Activity of Meropenem with a Novel Broader-Spectrum β-Lactamase Inhibitor, WCK 4234, against Gram-Negative Pathogens Endemic to New York City

WCK 4234 is a novel diazabicyclooctane with potent inhibitory activity against class A and D carbapenemases and class C enzymes. We examined the in vitro activity of meropenem plus WCK 4234 (4 or 8 μg/ml) against Gram-negative pathogens from New York City. Three groups of isolates were analyzed: a contemporary collection of isolates, a collection of known carbapenem-resistant isolates, and a collection of isolates with defined resistance mechanisms.

ABSTRACT

WCK 4234 is a novel diazabicyclooctane with potent inhibitory activity against class A and D carbapenemases and class C enzymes. We examined the in vitro activity of meropenem plus WCK 4234 (4 or 8 μg/ml) against Gram-negative pathogens from New York City. Three groups of isolates were analyzed: a contemporary collection of isolates, a collection of known carbapenem-resistant isolates, and a collection of isolates with defined resistance mechanisms. From the contemporary collection, we found (i) that all Enterobacteriaceae were susceptible to meropenem plus WCK 4234, (ii) that susceptibility rates for Acinetobacter baumannii were 56.5% for meropenem alone, 82.6% with 4 μg/ml WCK 4234, and 95.7% with 8 μg/ml WCK 4234, and (iii) that WCK 4234 had a modest effect on susceptibility of Pseudomonas aeruginosa. Against a collection of carbapenem-resistant isolates, the addition of WCK 4234 to meropenem (i) restored meropenem susceptibility against Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae isolates, (ii) improved susceptibility against A. baumannii, and (iii) had a negligible effect against P. aeruginosa. When tested against isolates with defined mechanisms of resistance, MICs of meropenem plus WCK 4234 were higher for K. pneumoniae with bla_KPC albeit well below the susceptibility breakpoint; efflux systems or porins did not correlate with susceptibility. For A. baumannii, MICs of meropenem plus WCK 4234 did not correlate with efflux systems, outer membrane protein, bla_ampC, or bla_oxa-51; however, MICs were higher in isolates with extended-spectrum β-lactamases (ESBLs). For P. aeruginosa, isolates with relatively higher MICs of meropenem plus WCK 4234 had increased expression of ampC. WCK 4234 is a potent β-lactamase inhibitor that, when combined with meropenem, displays promising activity against multidrug-resistant pathogens.

INTRODUCTION

The initial development of β-lactamase inhibitors (clavulanic acid, sulbactam, and tazobactam) greatly expanded the β-lactam spectrum of activity. However, the subsequent widespread emergence of metallo-β-lactamases and certain serine carbapenemases (e.g., Klebsiella pneumoniae carbapenemase [KPC] and OXA-48) diminished the clinical utility of these agents. The introduction of the diazabicyclooctane class of β-lactamase inhibitors has once again expanded the spectrum of β-lactam activity against many serine β-lactamases (1, 2). The first clinically available diazabicyclooctane, avibactam, has demonstrated effective inhibition of class A carbapenemases (particularly KPC) and class C β-lactamases (1, 2). However, avibactam has poor activity against class B and D β-lactamases (1, 2). In addition, the development of resistance to ceftazidime-avibactam has been reported during the treatment of infections due to KPC-producing pathogens (3). WCK 4234 is a novel diazabicyclooctane possessing a nitrile side chain at the C-2 position (4). WCK 4234 has been shown to be a potent inactivator of class A, C, and D β-lactamases (4, 5). The addition of WCK 4234 potentiated carbapenem activity against isolates producing KPC, AmpC, and OXA β-lactamases (5). In addition, the combination of meropenem with WCK 4234 displayed efficacy in murine models of infection involving carbapenem-hydrolyzing OXA-possessing Acinetobacter baumannii (4).

In this study, we describe the activity of meropenem with either 4 or 8 μg/ml of WCK 4234 against (i) a contemporary collection of clinical isolates gathered from hospitals in New York City (group 1), (ii) a collection of carbapenem-resistant clinical isolates of Klebsiella pneumoniae, A. baumannii, and Pseudomonas aeruginosa from 2013 to 2014 (group 2), and (iii) a collection of K. pneumoniae, A. baumannii, and P. aeruginosa isolates with defined mechanisms of resistance (group 3).

RESULTS

Group 1 isolates.

From a surveillance study conducted in 2017, there were 1,877 isolates of Escherichia coli, including three isolates carrying bla_KPC. Overall susceptibility rates included 92.5% for ceftazidime, 98.8% for piperacillin-tazobactam, 99.8% for amikacin, and 98.2% for polymyxin B. Overall, 99.9% were susceptible to meropenem (Table 1), and 100% (using the breakpoint for meropenem) were susceptible to the combination of meropenem and WCK 4234 (either 4 or 8 μg/ml). MICs of meropenem plus WCK 4234 were similar for isolates with and without extended-spectrum β-lactamases (ESBLs) and remained well below the breakpoint of susceptibility. Among 521 isolates of K. pneumoniae, susceptibility rates were 83.8% for ceftazidime, 95.4% for piperacillin-tazobactam, 98.5% for amikacin, and 96.9% for polymyxin B. While 97.1% of isolates were susceptible to meropenem, all were susceptible to the combination of meropenem and WCK 4234 (either 4 or 8 μg/ml). There were 18 isolates of K. pneumoniae with bla_KPC, and meropenem MICs were ≤0.25 μg/ml when meropenem was combined with 4 μg/ml WCK 4234 and ≤0.12 μg/ml when it was combined with 8 μg/ml WCK 4234. MICs of meropenem plus WCK 4234 were similar for isolates with ESBLs (but without KPC) and without ESBLs. Finally, there were 172 isolates of Enterobacter spp., including 59 Enterobacter aerogenes and 105 Enterobacter cloacae isolates. Three isolates possessed bla_KPC. Overall susceptibility rates included 83.0% for ceftazidime, 89.5% for piperacillin-tazobactam, 99.4% for amikacin, and 75% polymyxin B. Overall, 98.8% of these isolates were susceptible to meropenem, and all were susceptible to the combination of meropenem and WCK 4234 (either 4 or 8 μg/ml).

TABLE 1.

MICs of meropenem and meropenem plus WCK 4234 for the group 1 contemporary collection of Gram-negative clinical isolates

Organism and drug(s)a	MIC50 (μg/ml)	MIC90 (μg/ml)	MIC range (μg/ml)	Susceptibility (%)
E. coli (n =1,877)
Meropenem	≤0.03	0.06	≤0.03 to 4	99.9
Meropenem + 4 μg/ml WCK 4234	≤0.03	≤0.03	≤0.03 to 0.12	100
Meropenem + 8 μg/ml WCK 4234	≤0.03	≤0.03	≤0.03 to 0.12	100
K. pneumoniae (n = 521)
Meropenem	≤0.03	0.06	≤0.03 to >16	97.1
Meropenem + 4 μg/ml WCK 4234	≤0.03	≤0.03	≤0.03 to 0.25	100
Meropenem + 8 μg/ml WCK 4234	≤0.03	≤0.03	≤0.03 to 0.12	100
Enterobacter spp. (n =172)
Meropenem	≤0.03	0.06	≤0.03 to 8	98.8
Meropenem + 4 μg/ml WCK 4234	≤0.03	≤0.03	≤0.03 to 0.25	100
Meropenem + 8 μg/ml WCK 4234	≤0.03	≤0.03	≤0.03 to 0.12	100
A. baumannii (n = 46)
Meropenem	1	>16	≤0.03 to >16	56.5
Meropenem + 4 μg/ml WCK 4234	0.5	4	0.25 to 16	82.6
Meropenem + 8 μg/ml WCK 4234	0.5	2	≤0.03 to 8	95.7
P. aeruginosa (n = 271)
Meropenem	1	8	0.12 to >16	71.2
Meropenem + 4 μg/ml WCK 4234	1	8	≤0.03 to >16	79.3
Meropenem + 8 μg/ml WCK 4234	1	4	≤0.03 to >16	79.3

^an, number of isolates.

There were 46 isolates of A. baumannii. Overall susceptibility rates included 54% for ceftazidime, 43.5% for piperacillin-tazobactam, 73.9% for amikacin, and 93.5% for polymyxin B. While 56.5% of isolates were susceptible to meropenem, 82.6% were susceptible to meropenem plus 4 μg/ml WCK 4234, and 95.7% were susceptible to meropenem plus 8 μg/ml WCK 4234. The MICs for eight meropenem-resistant isolates that possessed bla_OXA-23 were 0.5 to 4 μg/ml (87.5% susceptible) for meropenem plus 4 μg/ml WCK 4234 and 0.5 to 2 μg/ml (100% susceptible) for meropenem 8 μg/ml WCK 4234. Two isolates possessed bla_OXA-24 and had MICs of 8 and 16 for meropenem plus 4 μg/ml WCK 4234 and MICs of 2 and 8 μg/ml for meropenem plus 8 μg/ml WCK 4234. Finally, there were 271 isolates of P. aeruginosa, with overall susceptibility rates of 82.5% for ceftazidime 75.1% for piperacillin-tazobactam, and 97.8% for amikacin. While 71.2% were susceptible to meropenem, 79.3% were susceptible to meropenem plus WCK 4234 (either 4 or 8 μg/ml).

Group 2 isolates.

From the surveillance isolates gathered in 2013 to 2014, there were 87 KPC-producing K. pneumoniae isolates. Overall, MIC₅₀ and MIC₉₀ values for meropenem, meropenem plus 4 μg/ml WCK 4234, and meropenem plus 8 μg/ml WCK 4234 were 8 and >16, ≤0.03 and 1, and ≤0.03 and 1 μg/ml, respectively. Overall, 93.1% of these isolates were susceptible to meropenem plus 4 μg/ml WCK 4234, and all were susceptible to meropenem plus 8 μg/ml WCK 4234.

There were 76 carbapenem-resistant A. baumannii isolates gathered, with all isolates having meropenem MICs of ≥16 μg/ml. Addition of WCK 4234 at 4 and 8 μg/ml resulted in substantial decreases in MIC₅₀ and MIC₉₀ values of meropenem to 4 and 8 μg/ml, respectively, and 2 and 4 μg/ml, respectively. Overall, 30.3% of isolates were susceptible to meropenem plus 4 μg/ml WCK 4234, and 59.2% were susceptible to meropenem plus 8 μg/ml WCK 4234. Of 47 isolates that possessed bla_OXA-23, the MIC₅₀ and MIC₉₀ values were 4 and 4 μg/ml, respectively, for meropenem plus 4 μg/ml WCK 4234 and 2 and 4 μg/ml, respectively, for meropenem plus 8 μg/ml WCK 4234. One isolate possessed bla_OXA-24 had a MIC of 2 μg/ml for meropenem plus WCK 4234 (either 4 or 8 μg/ml). One isolate possessed both bla_OXA-23 and bla_OXA-24 and had MICs of >16 μg/ml for meropenem plus WCK 4234 (both 4 and 8 μg/ml).

There were 119 carbapenem-resistant P. aeruginosa isolates gathered in 2013 to 2014. Nearly all had meropenem MICs of ≥16 μg/ml. The MIC₅₀ and MIC₉₀ values for meropenem plus WCK 4234 at 4 μg/ml and 8 μg/ml were both 8 and >16 μg/ml, respectively; 5.9% of isolates were susceptible to meropenem plus 4 μg/ml WCK 4234, and 9.2% were susceptible to meropenem plus 8 μg/ml WCK 4234.

Group 3 isolates.

There were 34 characterized isolates of K. pneumoniae, including 14 with bla_KPC. Isolates with MICs of ≥0.25 μg/ml for meropenem plus WCK 4234 (either 4 or 8 μg/ml) had levels of expression of mar, sox, acrB, ram, and ompK36 similar to those of isolates for which the MICs were ≤0.12 μg/ml. Similarly, the presence of a frameshift mutation in ompK35 did not affect MICs of meropenem plus WCK 4234. However, the presence of bla_KPC did affect MICs although all remained well below the breakpoint for meropenem. The MICs of meropenem plus 4 μg/ml WCK 4234 for isolates with bla_KPC were 0.36 ± 0.58 μg/ml, in contrast to 0.021 ± 0.0075 μg/ml (P = 0.05). for those without bla_KPC. Similarly, the MICs of meropenem plus 8 μg/ml WCK 4234 for isolates with bla_KPC were 0.17 ± 0.20 μg/ml whereas they were 0.022 ± 0.011 μg/ml (P = 0.02) for isolates without bla_KPC. Five of 5 isolates with MICs of meropenem plus WCK 4234 (either 4 or 8 μg/ml) that were ≥0.25 μg/ml possessed bla_KPC, compared to 9 of 29 with MICs of ≤0.125 μg/ml (P = 0.007). Of the isolates with meropenem MICs of ≥4 μg/ml (all with KPC), the MICs dropped at least 4-fold with the addition of 8 μg/ml WCK 4234. For isolates lacking KPC, MICs of meropenem plus WCK 4234 were similar for isolates with and without ESBLs (0.021 ± 0.008 versus 0.021 ± 0.008 μg/ml with 4 μg/ml WCK 4234 [P not significant] and 0.024 ± 0.014 versus 0.021 ± 0.007 μg/ml with 8 μg/ml WCK 4234 [P not significant]).

There were 34 characterized isolates of A. baumannii examined. Seventeen isolates had SHV-type ESBLs. The MICs of meropenem plus WCK 4234 were higher for these isolates than for the isolates lacking ESBLs (5.9 ± 1.1 versus 1.3 ± 0.2 μg/ml for meropenem plus 4 μg/ml WCK 4234 [P = 0.001] and 3.8 ± 0.7 versus 1.0 ± 0.1 μg/ml for 8 μg/ml WCK 4234 [P = 0.002]). Isolates for with the MICs of meropenem plus WCK 4234 (either 4 or 8 μg/ml) were ≥4 μg/ml did not have significantly increased expression of ampC, oxa-51, adeB, or abeM or decreased expression of oprF compared to expression levels in isolates with MICs of ≤2 μg/ml. Of the isolates for which the meropenem MICs were ≥4 μg/ml, those with least a 4-fold decrease in the MIC with the addition of 8 μg/ml WCK 4234 had higher levels of expression of ampC than those without a 4-fold decrease (76.5 ± 111 versus 15.9 ± 9.0 μg/ml; P = 0.01).

There were 33 isolates of P. aeruginosa analyzed. Isolates for which MICs of meropenem plus WCK 4234 (either 4 or 8 μg/ml) were ≥4 μg/ml had expression levels of mexA, mexC, mexE, mexX, and oprD similar to those in isolates with MICs of ≤2 μg/ml. However, isolates with MICs ≥4 μg/ml were more likely to have increased expression (>10 times control) of ampC (59% versus 19%; P = 0.03). Of the isolates with meropenem MICs of ≥4 μg/ml, those without a 4-fold decrease in the MIC with the addition of 8 μg/ml WCK 4234 had higher levels of expression of mexA than those with a 4-fold decrease (2.85 ± 3.01 versus 1.06 ± 0.54 μg/ml; P = 0.04), suggesting that increased expression of mexA may blunt the effect of WCK 4234.

DISCUSSION

Because of the associated mortality and health care burden, the WHO has listed carbapenem-resistant Enterobacteriaceae, A. baumannii, and P. aeruginosa as “critical-priority” pathogens (6). Novel therapies, effective against the myriad of resistance mechanisms, are urgently needed. K. pneumoniae has emerged as the most important pathogen among the Enterobacteriaceae, and isolates producing KPC, OXA-48, and metallo-β-lactamases have been especially widespread. The combination of WCK 4234 with a carbapenem has been previously shown to be active against multidrug-resistant Enterobacteriaceae producing extended-spectrum, AmpC, KPC, and OXA β-lactamases (5). In our report, the combination of meropenem and WCK 4234 demonstrated remarkable activity against Enterobacteriaceae, including a large number of KPC-producing K. pneumoniae isolates. Although isolates with bla_KPC had higher MICs than isolates lacking this enzyme, the MICs remained well below the meropenem susceptibility breakpoint. We found no correlation between the expression levels of genes encoding efflux systems or ompK36 or the presence of frameshift mutations in ompK35 and the MICs of meropenem plus WCK 4234.

Novel therapies are also urgently needed for multidrug-resistant A. baumannii as currently available β-lactamase inhibitors lack activity against class D carbapenemases. WCK 4234 is a potent inactivator of these enzymes, which are especially problematic in multidrug-resistant A. baumannii. WCK 4234 restored carbapenem activity against multidrug-resistant A. baumannii expressing OXA-23, OXA-24/40, and OXA-58 and hyperproducing the chromosomal OXA-51 carbapenemases (5). The combination of meropenem plus WCK 4234 was efficacious in vivo against A. baumannii isolates producing OXA-23 or OXA-26 (4). In our report, increasing the concentration of WCK 4234 led to progressive declines in the meropenem MIC₅₀ and MIC₉₀ values for carbapenem-resistant isolates, suggesting considerable inhibitory activity against Acinetobacter-derived carbapenemases. The activity of meropenem plus WCK 4234 was not affected by the presence of OXA-23. We again found no correlation between the expression levels of genes encoding efflux systems, chromosomal β-lactamases, or outer membrane proteins and the MICs of meropenem plus WCK 4234.

Because of a plethora of intrinsic mechanisms of resistance, multidrug-resistant P. aeruginosa remains a therapeutic challenge. WCK 4234 has potentiated the activity of carbapenems against OXA-expressing or AmpC-hyperproducing P. aeruginosa (5). In our collection of clinical isolates, none of which possessed an OXA carbapenemase, the addition of WCK 4234 had a modest effect on improving the MICs of meropenem. Although WCK 4234 is stable against the chromosomal AmpC β-lactamase (4), we found isolates with higher MICs of meropenem plus WCK 4234 were more likely to be hyperproducing this β-lactamase.

In summary, our results support the continued development of the β-lactamase inhibitor WCK 4234, particularly against multidrug-resistant strains of Enterobacteriaceae and A. baumannii. Further studies are needed to define the role of WCK 4234 for P. aeruginosa.

MATERIALS AND METHODS

Three groups of bacterial isolates were examined. The first group (group 1) involved a collection of clinical isolates of Escherichia coli, K. pneumoniae, Enterobacter spp., A. baumannii, and P. aeruginosa gathered from seven medical centers in Brooklyn, NY, in 2017, as previously described (7). The second group (group 2) consisted of a collection of KPC-producing K. pneumoniae isolates and carbapenem-resistant A. baumannii and P. aeruginosa isolates collected in a surveillance study conducted in 2013 to 2014 (8, 9). All cephalosporin-resistant isolates in group 1 and all isolates in group 2 were screened for the presence of carbapenemases, as previously described (7).

A third collection (group 3) consisted of characterized isolates of K. pneumoniae, A. baumannii, and P. aeruginosa. For K. pneumoniae, the expression levels of the bla_KPC, mar, sox, acrB, ram, and ompK36 genes were previously determined by real-time reverse transcription-PCR (RT-PCR) (10). In addition, frameshift mutations in ompK35 and the identification of β-lactamases were determined by DNA sequencing (10). For A. baumannii, expression levels of ampC, oxa-51, adeB, abeM, and oprF were examined by real-time RT-PCR, and the presence of β-lactamases was determined by DNA sequencing (11). Last, for P. aeruginosa, the expression levels of ampC, mexA, mexC, mexE, mexX, and oprD were examined, along with the identification of β-lactamases (12).

MICs of ceftazidime, piperacillin-tazobactam, amikacin, and polymyxin B were determined by the agar dilution method with Mueller-Hinton agar according to established methods (13). MICs of meropenem and meropenem plus 4 μg/ml and 8 μg/ml of WCK 4234 (Wockhardt Bio AG, India) were determined using the broth microdilution method using cation-supplemented Mueller-Hinton broth. The quality control (QC) isolates included E. coli ATCC 25922 and ATCC 35218, K. pneumoniae ATCC 700603, and P. aeruginosa ATCC 27853. Susceptibility rates were determined using CLSI-established breakpoints (13).

Statistical methods.

Student's t test and Fisher’s exact test were used to compare groups with various resistance mechanisms. A two-tailed P value of ≤0.05 was considered significant.