Abstract
Multidrug-resistant Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae are endemic to hospitals in New York City and other regions. RPX7009 is a novel β-lactamase inhibitor with activity against serine carbapenemases. We tested the activity of meropenem plus RPX7009 against 4,500 recent Gram-negative clinical isolates from 11 New York City hospitals. The meropenem-RPX7009 combination was found to have excellent in vitro activity against Escherichia coli, K. pneumoniae, and Enterobacter spp., including multidrug-resistant (MDR) KPC-producing strains. Overall, 131/133 (98.5%) KPC-producing Enterobacteriaceae strains were inhibited by meropenem (≤1 μg/ml) plus RPX7009 (8 μg/ml). In a limited number of strains, the combination appeared to have reduced activity against KPC-producing K. pneumoniae isolates with diminished ompK35 and ompK36 expression. The addition of RPX7009 did not affect the activity of meropenem against Acinetobacter baumannii and Pseudomonas aeruginosa. The meropenem-RPX7009 combination shows promise as a novel agent against KPC-producing Enterobacteriaceae and deserves further study. Other approaches will be needed to address multidrug-resistant A. baumannii and P. aeruginosa, which typically possess different mechanisms of carbapenem resistance.
INTRODUCTION
Carbapenem resistance has been reported worldwide among the Enterobacteriaceae, in Acinetobacter baumannii, and in Pseudomonas aeruginosa (1–3). Mechanisms of carbapenem resistance include production of carbapenem-hydrolyzing enzymes, loss or alteration of porins, and increased efflux system activity (4–6). Historically, the old β-lactamase inhibitors (clavulanate, sulbactam, and tazobactam), in combination with β-lactams, have been useful against organisms producing various types of β-lactamases. However, these compounds have not been found to be active against many carbapenemases, including Klebsiella pneumoniae carbapenemase (KPC) and metalloenzymes (7). Several new β-lactamase inhibitors are being developed in the hope of preserving β-lactam activity (7, 8).
RPX7009 is a boron-containing serine β-lactamase inhibitor. Unlike the old β-lactamase inhibitors, boronic acid β-lactamase inhibitors work via formation of a covalent bond between the boron moiety and the serine side chain of the β-lactamase (8). They are also resistant to hydrolysis by serine β-lactamases (8). Activity of RPX7009, in combination with biapenem (formerly RPX2003) or meropenem, has been demonstrated against a variety of carbapenem-resistant Enterobacteriaceae. The biapenem-RPX7009 combination has been shown to overcome resistance in the majority of KPC-producing isolates tested, but no inhibition was shown for class B- and D-producing isolates (9). RPX7009 also has activity against some AmpC β-lactamases and restored the activity of cefepime against some Enterobacter cloacae isolates with hyperproduction of AmpC (8).
This study evaluated the activity of the meropenem-RPX7009 combination compared to meropenem alone against clinical isolates of Enterobacteriaceae, A. baumannii, and P. aeruginosa collected from various New York City hospitals during 2013 and 2014. The activity of meropenem with and without RPX7009 was also evaluated against isolates of K. pneumoniae, A. baumannii, and P. aeruginosa with previously characterized mechanisms of resistance.
MATERIALS AND METHODS
Single patient clinical isolates of Escherichia coli, Enterobacter spp., K. pneumoniae, A. baumannii, and P. aeruginosa were obtained from hospitals located in Brooklyn and Queens, NY, from November 2013 through January 2014. Susceptibility testing was done by either the broth microdilution method (for meropenem with and without RPX7009) or the agar dilution method (for all other antibiotics), as described previously (10), and the MICs were interpreted according to CLSI guidelines (11). RPX7009 (Rempex, San Diego, CA, USA) was combined with meropenem at fixed concentrations of 4 and 8 μg/ml, which were previously shown to optimally enhance the activity of biapenem (9). E. coli ATCC 25922, E. coli ATCC 35218, and P. aeruginosa ATCC 27853 were used as control organisms. Cephalosporin-resistant isolates were screened by PCR, using previously described primers and conditions (12–15), for the following carbapenemase genes: blaKPC, blaNDM, blaVIM, blaIMP, blaOXA48, blaOXA58, blaOXA23-like, and blaOXA24-like. The MICs of meropenem with and without RPX7009 were also determined for K. pneumoniae, A. baumannii, and P. aeruginosa isolates previously characterized for mechanisms of resistance (5, 6, 16).
RESULTS
Escherichia coli.
A total of 2,770 isolates of E. coli were tested. Almost 100% of the isolates were susceptible to meropenem (99.9%), with all the isolates inhibited by meropenem (≤1 μg/ml) in combination with RPX7009. Five isolates were found to possess blaKPC. The meropenem MICs were 0.125, 0.25, 0.5, 2, and 4 μg/ml. These MICs decreased to 0.03, 0.015, 0.03, 0.03, and 0.03 μg/ml, respectively, with RPX7009 at 4 μg/ml and to 0.015, 0.015, 0.03, 0.015, and 0.015 μg/ml, respectively, with RPX7009 at 8 μg/ml. Except for blaKPC, no carbapenemase genes were detected.
Klebsiella pneumoniae.
Antibiotic susceptibilities were determined for 894 isolates of K. pneumoniae. Eighty-eight percent of the isolates were susceptible to meropenem alone; this increased to 98.8% inhibited by meropenem at 1 μg/ml combined with RPX7009 at 4 μg/ml and to 99.8% combined with RPX7009 at 8 μg/ml. There were 121 isolates of K. pneumoniae found to possess blaKPC (Table 1). The addition of RPX7009 resulted in a 64- to 512-fold decrease in the meropenem MIC in the majority of KPC-positive isolates (range, 0- to 2,048-fold decrease). All but 2 of these isolates (98.3%) were inhibited by 1 μg/ml meropenem combined with RPX7009 at 8 μg/ml. No carbapenemase genes other than blaKPC were detected.
TABLE 1.
Susceptibility patterns of meropenem in combination with RPX7009 and other antibiotics against carbapenem-resistant Gram-negative organisms in New York City
| Drug(s) | MIC50 (μg/ml) | MIC90 (μg/ml) | Range (μg/ml) | % Susceptiblea |
|---|---|---|---|---|
| K. pneumoniae (KPC+) (n = 121) | ||||
| Piperacillin-tazobactam | >128/4 | >128/4 | 2/4 to >128/4 | 1 |
| Ceftazidime | >16 | >16 | 8 to >16 | 0 |
| Gentamicin | >8 | >8 | 0.5 to >8 | 37 |
| Amikacin | 32 | 64 | ≤0.5 to >64 | 46 |
| Ciprofloxacin | >4 | >4 | ≤0.125 to >4 | 9 |
| Trimethoprim-sulfamethoxazole | >4 | >4 | ≤0.5 to >4 | 13 |
| Meropenem | 8 | 64 | 0.25 to >64 | 7 |
| Meropenem-RPX7009 (4 μg/ml) | 0.06/4 | 2/4 | 0.008/4 to >64/4 | NA |
| Meropenem-RPX7009 (8 μg/ml) | 0.03/8 | 0.5/8 | ≤0.004/8 to >64/8 | NA |
| A. baumannii (n = 84) | ||||
| Ampicillin-sulbactam | 32/16 | >32/16 | 4/2 to >32/16 | 17 |
| Piperacillin-tazobactam | >128/4 | >128/4 | >128/4 to >128/4 | 4 |
| Ceftazidime | >16 | >16 | 4 to >16 | 2 |
| Gentamicin | >8 | >8 | ≤0.25 to >8 | 20 |
| Amikacin | 4 | >64 | 1 to >64 | 59 |
| Ciprofloxacin | >4 | >4 | ≤0.125 to >4 | 4 |
| Trimethoprim-sulfamethoxazole | >4 | >4 | ≤0.5 to >4 | 10 |
| Meropenem | 32 | 64 | 4 to >64 | 0 |
| Meropenem-RPX7009 (4 μg/ml) | 32/4 | 64/4 | 0.25/4 to >64/4 | NA |
| Meropenem-RPX7009 (8 μg/ml) | 32/8 | 64/8 | 1/8 to >64/8 | NA |
| P. aeruginosa (n = 98) | ||||
| Piperacillin-tazobactam | 16/4 | >128/4 | 16/4 to >128/4 | 52 |
| Ceftazidime | 8 | >16 | 1 to >16 | 37 |
| Amikacin | 4 | 16 | ≤0.5 to >64 | 94 |
| Ciprofloxacin | >4 | >4 | ≤0.125 to >4 | 35 |
| Meropenem | 8 | 32 | 4 to >64 | 0 |
| Meropenem-RPX7009 (4 μg/ml) | 8/4 | 32/4 | 0.125/4 to >64/4 | NA |
| Meropenem-RPX7009 (8 μg/ml) | 8/8 | 32/8 | 0.25/8 to 64/8 | NA |
NA, not available (breakpoint not established).
Isolates previously characterized for mechanisms of resistance were also tested (Table 2). The isolates were previously screened for the presence of blaKPC, blaSHV, and blaTEM and for expression of blaKPC, the porin genes ompK35 and ompK36, and the efflux system gene acrB. A total of 26 isolates representing 7 clonal groups were available for testing. The KPC-negative isolates were all susceptible to meropenem. Among the isolates that possessed blaKPC (n = 12), meropenem MICs ranged from 0.015 to 8 μg/ml (with 4 μg/ml of RPX7009) and 0.008 to 2 μg/ml (with 8 μg/ml of RPX7009). OmpK35 was not produced by nearly all of the KPC-producing isolates and was not included in further analysis. No correlation was found between MICs of meropenem combined with RPX7009 at 8 μg/ml and relative expression of blaKPC, ompK36, or acrB. However, two isolates (KC850 and CI855) (Table 2) had relatively high meropenem MICs despite the addition of RPX7009 (MIC = 8 μg/ml with 4 μg/ml of RPX7009 and MIC = 1 and 2 μg/ml with 8 μg/ml of RPX7009). Both of these isolates had decreased expression of ompK36 compared to only 1 of 10 isolates with meropenem-RPX7009 (8 μg/ml) MICs ≤ 0.5 μg/ml (P = 0.046). The significance of this finding requires further investigation given the small number of isolates studied.
TABLE 2.
Susceptibilities to meropenem in combination with RPX7009 of characterized isolates of K. pneumoniae
| Isolate | Clonal group | Other β-lactamase(s) | Relative expressionb |
MIC (μg/ml)c |
||||
|---|---|---|---|---|---|---|---|---|
| blaKPC | acrB | ompK36 | MEM | MEM + R4 | MEM + R8 | |||
| CI512 | B | SHV12; TEM1 | 1.07 | 8.22 | 0.015 | 0.015 | 0.008 | |
| VA302 | C | SHV5; TEM1 | 0.21 | 1.32 | 0.015 | 0.015 | 0.015 | |
| DM152 | I | None | 1.71 | 1.92 | 0.015 | 0.015 | 0.008 | |
| KB351 | U | SHV12 | 0.34 | 1.97 | 0.015 | 0.015 | 0.008 | |
| MA340 | H | SHV11; TEM1 | 0.24 | 1.04 | 0.015 | 0.015 | 0.015 | |
| CI504 | A | SHV11; SHV12 | 0.45 | 0.25 | 0.015 | 0.015 | 0.008 | |
| KB417 | J | SHV12; TEM1 | 1.24 | 1.36 | 0.03 | 0.03 | 0.03 | |
| CI505 | A | SHV11 | 0.02 | 1.31 | 0.03 | 0.015 | 0.008 | |
| CI518 | A | SHV12 | 0.02 | 1.01 | 0.03 | 0.015 | 0.015 | |
| VM522 | A | SHV12 | 0.003 | 0.88 | 0.03 | 0.008 | 0.008 | |
| CI511 | B | SHV12; TEM1 | 0.5 | 3.11 | 0.06 | 0.06 | 0.06 | |
| WH307 | C | TEM1 | 0.38 | 1.41 | 0.06 | 0.015 | 0.008 | |
| KB370 | C | TEM1 | 0.07 | 0.86 | 0.06 | 0.015 | 0.015 | |
| CI806 | B | SHV12; TEM1 | 0.11 | 1.81 | 0.12 | 0.06 | 0.03 | |
| VM9a | A | SHV11; TEM1 | 1.1 | 0.05 | 1.7 | 2 | 0.015 | 0.015 |
| CI302a | D | SHV5; TEM1 | 40 | 1.6 | 8.19 | 2 | 0.015 | 0.008 |
| CI516a | A | SHV11; TEM1 | 1 | 0.2 | 4.19 | 4 | 0.015 | 0.015 |
| DM834a | A | SHV12; TEM1 | 1.25 | 0.32 | 2.94 | 4 | 0.015 | 0.015 |
| CI513a | B | SHV12; TEM1 | 18.75 | 1.64 | 7.71 | 4 | 0.12 | 0.06 |
| CI839a | B | SHV12; TEM1 | 5 | 0.74 | 7.59 | 8 | 0.25 | 0.12 |
| MA31a | A | SHV12 | 1 | 0.49 | 0.25 | 16 | 0.12 | 0.06 |
| KB528a | B | SHV11; TEM1 | 22.5 | 0.56 | 3.28 | 32 | 2 | 0.5 |
| BD503a | D | SHV5; TEM1 | 198.75 | 6.01 | 4.51 | 32 | 0.06 | 0.06 |
| WO822a | B | SHV12; TEM1 | 1.1 | 0.52 | 5.7 | 64 | 0.5 | 0.12 |
| KC850a | A | SHV12; TEM1 | 2.5 | 0.04 | 0.004 | >64 | 8 | 1 |
| CI855ia | C | SHV12; TEM1 | 15 | 0.17 | 0.18 | >64 | 8 | 2 |
KPC-positive.
Relative expression compared with control (set to 1).
MEM meropenem; MEM + R4 meropenem, + RPX7009 (4 μg/ml); MEM + R8, meropenem + RPX7009 (8 μg/ml).
Enterobacter species.
A total of 211 Enterobacter species isolates were collected, including 121 E. cloacae and 88 Enterobacter aerogenes. Seven isolates were found to have blaKPC on screening: three were E. aerogenes, while four were E. cloacae. For meropenem alone, the MICs were 0.06, 2, 4, >8, >8, >8, and >8 μg/ml; the MICs decreased to 0.03, 0.06, 0.06, 0.03, 0.03, 0.06, and 2 μg/ml, respectively, with RPX7009 at 4 μg/ml and to 0.015, 0.03, 0.03, 0.03, 0.03, 0.03, and 0.5 μg/ml, respectively, with RPX7009 at 8 μg/ml. Genes for carbapenemases other than blaKPC were not detected. One isolate of E. aerogenes lacking blaKPC had a meropenem MIC of 2 μg/ml; the MICs for this isolate were 1 and 0.5 μg/ml with the addition of RPX7009 at 4 and 8 μg/ml, respectively. All the remaining KPC-negative isolates were susceptible to meropenem alone (MIC50/MIC90 = 0.06/0.125 μg/ml; range, 0.03 to 0.5 μg/ml).
Acinetobacter baumannii.
The susceptibilities of 158 isolates of A. baumannii were determined. Overall, 47% of the isolates were susceptible to meropenem. Among the meropenem-nonsusceptible isolates, MICs were largely unchanged by the addition of RPX7009 (Table 1). A 4-fold or greater decrease in the meropenem MIC was seen in only 4/84 and 2/84 meropenem-nonsusceptible isolates with RPX7009 at 4 and 8 μg/ml, respectively. One isolate was found to possess blaKPC. The MIC for meropenem alone was >64 μg/ml; this decreased to 16 μg/ml and 8 μg/ml with RPX7009 at 4 μg/ml and 8 μg/ml, respectively. Of the isolates screened, 58 were found to possess blaOXA23-like. The MIC50 and MIC90 for meropenem were 32 and 64 μg/ml, respectively, with or without RPX7009. Two isolates were found to have blaOXA24-like. For one isolate, the MICs for meropenem were >64 μg/ml with or without RPX7009; for the other isolate, the MICs were 32, 64, and 32 μg/ml, respectively, for meropenem alone, with RPX7009 at 4 μg/ml, and with RPX7009 at 8 μg/ml. Genes for the carbapenemases NDM, IMP, VIM, and OXA58 were not detected.
Thirty isolates previously characterized for mechanisms of resistance were also tested (data not shown). Expression of the β-lactamase genes ampC and blaOXA51, the porin gene ompA, and the efflux system genes adeB and abeM were previously determined. Twenty-five isolates were found to be nonsusceptible to meropenem; for these isolates, there was no change in meropenem MICs with the addition of RPX7009 at 8 μg/ml, regardless of the expression of ampC, blaOXA51, ompA, adeB, and abeM.
Pseudomonas aeruginosa.
A total of 467 isolates of P. aeruginosa were collected, and their susceptibilities were determined. Overall, 79% of the isolates were susceptible to meropenem. Among the meropenem-nonsusceptible isolates, the MIC50 and MIC90 were largely unchanged by the addition of RPX7009 (Table 1). A 4-fold or greater decrease in the meropenem MIC was seen in 9/98 and 6/98 meropenem-nonsusceptible isolates with RPX7009 at 4 and 8 μg/ml, respectively. None of the isolates possessed any of the carbapenemase genes screened.
Thirty isolates previously characterized for mechanisms of resistance were also tested (data not shown). Expression of the β-lactamase gene ampC; the porin gene oprD; and the efflux system genes mexA, mexC, mexE, and mexX were previously determined. Twenty-three isolates were found to be nonsusceptible to meropenem; for these isolates, there was no change in meropenem MICs with the addition of RPX7009 at 8 μg/ml, regardless of the expression of ampC, oprD, mexA, mexC, mexE, and mexX.
DISCUSSION
RPX7009, a new boronic acid β-lactamase inhibitor, is one of several β-lactamase inhibitors currently being developed to address the growing carbapenem resistance in Enterobacteriaceae, A. baumannii, and P. aeruginosa. It has been previously shown to preserve meropenem activity against KPC-producing isolates (17, 18). The meropenem-RPX7009 combination was also tested using an in vitro hollow-fiber model to simulate human exposure (19). Simulating human exposures of 2 g meropenem plus 2 g RPX7009 dosed every 8 h and infused over 3 h, the combination demonstrated bactericidal activity against KPC-producing isolates of Enterobacteriaceae. The meropenem-RPX7009 combination also showed efficacy in vivo against KPC-producing isolates of K. pneumoniae, E. coli, and E. cloacae with meropenem-RPX7009 (8 μg/ml) MICs ranging from ≤0.06 to 8 μg/ml using a murine thigh infection model (20). A study evaluating the efficacy, safety, and tolerability of the combination in adults with serious infections due to carbapenem-resistant Enterobacteriaceae is under way (http://clinicaltrials.gov/ct2/show/NCT02168946).
Our study confirmed the in vitro activity of meropenem plus RPX7009 against a large recent collection of Gram-negative clinical isolates from New York City, a region particularly affected by KPC-producing Enterobacteriaceae. The addition of RPX7009 resulted in a significant decrease in meropenem MICs in all but one of the blaKPC-possessing Enterobacteriaceae, regardless of species. Although the number of characterized isolates was small, the activity of meropenem-RPX7009 against KPC-producing K. pneumoniae isolates did not appear to be affected by expression of blaKPC or acrB or by diminished ompK35 alone. However, meropenem-RPX7009 MICs were relatively high in isolates with concomitantly diminished ompK35 and ompK36 expression, particularly when the lower RPX7009 concentration of 4 μg/ml was used. Previous studies have shown an association between carbapenem resistance and reduced ompK36 expression in K. pneumoniae (16), although the concomitant presence of both reduced ompK35 and ompK36 may be necessary (21). The combined presence of outer membrane porin deficiency along with β-lactamases has been previously reported to diminish the effect of novel β-lactamase inhibitors (9, 22) and might limit the utility of these agents against some strains. Additional studies of the effect of porin loss on the activity of meropenem-RPX7009 are needed.
The enhanced activity of meropenem in the presence of RPX7009 was limited to isolates where KPC production was the main mechanism of carbapenem resistance. Despite having activity against some class C β-lactamases, RPX7009 did not enhance the effect of meropenem against most A. baumannii and P. aeruginosa isolates in this study. Differing mechanisms of carbapenem resistance, including production of class D β-lactamases, loss or alteration of porins, and increased activity of efflux systems, may explain the lack of effect of RPX7009 against A. baumannii strains from this region. Decreased permeability and increased efflux, the primary mechanisms of carbapenem resistance among P. aeruginosa isolates from this region (5), would also not be affected by the addition of RPX7009. Other approaches will be necessary to address the problem of multidrug-resistant (MDR) A. baumannii and P. aeruginosa.
The meropenem-RPX7009 combination shows promise as a potential therapeutic agent against Gram-negative organisms, including KPC-producing strains, given the limited treatment options currently available. Additional studies are warranted to determine the clinical efficacy of the combination.
ACKNOWLEDGMENT
The study was supported by Rempex Pharmaceuticals, a wholly owned subsidiary of The Medicines Company.
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