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. 2018 Jul 27;62(8):e00533-18. doi: 10.1128/AAC.00533-18

In Vitro Activity of Imipenem-Relebactam and Ceftolozane-Tazobactam against Resistant Gram-Negative Bacilli

Suzannah M Schmidt-Malan a, Avisya J Mishra a, Ammara Mushtaq a, Cassandra L Brinkman a, Robin Patel a,b,✉
PMCID: PMC6105828  PMID: 29760145

Understanding which antimicrobial agents are likely to be active against Gram-negative bacilli can guide selection of antimicrobials for empirical therapy as mechanistic rapid diagnostics are adopted. In this study, we determined the MICs of a novel β-lactam–β-lactamase inhibitor combination, imipenem-relebactam, along with ceftolozane-tazobactam, imipenem, ertapenem, meropenem, ceftriaxone, and cefepime, against 282 drug-resistant isolates of Gram-negative bacilli.

KEYWORDS: antimicrobial resistance, Gram-negative bacilli

ABSTRACT

Understanding which antimicrobial agents are likely to be active against Gram-negative bacilli can guide selection of antimicrobials for empirical therapy as mechanistic rapid diagnostics are adopted. In this study, we determined the MICs of a novel β-lactam–β-lactamase inhibitor combination, imipenem-relebactam, along with ceftolozane-tazobactam, imipenem, ertapenem, meropenem, ceftriaxone, and cefepime, against 282 drug-resistant isolates of Gram-negative bacilli. For isolates harboring blaKPC (n = 110), the addition of relebactam to imipenem lowered the MIC50/MIC90 from 16/>128 μg/ml for imipenem alone to 0.25/1 μg/ml. For isolates harboring blaCTX-M (n = 48), the MIC50/MIC90 of ceftolozane-tazobactam were 0.5/16 μg/ml (83% susceptible). For isolates harboring blaCMY-2 (n = 17), the MIC50/MIC90 of ceftolozane-tazobactam were 4/8 μg/ml (47% susceptible). Imipenem-relebactam was active against most KPC-producing (but not NDM- or IMP-producing) Enterobacteriaceae and is an encouraging addition to the present antibiotic repertoire.

INTRODUCTION

Multidrug-resistant Gram-negative bacilli are an increasing health care concern. Over the past decade, there has been a proliferation of resistance mechanisms, accompanied by an increase in overall levels of resistance (1, 2). This poses a challenge for treatment of infectious diseases due to the limitations in the activity of some currently available antimicrobial agents (3–6). In addition, newer agents are typically not active against all multidrug-resistant Gram-negative bacilli; their activity is influenced by the resistance mechanism(s) present. Of particular challenge are the Enterobacteriaceae and Pseudomonas aeruginosa, many isolates of which have developed resistance to target antimicrobials through acquisition of β-lactamases, including carbapenemases, extended-spectrum β-lactamases (ESBLs), and plasmid-mediated AmpC production (7–12). Additionally, mutations leading to loss of outer membrane porins or upregulation of efflux pumps can confer β-lactam resistance (2, 4, 13).

As multidrug-resistant Gram-negative bacilli often harbor β-lactamases, β-lactamase inhibitors can be used to restore activity of β-lactam antibiotics; however, their ability to do so depends on the particular β-lactamase present, alongside the activity of the individual β-lactam (14, 15).

Ceftolozane-tazobactam is a cephalosporin–β-lactamase inhibitor combination that is FDA cleared/approved for treatment of complicated intra-abdominal infections and complicated urinary tract infections caused by Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, and P. aeruginosa, as well as for treatment of complicated intra-abdominal infections caused by Enterobacter cloacae and Klebsiella oxytoca (16, 17). Ceftolozane-tazobactam is not active against serine carbapenemases such as K. pneumoniae carbapenemase (KPC) or against metallo-β-lactamases (8).

Imipenem-relebactam is an investigational carbapenem–β-lactamase inhibitor combination; relebactam is a diazabicyclooctane non-β-lactam β-lactamase inhibitor, in the same category as avibactam. However, although relebactam and avibactam are similar in chemical structure, relebactam has an additional piperidine ring. Relebactam shows activity against Ambler classes A and C β-lactamases; when paired with imipenem against KPC-producing K. pneumoniae, MIC values have been reported to decrease 64-fold (3, 7).

With the emergence of multidrug-resistant Gram-negative bacilli and the multitude of mechanisms by which resistance is affected, predicting the activity of novel antimicrobial agents using emerging diagnostic tools capable of identifying resistance genes and mutations is becoming increasingly clinically important. In this study, 282 drug-resistant Gram-negative bacilli, characterized using molecular methods, were tested for susceptibility to imipenem-relebactam, with ceftolozane-tazobactam, imipenem, ertapenem, meropenem, ceftriaxone, and cefepime studied as comparators.

RESULTS

Cumulative MICs for the study isolates are shown in Tables 1 and 2.

TABLE 1.

Cumulative MIC results for CTX-M and CMY-2 gene-positive isolates for all antimicrobials testeda

Type of isolate and drug No. of isolates (cumulative %) inhibited at specified concn (μg/ml)
 MIC50 (μg/ml) MIC90 (μg/ml) % S % I % R
0.0018 0.0037 0.008 0.015 0.03 0.06 0.125 0.25 0.5 1 2 4 8 16 32 64 128 >128
CTX-M gene-positive isolates (n = 48)
    Imipenem (4 μg/ml relebactam) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 3 (6.3) 16 (39.6) 20 (81.3) 6 (93.8) 2 (97.9) 0 (100.0) 0 (100.0) 0 (100.0) 0 (100.0) 0 (100.0) 0 (100.0) 0 (100.0) 0 (100.0) 0 (100.0) 0.125 0.25 100.0b 0.0b 0.0b
    Imipenem 0 (0.0) 0 (0.0) 0 (0.0) 2 (4.2) 3 (10.4) 8 (27.1) 26 (81.3) 7 (95.8) 2 (100.0) 0 (100.0) 0 (100.0) 0 (100.0) 0 (100.0) 0 (100.0) 0 (100.0) 0 (100.0) 0 (100.0) 0 (100.0) 0.125 0.25 100.0 0.0 0.0
    Ertapenem 1 (2.1) 0 (0.0) 5 (12.5) 10 (33.3) 12 (58.3) 6 (70.8) 3 (77.1) 5 (87.5) 4 (95.8) 1 (97.9) 0 (100.0) 1 (100.0) 0 (100.0) 0 (100.0) 0 (100.0) 0 (100.0) 0 (100.0) 0 (100.0) 0.03 0.5 95.8 2.1 2.1
    Meropenem 0 (0.0) 2 (4.2) 4 (12.5) 25 (64.6) 11 (87.5) 5 (97.9) 0 (97.9) 1 (100.0) 0 (100.0) 0 (100.0) 0 (100.0) 0 (100.0) 0 (100.0) 0 (100.0) 0 (100.0) 0 (100.0) 0 (100.0) 0 (100.0) 0.015 0.06 100.0 0.0 0.0
    Ceftolozane (4 μg/ml tazobactam) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 3 (6.3) 17 (41.7) 9 (60.4) 8 (77.1) 3 (83.3) 0 (83.3) 2 (87.5) 3 (93.8) 1 (95.8) 0 (95.8) 0 (95.8) 2 (100.0) 0.5 16 83.3 0.0 16.7
    Cefepime 0 (0.0) 0 (0.0) 2 (4.2) 0 (4.2) 0 (4.2) 0 (4.2) 1 (6.3) 0 (6.3) 0 (6.3) 0 (6.3) 3 (12.5) 1 (14.6) 0 (14.6) 7 (29.2) 1 (31.3) 0 (31.3) 2 (35.4) 31 (100.0) >128 >128 12.5 NA 85.4
    Ceftriaxone 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 1 (2.1) 1 (4.2) 0 (4.2) 1 (6.3) 1 (8.3) 1 (10.4) 43 (100.0) >128 >128 0.0 0.0 100.0
CMY-2 gene-positive isolates (n = 17)
    Imipenem (4 μg/ml relebactam) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 2 (11.8) 0 (11.7) 6 (47.0) 5 (76.5) 2 (88.2) 0 (88.2) 0 (88.2) 2 (100.0) 0 (100.0) 0 (100.0) 0 (100.0) 0 (100.0) 0 (100.0) 0 (100.0) 0.25 0.5 88.2b 0.0b 11.8b
    Imipenem 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 3 (17.6) 6 (52.9) 3 (70.6) 3 (88.2) 0 (88.2) 2 (100.0) 0 (100.0) 0 (100.0) 0 (100.0) 0 (100.0) 0 (100.0) 0 (100.0) 0.25 1 88.2 0.0 11.8
    Ertapenem 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 5 (29.4) 1 (35.3) 2 (47.0) 3 (64.7) 3 (82.4) 1 (88.2) 0 (88.2) 2 (100.0) 0 (100.0) 0 (100.0) 0 (100.0) 0 (100.0) 0 (100.0) 0 (100.0) 0.25 1 82.4 5.9 11.8
    Meropenem 0 (0.0) 0 (0.0) 0 (0.0) 9 (52.9) 5 (82.3) 1 (88.2) 2 (100.0) 0 (100.0) 0 (100.0) 0 (100.0) 0 (100.0) 0 (100.0) 0 (100.0) 0 (100.0) 0 (100.0) 0 (100.0) 0 (100.0) 0 (100.0) 0.015 0.06 100.0 0.0 0.0
    Ceftolozane (4 μg/ml tazobactam) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 1 (5.9) 3 (23.5) 2 (35.3) 2 (47.0) 4 (70.6) 3 (88.2) 2 (100.0) 0 (100.0) 0 (100.0) 0 (100.0) 0 (100.0) 4 8 47.0 23.5 29.4
    Cefepime 0 (0.0) 0 (0.0) 1 (5.9) 2 (17.6) 0 (17.6) 0 (17.6) 2 (29.4) 5 (58.8) 2 (70.6) 1 (76.5) 2 (88.2) 0 (88.2) 0 (88.2) 0 (88.2) 1 (94.1) 0 (94.1) 0 (94.1) 1 (100.0) 0.25 2 88.2 NA 11.8
    Ceftriaxone 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 2 (11.8) 0 (118) 0 (11.8) 0 (11.8) 0 (11.8) 0 (11.8) 0 (11.8) 1 (17.6) 4 (41.2) 1 (47.0) 4 (70.6) 4 (94.1) 1 (100.0) 64 128 11.8 0.0 88.2
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a

S, susceptible; I, intermediate; R, resistant; NA, not applicable.

b

Using imipenem breakpoints.

TABLE 2.

Cumulative MIC results for NDM, IMP, and KPC gene-positive isolates for all antimicrobial agents testeda

Type of isolate and drug No. of isolates (cumulative %) inhibited at specified concn (μg/ml)
 MIC50 (μg/ml) MIC90 (μg/ml) % S % I % R
0.0018 0.0037 0.008 0.015 0.03 0.06 0.125 0.25 0.5 1 2 4 8 16 32 64 128 >128
NDM gene-positive isolates (n = 31)
    Imipenem (4 μg/ml relebactam) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 1 (3.2) 4 (16.1) 10 (48.4) 9 (77.4) 6 (96.7) 1 (100.0) 32 128 0b 0b 100b
    Imipenem 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 1 (3.2) 4 (16.1) 12 (54.8) 7 (77.4) 5 (93.5) 2 (100.0) 32 128 0 0 100
    Ertapenem 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 1 (3.2) 3 (12.9) 9 (41.9) 10 (74.2) 8 (100.0) 128 >128 0 0 100
    Meropenem 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 4 (12.9) 4 (25.8) 9 (54.8) 10 (87.1) 4 (100.0) 64 >128 0 0 100
    Ceftolozane (4 μg/ml tazobactam) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 31 (100.0) >128 >128 0 0 100
    Cefepime 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 2 (6.4) 2 (12.9) 27 (100.0) >128 >128 0 NA 100
    Ceftriaxone 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 31 (100.0) >128 >128 0 0 100
IMP gene-positive isolates (n = 11)
    Imipenem (4 μg/ml relebactam) 0 (0.0) 1 (9.1) 0 (9.1) 0 (9.1) 0 (9.1) 1 (18.2) 1 (27.3) 3 (54.5) 1 (63.6) 1 (72.7) 0 (72.7) 0 (72.7) 2 (90.9) 1 (100.0) 4 128 18.2b 9.1b 72.7b
    Imipenem 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 1 (9.1) 1 (18.2) 1 (27.3) 2 (45.5) 2 (63.6) 1 (72.7) 0 (72.7) 0 (72.7) 2 (90.9) 1 (100.0) 8 128 18.2 9.0 72.8
    Ertapenem 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 2 (18.2) 1 (27.3) 0 (27.3) 4 (63.6) 0 (63.6) 1 (72.7) 1 (81.8) 2 (100.0) 16 >128 0 0 100
    Meropenem 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 1 (9.1) 2 (27.3) 0 (27.3) 0 (27.3) 3 (54.5) 1 (63.6) 1 (72.7) 1 (81.8) 2 (100.0) 0 (100.0) 8 128 27.3 0 72.7
    Ceftolozane (4 μg/ml tazobactam) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 1 (9.1) 0 (9.1) 0 (9.1) 1 (18.2) 9 (100.0) >128 >128 0 0 100
    Cefepime 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 1 (9.1) 2 (27.3) 1 (36.4) 1 (45.5) 0 (45.5) 6 (100.0) >128 >128 0 NA 91.9
    Ceftriaxone 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 1 (9.1) 2 (27.3) 8 (100.0) >128 >128 0 0 100
KPC gene-positive isolates (n = 110)
    Imipenem (4 μg/ml relebactam) 0 (0.0) 0 (0.0) 0 (0.0) 1 (1.0) 5 (5.4) 15 (18.9) 24 (40.5) 20 (58.6) 22 (78.4) 14 (90.9) 4 (94.6) 1 (95.5) 1 (96.4) 2 (98.2) 1 (99.1) 0 (99.1) 0 (99.1) 1 (100.0) 0.25 1 90.9b 3.6b 5.4b
    Imipenem 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 2 (1.8) 1 (2.7) 2 (4.5) 3 (7.2) 16 (21.6) 15 (35.1) 20 (53.2) 20 (71.2) 11 (81.1) 7 (87.4) 14 (100.0) 16 >128 4.5 2.7 92.8
    Ertapenem 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 1 (1.0) 0 (1.0) 0 (1.0) 0 (1.0) 2 (2.7) 1 (3.6) 4 (7.2) 9 (15.3) 13 (27.0) 16 (41.4) 20 (59.5) 22 (79.3) 23 (100.0) 64 >128 1.8 1.0 97.2
    Meropenem 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 1 (1.0) 1 (1.8) 0 (1.8) 0 (1.8) 1 (2.7) 3 (5.4) 3 (8.1) 11 (18.0) 13 (29.7) 13 (41.4) 23 (62.2) 9 (70.3) 3 (73.0) 30 (100.0) 32 >128 5.4 2.7 91.9
    Ceftolozane (4 μg/ml tazobactam) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 1 (1.0) 2 (2.7) 1 (3.6) 1 (4.5) 1 (5.4) 6 (10.8) 13 (22.5) 21 (41.4) 37 (74.8) 28 (100.0) 128 >128 3.6 1 95.4
    Cefepime 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 1 (1.0) 0 (1.0) 1 (1.8) 1 (2.7) 4 (6.3) 7 (12.6) 9 (20.7) 6 (26.1) 9 (34.2) 10 (43.2) 63 (100.0) >128 >128 2.7 NA 97.3
    Ceftriaxone 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 1 (1.0) 1 (1.8) 0 (1.8) 1 (2.7) 1 (3.6) 0 (3.6) 6 (9.0) 8 (16.2) 93 (100.0) >128 >128 1.8 1.0 97.2
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a

S, susceptible; I, intermediate; R, resistant; NA, not applicable.

b

Using imipenem breakpoints.

Carbapenemase gene-negative isolates.

There were 48 isolates harboring genes for cefotaxime-hydrolyzing β-lactamase (CTX-M) and 17 harboring genes for cephamycin-hydrolyzing β-lactamase (CMY-2) (see Table S1 in the supplemental material).

The MIC50/MIC90 of the CTX-M-positive isolates were 0.125/0.25 μg/ml for both imipenem-relebactam and imipenem. All CTX-M-positive isolates were susceptible to imipenem (as well as to imipenem-relebactam if using the imipenem breakpoint). Eighty-three percent of these isolates were susceptible to ceftolozane-tazobactam, while 12 and 0% were susceptible to cefepime and ceftriaxone, respectively. The MIC50/MIC90 for the same isolates were 0.5/16, >128/>128, and >128/>128 μg/ml for ceftolozane-tazobactam, cefepime, and ceftriaxone, respectively (Table 1).

Eighty-eight percent of CMY-2-positive isolates were susceptible to imipenem (as well as to imipenem-relebactam if using the imipenem breakpoint). The MIC50/MIC90 for the CMY-2-positive isolates were 0.25/0.5 and 0.25/1 μg/ml for imipenem-relebactam and imipenem, respectively. Forty-seven percent of CMY-2-positive isolates were susceptible to ceftolozane-tazobactam, 88% were susceptible to cefepime, and 12% were susceptible to ceftriaxone. The MIC50/MIC90 for the CMY-2-positive isolates were 4/8, 0.25/2, and 64/128 μg/ml for ceftolozane-tazobactam, cefepime, and ceftriaxone, respectively (Table 1).

For the other resistance mechanisms tested, there were fewer than 10 isolates per group, so MIC50/MIC90 values were not calculated. Nonetheless, all isolates harboring cefoxitin-hydrolyzing β-lactamase (FOX-5), SHV, TEM, TEM plus SHV (11 of which were ESBL producers as determined by clavulanate disc augmentation), or other combined mechanisms of resistance were susceptible to imipenem, meropenem, and ertapenem, and the addition of relebactam to imipenem did not change their MIC values compared to those for imipenem alone (Table S1). For ceftolozane-tazobactam, there were some differences noted compared to the other cephalosporins. The FOX-5 isolate was susceptible to ceftolozane-tazobactam and cefepime and intermediate to ceftriaxone. The SHV-positive isolates were all resistant to ceftriaxone, 3 of 6 were resistant to cefepime, and 4 of 6 were resistant to ceftolozane-tazobactam. For the TEM-positive isolates, 7/8 and 4/8 were resistant to ceftriaxone and cefepime, respectively, while 4, 2, and 2 were susceptible, intermediate, and resistant, respectively, to ceftolozane-tazobactam. Thirty-three percent of dually TEM- and SHV-positive isolates were susceptible to ceftolozane-tazobactam, while 25 and 0% were susceptible to cefepime and ceftriaxone, respectively. For the 19 isolates with other combined mechanisms of resistance, 17, 12, and 9 were resistant to ceftriaxone, cefepime, and ceftolozane-tazobactam, respectively.

Carbapenemase gene-positive isolates.

The carbapenemase gene-positive isolates included 31 positive for New Delhi metallo-β-lactamase (NDM), 11 positive for imipenem metallo-β-lactamase (IMP), and 110 positive for KPC (Table S2). All NDM-positive isolates were resistant to imipenem, and their MIC50/MIC90 were 32/128 μg/ml for both imipenem and imipenem-relebactam; if using the imipenem breakpoint, all isolates would be considered resistant to imipenem-relebactam. All NDM-positive isolates were also resistant to ceftolozane-tazobactam, cefepime, and ceftriaxone, with MIC50 and MIC90 values all being >128 μg/ml (Table 2). Eighteen percent of IMP-positive isolates were susceptible to imipenem (as well as to imipenem-relebactam if using the imipenem breakpoint). The MIC50/MIC90 for the IMP isolates were 4/128 and 8/128 μg/ml for imipenem-relebactam and imipenem, respectively. All IMP-positive isolates were resistant to ceftolozane-tazobactam, with MIC50 and MIC90 values of >128 μg/ml; they exhibited 8 and 0% susceptibility to cefepime and ceftriaxone, respectively (Table 2). Five percent of the KPC-positive isolates were susceptible to imipenem; 91% would be considered susceptible to imipenem-relebactam if using the imipenem breakpoints. The MIC50/MIC90 for the KPC-positive isolates were 0.25/1 and 16/>128 μg/ml for imipenem-relebactam and imipenem, respectively. Three percent and 2% of the KPC-positive isolates were susceptible to cefepime and ceftriaxone, respectively, and 4% were susceptible to ceftolozane-tazobactam. The MIC50/MIC90 for ceftolozane-tazobactam for the KPC-positive isolates were 128/>128 μg/ml (Table 2).

All imipenem-hydrolyzing β-lactamase (IMI)-positive isolates were resistant to imipenem and susceptible to ceftolozane-tazobactam, cefepime, and ceftriaxone; addition of relebactam to imipenem lowered the MIC values up to 6 doubling dilutions compared to those for imipenem alone (Table S2). For oxacillin-hydrolyzing β-lactamase (OXA)-positive isolates (including OXA-48, -181, and -232), addition of relebactam to imipenem did not change MIC values compared to those for imipenem alone; isolates which were resistant to ceftriaxone and cefepime were also resistant to ceftolozane-tazobactam. One isolate was susceptible to ceftriaxone but resistant to ceftolozane-tazobactam and cefepime. For the OXA-48-positive isolates, 4 of 8 had imipenem-relebactam MICs of ≤1 μg/ml. In the case of two Serratia marcescens enzyme (SME)-positive isolates, the addition of relebactam to imipenem dropped the MIC from 128 μg/ml for imipenem alone to 0.5 μg/ml for one and from >128 μg/ml to 16 for the other; it did not change for the MIC of the third SME-positive isolate. Finally, for the Verona integrin-encoded metallo-β-lactamase (VIM)-positive isolates, all of which were resistant to imipenem, ceftolozane-tazobactam, cefepime, and ceftriaxone, the addition of relebactam to imipenem did not change the MICs compared to those for imipenem alone (Table S2).

DISCUSSION

For the isolates which had tested positive for CTX-M or CMY-2, the addition of relebactam to imipenem did not change the MIC50/MIC90; 100 and 88% of these isolates, respectively, were susceptible to imipenem alone, as expected (1). The addition of tazobactam to ceftolozane inhibited most CTX-M-positive isolates, rendering 83% susceptible. These results are not unexpected given that tazobactam inhibits most class A β-lactamases, including common ESBL enzymes, such as CTX-M (3, 11). There are CTX-M variants that have amino acid substitutions which improve recognition of ceftazidime (namely, D240G and P167S/T) but may result in attenuated bioactivity of cefotaxime and/or cefepime (18); this could explain the high rate of resistance to cefepime noted.

For isolates harboring CMY-2, the addition of tazobactam to ceftolozane appears to slightly increase the susceptibility compared to that for ceftriaxone, although fewer than half were susceptible to ceftolozane-tazobactam. This is not surprising given that tazobactam is only a moderate inhibitor of class C β-lactamases and has strain-dependent activity (19). For isolates harboring CMY-2, first- through fourth-generation cephalosporins are expected to be hydrolyzed. However, cefepime may be active (1), which is consistent with our findings.

The addition of relebactam to imipenem lowered the MIC50/MIC90 from 16/>128 μg/ml to 0.25/1 μg/ml for the KPC-positive isolates, 93% of which were resistant to imipenem alone. Currently, there are no established breakpoints for imipenem-relebactam, but using the breakpoint for imipenem alone, relebactam restored the activity of imipenem in isolates testing positive for KPC. As expected, this increased susceptibility to imipenem-relebactam compared to imipenem alone was not noted for the NDM-positive or IMP-positive isolates; relebactam overcomes class A and C (20) and not class B β-lactamases, which include NDM and IMP (1). Among the NDM-, IMP-, and KPC-harboring isolates, more than 90% were resistant to the cephalosporins tested (ceftolozane-tazobactam, cefepime, and ceftriaxone), which is expected given that class A carbapenemases and class C β-lactamases hydrolyze first- through fourth-generation cephalosporins (1, 8, 21). Our findings agree with a prior study showing that ceftolozane-tazobactam is inactive against Enterobacteriaceae harboring class A serine carbapenemases (e.g., KPC) and class B metallo-β-lactamases (e.g., NDM, IMP, and VIM) (8). Interestingly, 4 of the 8 OXA-48-positive isolates had imipenem-relebactam MICs of ≤1 μg/ml. This has not been well described in the literature thus far, but one study by Livermore et al. tested imipenem-relebactam against 5 K. pneumoniae OXA-48-positive isolates and found that 3 out of 5 isolates had MICs of ≤1 μg/ml (20).

One limitation to our study is that although the study isolates were fairly well characterized, many of the resistance mechanisms were present in only a limited number of isolates. We can deduce trends from only some groups of isolates and are not able to provide mechanism-specific MIC50/MIC90 data for all resistance types. A second limitation is that resistance in Gram-negative bacilli is complex and we evaluated the impact of only selected β-lactamases. For example, the KPC- and NDM-positive isolates in this study were not further characterized, and there could be other β-lactamases present. We did not evaluate other potentially concomitant mechanisms of resistance, such as outer membrane permeability and efflux, which, when present in combination with β-lactamases, can affect in vitro activity. Our identification of isolates with AmpCs that were nonsusceptible to ceftolozane-tazobactam suggests the presence of other resistance mechanisms (e.g., porin mutations). K. pneumoniae IDRL-10648, reported only as having a non-ESBL SHV-1, must have additional resistance mechanisms because the MICs of ceftolozane-tazobactam, ceftriaxone, and cefepime were very high. Another limitation is that at the time of this writing, there are no established breakpoints for imipenem-relebactam.

Overall, results of this study show that imipenem-relebactam is active against most KPC-positive (but not NDM- or IMP-positive) Enterobacteriaceae. Imipenem-relebactam is a promising addition to the existing antibiotic armamentarium; however, clinicians will need to be aware of geographical epidemiology and the genetic makeup of strains.

MATERIALS AND METHODS

Bacterial strains.

A total of 282 previously characterized isolates of Gram-negative bacilli collected from across the United States, Canada, and Singapore were studied. The isolates exhibited one or more of the following resistance mechanisms previously or during this study genotypically characterized by PCR with or without sequencing (9, 12, 22–31): TEM, SHV, CMY, IMP, NDM, KPC, OXA, VIM, CTX-M, SME, FOX, and/or IMI. One hundred five isolates were carbapenemase gene negative and included E. coli (74), K. pneumoniae (28), Morganella morganii (2), and Enterobacter cloacae complex (1) (Table S3). A total of 177 harbored carbapenemase genes, including 4 Citrobacter freundii, 3 Citrobacter koseri, 1 Citrobacter sedlakii, 3 Enterobacter aerogenes, 20 E. cloacae, 15 E. coli, 119 K. pneumoniae, 4 P. aeruginosa, 1 P. mirabilis, 2 Providencia stuartii, and 5 Serratia marcescens isolates (Table S4). Six quality control strains were tested with each trial: Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, E. coli ATCC 25922, P. aeruginosa ATCC 27853, E. coli ATCC 35218, and K. pneumoniae ATCC 700603. All isolates were stored in MicroBank vials (Pro-Lab Diagnostics, Round Rock, TX) at −80°C.

Antimicrobial agent preparation.

Seven antibiotics, including cefepime, ceftriaxone, and meropenem (provided by USP, Rockville, MD) and ertapenem, imipenem, imipenem-relebactam, and ceftolozane-tazobactam (provided by Merck & Co., Inc.) were studied. Stock solutions of each antibiotic were prepared according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (32, 33). Prepared stock solutions were aliquoted into 1.5-ml microcentrifuge tubes and stored at −80°C.

Broth microdilution susceptibility testing.

Bacteria were grown for 18 to 20 h on BBL Trypticase soy agar with 5% sheep blood (Becton Dickinson, Franklin Lakes, NJ) at 37°C and then subcultured for study of F2 generation colonies. MICs were determined by broth microdilution, using the same inoculum for all antimicrobials, following CLSI guidelines (33). Antimicrobial concentrations tested ranged from 0.0018 to 128 μg/ml, in 2-fold serial dilutions, using a constant concentration of 4 μg/ml of relebactam and tazobactam when combined with imipenem or ceftolozane, respectively. Plates were incubated for 16 to 20 h at 37°C, and the MIC was recorded as the first well with no growth. The MIC50/MIC90, cumulative MICs, and percent of isolates that were susceptible, intermediate, and resistant for each antimicrobial agent were interpreted using CLSI breakpoints (33).

Supplementary Material

Supplemental file 1
zac008187305s1.docx (87.9KB, docx)

ACKNOWLEDGMENTS

Research reported in this publication was supported by Merck & Co.

We acknowledge Kerryl E. Greenwood-Quaintance, M.S., Melissa J. Karau, Audrey N. Schuetz, Peggy C. Kohner, Nicolynn C. Cole, and Scott A. Cunningham for technical advice. We thank Mayo Clinic Clinical Bacteriology Lab, Donna J. Hata from Mayo Clinic in Jacksonville, FL, James R. Johnson from the VA Medical Center in Minneapolis, MN, Hennepin County Medical Center in Minneapolis, MN, Mary K. Hayden and Karen Lolans from Rush University Medical Center, and Paul C. Schreckenberger from Loyola, both in Chicago, IL, Patricia J. Simner with the Canadian Antimicrobial Resistance Alliance and the CANWARD study, George G. Zhanel and Daryl J. Hoban from the University of Manitoba, Partha Pratim De, Sanjay Ryan Menon, and Shawn Vasoo from Tan Tock Seng Hospital, and Koh Tse Hsien from Singapore General Hospital in Singapore for isolates included in this study.

Footnotes

Supplemental material for this article may be found at https://doi.org/10.1128/AAC.00533-18.

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Supplementary Materials

Supplemental file 1
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