin-tazobactam showed improved activity compared to that of piperacillin tested alone against the penicillin-intermediate or -resistant S. pneumoniae isolates. Levofloxacin, vancomycin, and linezolid, with MIC90s of 1, 0.25, and 1 μg/ml, respectively, showed similar activity regardless of penicillin susceptibility. The Streptococcus pyogenes and Streptococcus agalactiae isolates tested were highly susceptible to all antibacterial agents evaluated (MICs, ≤2 μg/ml).

The in vitro activity of the combination of piperacillin-BLI-489 and comparative antibacterial agents against anaerobic clinical isolates is displayed in Table 4. The gram-negative anaerobic isolates (Bacteroides fragilis, Bacteroides thetaiotaomicron, and Bacteroides uniformis) were equally susceptible to low levels of piperacillin-BLI-489 and piperacillin-tazobactam (MIC90s, 0.25 to 16 μg/ml). The combinations demonstrated a significant improvement in activity compared to that of piperacillin tested as a single agent (MIC90s of >128, >128, and 32 μg/ml, respectively). Cefoxitin and imipenem also showed moderate to good activity against these gram-negative anaerobic isolates (MIC90s of 16 to 32 and 0.25 to 1 μg/ml, respectively). Piperacillin tested alone demonstrated potent activity against the gram-positive anaerobic isolates (Clostridium perfringens, Peptostreptococcus micros, and Peptostreptococcus anaerobius; MIC90s of 0.25, 0.25, and 0.06 μg/ml, respectively). However, the addition of the β-lactamase inhibitors BLI-489 and tazobactam further enhanced this activity by up to four twofold dilutions (MIC90s of ≤0.015 to 0.25 μg/ml). Cefoxitin and imipenem also were highly active against these gram-positive anaerobic isolates (MIC90s of 0.5 to 2 and 0.06 to 0.12 μg/ml, respectively).

TABLE 4.

In vitro activities of piperacillin in combination with BLI-489 and comparative antibacterial agents against recently identified anaerobic clinical isolates

Organism (no. of isolates) Antibiotic MIC (μg/ml)
Range 50% 90%
Bacteroides fragilis (10) Piperacillin 1->128 8 >128
Piperacillin-tazobactam ≤0.015-0.5 0.06 0.25
Piperacillin-BLI-489 0.06-1 0.25 0.5
Cefoxitin 4-64 8 16
Imipenem 0.03-4 0.25 0.5
Bacteroides thetaiotaomicron (10) Piperacillin 32->128 32 >128
Piperacillin-tazobactam 1->128 4 8
Piperacillin-BLI-489 8-16 16 16
Cefoxitin 16-32 16 32
Imipenem 0.25-16 0.25 1
Bacteroides uniformis (10) Piperacillin 8-64 16 32
Piperacillin-tazobactam 0.12-8 0.5 4
Piperacillin-BLI-489 0.5-16 4 8
Cefoxitin 2-32 16 16
Imipenem 0.12-0.25 0.12 0.25
Clostridium perfringens (10) Piperacillin 0.03-0.25 0.06 0.25
Piperacillin-tazobactam ≤0.015-0.25 ≤0.015 0.03
Piperacillin-BLI-489 ≤0.015 ≤0.015 ≤0.015
Cefoxitin 0.5-2 1 2
Imipenem 0.03-0.06 0.06 0.06
Peptostreptococcus micros (10) Piperacillin 0.03-0.25 0.12 0.25
Piperacillin-tazobactam ≤0.015-0.5 0.03 0.25
Piperacillin-BLI-489 ≤0.015-0.12 ≤0.015 ≤0.015
Cefoxitin 0.03-8 0.5 0.5
Imipenem ≤0.015-0.12 0.06 0.12
Peptostreptococcus anaerobius (10) Piperacillin ≤0.015-0.12 0.06 0.06
Piperacillin-tazobactam ≤0.015 ≤0.015 ≤0.015
Piperacillin-BLI-489 ≤0.015 ≤0.015 ≤0.015
Cefoxitin 0.12-1 0.25 1
Imipenem ≤0.015-0.06 0.03 0.06
Open in a new tab

DISCUSSION

The historical issue of whether susceptibility testing should be performed using β-lactamase inhibitors (tazobactam, clavulanic acid, and sulbactam) at fixed concentration or titrated in a fixed ratio had been a source of much debate during the early development of these inhibitors (24, 27). Tazobactam has been used at a fixed concentration (4 μg/ml) in combination with piperacillin, whereas sulbactam is used in a 2:1 ratio when tested with ampicillin. To further complicate the debate, clavulanic acid is tested in a 2:1 ratio in combination with amoxicillin and a fixed concentration (2 μg/ml) when tested in combination with ticarcillin. It had been postulated during the development of these inhibitors that human pharmacokinetics should dictate the reference MIC testing methodology (24, 27).

Predictor panels have been successfully used in the past to determine the interpretive criteria for β-lactam/β-lactamase inhibitor combinations (12-14, 40, 42). The predictor panel utilized for those analyses contained strains with well-characterized mechanisms of resistance to β-lactam antibiotics. In order to determine the combination of piperacillin-BLI-489 to be used in MIC tests that would most accurately separate susceptible from resistant strains, a contemporary predictor panel of β-lactamase-producing bacteria was assembled (Table 1). This evaluation utilized the panel to determine the reliability of susceptibility test methods of the combination of piperacillin-BLI-489 at a constant concentration of 2 or 4 μg/ml of BLI-489 and at PIP:BLI-489 ratios of 1:1, 2:1, 4:1, and 8:1. The results showed that a constant concentration of 4 μg/ml was the methodology that produced the most reliable separation of resistant and susceptible isolates and therefore the most accurate results.

Using a mouse model of intraperitoneal sepsis, it was shown that dosing piperacillin-BLI-489 at an 8:1 ratio was efficacious in infections caused by a number of β-lactamase-producing pathogens (35). This is the same ratio that is currently approved for the dosing of piperacillin-tazobactam. In addition, studying the pharmacokinetics in animals indicated that the results for exposure to BLI-489 was similar to those of exposure to tazobactam (48). Given the similarities of pharmacokinetics and the ratio for efficacy in rodents between piperacillin-tazobactam and piperacillin-BLI-489, the results of this study indicating that piperacillin-BLI-489 also should be tested with a constant concentration of 4 μg/ml in MIC tests, as is the case for piperacillin-tazobactam, were not unexpected. Although increased in vitro activity was observed with piperacillin-BLI-489 at the 1:1 and 2:1 ratios, achieving comparable concentrations in vivo may not be possible for dosing at an 8:1 ratio of piperacillin to BLI-489.

The increasing development of bacterial resistance and the increasing prevalence of ESBL- and AmpC-producing strains are challenging the current therapeutic options. The mobile nature and broad resistance profile of plasmid-carried AmpC enzymes are certain to become a major public health issue (49). These organisms confer broad-spectrum resistance to β-lactam-inhibitor combinations, expanded-spectrum and “newer generation” cephalosporins, and aztreonam. In addition, they are frequently associated with cross-resistance to fluoroquinolones, aminoglycosides, and trimethoprim-sulfamethoxazole (50). To confront the continued emergence of ESBL and AmpC resistance, a number of compounds, including β-lactam and non-β-lactam inhibitors, penems, cephalosporins, and carbapenems, are in development (3).

In this study, the novel 6-methylidene-penem β-lactamase inhibitor BLI-489, in combination with piperacillin, exceeded the activity of piperacillin-tazobactam against approximately one-third of the gram-negative isolates tested. Improved activity also was demonstrated against the problematic ESBL- and AmpC-containing strains of Enterobacteriaceae and P. aeruginosa. Although the predictor panel contained strains expressing the KPC-2 and inhibitor-resistant TEM-type β-lactamases, these strains also expressed up to three additional β-lactamases. Therefore, it was not possible to accurately assess the ability of BLI-489 to inhibit these enzymes and will require further study. The combination of piperacillin-BLI-489 also enhanced the activity of piperacillin against many staphylococcal and streptococcal isolates. The increased activity of the piperacillin-BLI-489 combination against some staphylococci, streptococci, and the more fastidious gram-negative bacteria may be due in part to the moderate intrinsic activity of BLI-489 against these strains (data not shown). The lack of improvement for enterococci and penicillin-intermediate or -resistant S. pneumoniae was not unexpected, as the production of a β-lactamase enzyme is not the causative means of piperacillin resistance in these species (25). The in vitro activity of piperacillin in combination with this new penem β-lactamase inhibitor and the clear advantage demonstrated over piperacillin-tazobactam against a wide variety of enzymes make it a strong candidate for further development.

Footnotes

▿

Published ahead of print on 10 November 2008.

REFERENCES

  • 1.Barnaud, G., Y. Benzerara, J. Gravisse, L. Raskine, M. J. Sanson-Le Pors, R. Labia, and G. Arlet. 2004. Selection during cefepime treatment of a new cephalosporinase variant with extended-spectrum resistance to cefepime in an Enterobacter aerogenes clinical isolate. Antimicrob. Agents Chemother. 48:1040-1042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Barnaud, G., R. Labia, L. Raskine, M. J. Sanson-Le Pors, A. Philippon, and G. Arlet. 2001. Extension of resistance to cefepime and cefpirome associated to a six amino acid deletion in the H-10 helix of the cephalosporinase of an Enterobacter cloacae clinical isolate. FEMS Microbiol. Lett. 195:185-190. [DOI] [PubMed] [Google Scholar]
  • 3.Bassetti, M., E. Righi, and C. Viscoli. 2008. Novel β-lactam antibiotics and inhibitor combinations. Expert Opin. Investig. Drugs 17:285-296. [DOI] [PubMed] [Google Scholar]
  • 4.Bhat, S. V., A. Y. Peleg, T. P. Lodise, Jr., K. A. Shutt, B. Capitano, B. A. Potoski, and D. L. Paterson. 2007. Failure of current cefepime breakpoints to predict clinical outcomes of bacteremia caused by gram-negative organisms. Antimicrob. Agents Chemother. 51:4390-4395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Bouchillon, S. K., B. M. Johnson, D. J. Hoban, J. L. Johnson, M. J. Dowzicky, D. H. Wu, M. A. Visalli, and P. A. Bradford. 2004. Determining incidence of extended spectrum β-lactamase producing Enterobacteriaceae, vancomycin-resistant Enterococcus faecium and methicillin-resistant Staphylococcus aureus in 38 centres from 17 countries: the PEARLS study 2001-2002. Intern. J. Antimicrob. Agents 24:119-124. [DOI] [PubMed] [Google Scholar]
  • 6.Bradford, P. A. 2001. Extended-spectrum β-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat. Clin. Microbiol. Rev. 14:933-951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Bradford, P. A., S. Bratu, C. Urban, M. Visalli, N. Mariano, D. Landman, J. J. Rahal, S. Brooks, S. Cebular, and J. Quale. 2004. Emergence of carbapenem-resistant Klebsiella species possessing the class A carbapenem-hydrolyzing KPC-2 and inhibitor-resistant TEM-30 β-lactamases in New York City. Clin. Infect. Dis. 39:55-60. [DOI] [PubMed] [Google Scholar]
  • 8.Bradford, P. A., C. E. Cherubin, V. Idemyor, B. A. Rasmussen, and K. Bush. 1994. Multiply resistant Klebsiella pneumoniae strains from two Chicago hospitals: identification of the extended-spectrum TEM-12 and TEM-10 ceftazidime-hydrolyzing β-lactamases in a single isolate. Antimicrob. Agents Chemother. 38:761-766. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Bradford, P. A., N. V. Jacobus, N. Bhachech, and K. Bush. 1996. TEM-28 from an Escherichia coli clinical isolate is a member of the His-164 family of TEM-1 extended-spectrum β-lactamases. Antimicrob. Agents Chemother. 40:260-262. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Bradford, P. A., P. J. Petersen, I. M. Fingerman, and D. G. White. 1999. Characterization of expanded-spectrum cephalosporin resistance in E. coli isolates associated with bovine calf diarrhoeal disease. J. Antimicrob. Chemother. 44:607-610. [DOI] [PubMed] [Google Scholar]
  • 11.Bradford, P. A., and C. C. Sanders. 1995. Development of test panel of β-lactamases expressed in a common Escherichia coli host background for evaluation of new β-lactam antibiotics. Antimicrob. Agents Chemother. 39:308-313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Bradford, P. A., and C. C. Sanders. 1992. Use of a predictor panel for development of a new disk for diffusion tests with cefoperazone-sulbactam. Antimicrob. Agents Chemother. 36:394-400. (Erratum, 36:1345.) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Bradford, P. A., and C. C. Sanders. 1993. Use of a predictor panel to evaluate susceptibility test methods proposed for piperacillin-tazobactam. Antimicrob. Agents Chemother. 37:2578-2583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Bradford, P. A., and C. C. Sanders. 1993. Use of a predictor panel to evaluate susceptibility testing methods for ampicillin-sulbactam. Antimicrob. Agents Chemother. 37:251-259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Bradford, P. A., C. Urban, A. Jaiswal, N. Mariano, B. A. Rasmussen, S. J. Projan, J. J. Rahal, and K. Bush. 1995. SHV-7, a novel cefotaxime-hydrolyzing β-lactamase, identified in Escherichia coli isolates from hospitalized nursing home patients. Antimicrob. Agents Chemother. 39:899-905. (Erratum, 39:1646.) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Bradford, P. A., Y. Yang, D. Sahm, I. Grope, D. Gardovska, and G. Storch. 1998. CTX-M-5, a novel cefotaxime-hydrolyzing β-lactamase from an outbreak of Salmonella typhimurium in Latvia. Antimicrob. Agents Chemother. 42:1980-1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Bush, K. 2002. The impact of β-lactamases on the development of novel antimicrobial agents. Curr. Opin. Investig. Drugs 3:1284-1290. [PubMed] [Google Scholar]
  • 18.Bush, K., G. Jacoby, and A. Medeiros. 1995. A functional classification scheme for β-lactamases and its correlation with molecular structure. Antimicrob. Agents Chemother. 39:1211-1233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.CLSI. 2006. Methods for antimicrobial susceptibility testing of anaerobic bacteria: approved standard M11-A7, 7th ed., vol. 27. Clinical Laboratory Standards Institute, Wayne, PA.
  • 20.CLSI. 2006. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard M7-A7, 7th ed., vol. 26. Clinical and Laboratory Standards Institute, Wayne, PA.
  • 21.CLSI. 2006. Performance standards for antimicrobial susceptibility testing, 16th informational supplement M100-S16, vol. 26. Clinical Laboratory Standards Institute, Wayne, PA.
  • 22.Giske, G. C., D. L. Monnet, O. Cars, and Y. Carmeli. 2008. Clinical and economic impact of common multidrug-resistant gram-negative bacilli. Antimicrob. Agents Chemother. 52:813-821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Gniadkowski, M. 2001. Evolution and epidemiology of extended-spectrum β-lactamases (ESBLs) and ESBL-producing microorganisms. Clin. Microbiol. Infect. 7:597-608. [DOI] [PubMed] [Google Scholar]
  • 24.Greenwood, D. 1996. Fixed or variable concentrations of β-lactamase inhibitors in in-vitro tests? J. Antimicrob. Chemother. 38:17-20. [DOI] [PubMed] [Google Scholar]
  • 25.Hakenbeck, R., and J. Coyette. 1998. Resistant penicillin-binding proteins. Cell Mol. Life Sci. 54:332-340. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Jones, C. H., and P. J. Petersen. 2005. Tigecycline: a review of preclinical and clinical studies of the first-in-class glycylcycline antibiotic. Drugs Today 41:637-659. [DOI] [PubMed] [Google Scholar]
  • 27.Jones, R. N., and A. L. Barry. 1989. Studies to optimize the in vitro testing of piperacillin combined with tazobactam (YTR 830). Diagn. Microbiol. Infect. Dis. 12:495-510. [DOI] [PubMed] [Google Scholar]
  • 28.Kotapati, S., L. J. Kuti, C. H. Nightingale, and D. P. Nicolau. 2005. Clinical implications of extended spectrum β-lactamases (ESBL) producing Klebsiella species and Escherichia coli on cefepime effectiveness. J. Infect. 51:211-217. [DOI] [PubMed] [Google Scholar]
  • 29.Kuck, N. A., N. V. Jacobus, M. D. Spengler, and R. T. Testa. 1996. In vitro activity of piperacillin-tazobactam against isolates from patients enrolled in clinical trials. Int. J. Antimicrob. Agents 7:15-21. [DOI] [PubMed] [Google Scholar]
  • 30.Li, J., R. L. Nation, J. D. Turnidge, R. W. Milne, K. Coulthard, C. R. Rayner, and D. L. Paterson. 2006. Colistin: the re-emerging antibiotic for multidrug-resistant gram-negative bacterial infections. Lancet Infect. Dis. 6:589-601. [DOI] [PubMed] [Google Scholar]
  • 31.Maiti, S. N., O. A. Phillips, R. G. Micetich, and D. M. Livermore. 1998. β-Lactamase inhibitors: agents to overcome bacterial resistance. Curr. Med. Chem. 5:441-456. [PubMed] [Google Scholar]
  • 32.Marchaim, D., S. Navon-Venezia, M. J. Schwaber, and Y. Carmeli. 2008. Isolation of imipenem-resistant Enterobacter species: emergence of KPC-2 carbapenemase, molecular characterization, epidemiology, and outcomes. Antimicrob. Agents Chemother. 52:1413-1418. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Miller, L. A., K. Ratnam, and D. J. Payne. 2001. β-Lactamase-inhibitor combinations in the 21st century: current agents and new developments. Curr. Opin. Pharm. 1:451-458. [DOI] [PubMed] [Google Scholar]
  • 34.Papanicolaou, G., A. A. Medeiros, and G. A. Jacoby. 1990. Novel plasmid-mediated β-lactamase (MIR-1) conferring resistance to oxyimino- and α-methoxy β-lactams in clinical isolates of Klebsiella pneumoniae. Antimicrob. Agents Chemother. 34:2200-2209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Petersen, P. J., C. H. Jones, and P. A. Bradford. 2007. Abstr. 47th Intersci. Conf. Antimicrob. Agents Chemother., abstr. F1-323.
  • 36.Rasheed, J. K., C. Jay, B. Metchock, F. Berkowitz, L. Weigel, J. Crellin, C. Steward, B. Hill, A. A. Medeiros, and F. C. Tenover. 1997. Evolution of extended-spectrum β-lactam resistance (SHV-8) in a strain of Escherichia coli during multiple episodes of bacteremia. Antimicrob. Agents Chemother. 41:647-653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Rasmussen, B. A., P. A. Bradford, J. P. Quinn, J. Wiener, R. A. Weinstein, and K. Bush. 1993. Genetically diverse ceftazidime-resistant isolates from a single center: biochemical and genetic characterization of TEM-10 β-lactamases encoded by different nucleotide sequences. Antimicrob. Agents Chemother. 37:1989-1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Rasmussen, B. A., Y. Gluzman, and F. P. Tally. 1990. Cloning and sequencing of the class B β-lactamase gene (ccrA) from Bacteroides fragilis TAL3636. Antimicrob. Agents Chemother. 34:1590-1592. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Rhomberg, P. R., T. R. Fritsche, H. S. Sader, and R. N. Jones. 2006. Antimicrobial susceptibility pattern comparisons among intensive care unit and general ward gram-negative isolates from the Meropenem Yearly Susceptibility Test Information Collection Program (USA). Diagn. Microbiol. Infect. Dis. 56:57-62. [DOI] [PubMed] [Google Scholar]
  • 40.Sanders, C. C., K. S. Thomson, and P. A. Bradford. 1993. Problems with detection of β-lactam resistance among nonfastidious gram-negative bacilli. Infect. Dis. Clin. N. Am. 7:411-424. [PubMed] [Google Scholar]
  • 41.Song, W., E. S. Moland, N. D. Hanson, J. S. Lewis, J. H. Jorgensen, and K. S. Thomson. 2005. Failure of cefepime therapy in treatment of Klebsiella pneumoniae bacteremia. J. Clin. Microbiol. 43:4891-4894. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Sutton, L. D., and R. N. Jones. 1995. Suggested modifications for disk diffusion susceptibility testing criteria for levofloxacin and sparfloxacin following tests with a predictor panel of ciprofloxacin-resistant clinical isolates. J. Clin. Microbiol. 33:146-148. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Urban, C., K. S. Meyer, N. Mariano, J. J. Rahal, R. Flamm, B. A. Rasmussen, and K. Bush. 1994. Identification of TEM-26 β-lactamase responsible for a major outbreak of ceftazidime-resistant Klebsiella pneumoniae. Antimicrob. Agents Chemother. 38:392-395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Venkatesan, A. M., A. Agarwal, T. Abe, H. Ushirogochi, I. Yamamura, T. Kumagai, P. J. Petersen, W. J. Weiss, E. Lenoy, Y. Yang, D. M. Shlaes, J. L. Ryan, and T. S. Mansour. 2004. Novel imidazole substituted 6-methylidene-penems as broad-spectrum β-lactamase inhibitors. Bioorg. Med. Chem. 12:5807-5817. [DOI] [PubMed] [Google Scholar]
  • 45.Venkatesan, A. M., Y. Gu, O. Dos Santos, T. Abe, A. Agarwal, Y. Yang, P. J. Petersen, W. J. Weiss, T. S. Mansour, M. Nukaga, A. M. Hujer, R. A. Bonomo, and J. R. Knox. 2004. Structure-activity relationship of 6-methylidene penems bearing tricyclic heterocycles as broad-spectrum β-lactamase inhibitors: crystallographic structures show unexpected binding of 1,4-thiazepine intermediates. J. Med. Chem. 47:6556-6568. [DOI] [PubMed] [Google Scholar]
  • 46.Walsh, T. R. 2008. Clinically significant carbapenemases: an update. Curr. Opin. Infect. Dis. 21:367-371. [DOI] [PubMed] [Google Scholar]
  • 47.Walther-Rasmussen, J., and N. Hoiby. 2002. Plasmid-borne AmpC β-lactamases. Can. J. Microbiol. 48:479-493. [DOI] [PubMed] [Google Scholar]
  • 48.Weiss, W. J., P. J. Petersen, T. M. Murphy, L. Tardio, Y. Yang, P. A. Bradford, A. M. Venkatesan, T. Abe, T. Isoda, A. Mihira, H. Ushirogochi, T. Takasake, S. Projan, J. O'Connell, and T. S. Mansour. 2004. In vitro and in vivo activities of novel 6-methylidene penems as β-lactamase inhibitors. Antimicrob. Agents Chemother. 48:4589-4596. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Woodford, N., S. Reddy, E. J. Fagan, R. L. Hill, K. L. Hopkins, M. E. Kaufmann, J. Kistler, M. F. Palepou, R. Pike, M. E. Ward, J. Cheesbrough, and D. M. Livermore. 2007. Wide geographic spread of diverse acquired AmpC β-lactamases among Escherichia coli and Klebsiella spp. in the UK and Ireland. J. Antimicrob. Chemother. 59:102-105. [DOI] [PubMed] [Google Scholar]
  • 50.Yang, K., and B. J. Guglielmo. 2007. Diagnosis and treatment of extended-spectrum and AmpC β-lactamase-producing organisms. Ann. Pharmacother. 41:1427-1435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Yang, Y., N. Bhachech, P. A. Bradford, B. D. Jett, D. F. Sahm, and K. Bush. 1998. Ceftazidime-resistant Klebsiella pneumoniae and Escherichia coli isolates producing TEM-10 and TEM-43 β-lactamases from St. Louis, Missouri. Antimicrob. Agents Chemother. 42:1671-1676. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Antimicrobial Agents and Chemotherapy are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES