Carbapenem-resistant Enterobacteriaceae (CRE) strains are an urgent public health threat. We evaluated the in vitro activities of 19 antimicrobial agents, including imipenem-relebactam, against (i) 106 CRE bloodstream isolates that primarily expressed Klebsiella pneumoniae carbapenemase (KPC) and (ii) 20 OXA-48-like-expressing CRE isolates. Ninety-five percent of CRE bloodstream isolates were susceptible to imipenem-relebactam.
KEYWORDS: carbapenem-resistant Enterobacteriaceae, ceftazidime-avibactam, imipenem-relebactam
ABSTRACT
Carbapenem-resistant Enterobacteriaceae (CRE) strains are an urgent public health threat. We evaluated the in vitro activities of 19 antimicrobial agents, including imipenem-relebactam, against (i) 106 CRE bloodstream isolates that primarily expressed Klebsiella pneumoniae carbapenemase (KPC) and (ii) 20 OXA-48-like-expressing CRE isolates. Ninety-five percent of CRE bloodstream isolates were susceptible to imipenem-relebactam. In contrast to their comparable activities against KPC-producing CRE strains, ceftazidime-avibactam was more active in vitro against OXA-48-like CRE strains than was imipenem-relebactam (90% susceptible versus 15% susceptible).
TEXT
Carbapenem-resistant Enterobacteriaceae (CRE) strains are an urgent public health threat (1). The emergence of CRE is due to the rapid expansion of plasmid-encoded, carbapenem-hydrolyzing enzymes (carbapenemases). Klebsiella pneumoniae carbapenemase (KPC) is the most common carbapenem resistance mechanism among Enterobacteriaceae strains in the United States, South America, Israel, Greece, and Italy. Other carbapenemase enzymes predominate in other countries, including OXA-48-type enzymes that are prevalent in Spain, France, Turkey, North Africa, and the Middle East (2–4). Relebactam and avibactam are novel diazabicyclooctanes that effectively inhibit KPC enzymes and inhibit some OXA-48-type carbapenemases (5, 6). Relebactam has been combined with imipenem and avibactam has been combined with ceftazidime to represent novel β-lactam/β-lactamase inhibitor combinations, which have great potential for the treatment of infections due to many CRE strains.
In this study, we compare the in vitro activity of imipenem-relebactam, ceftazidime-avibactam, and other potentially useful antimicrobial agents against a collection of 106 contemporary CRE bloodstream isolates and 20 OXA-48-like isolates that have been genetically characterized. Antimicrobial susceptibility testing was performed by reference broth microdilution to assess the activities of 19 antimicrobial agents, including penicillins (piperacillin-tazobactam and amoxicillin-clavulanate), cephalosporins (ceftazidime, ceftazidime-avibactam, and cefepime), a monobactam (aztreonam), carbapenems (ertapenem, imipenem, imipenem-relebactam, meropenem, and doripenem), a fluoroquinolone (ciprofloxacin), aminoglycosides (gentamicin and amikacin), a tetracycline (minocycline), a glycylcycline (tigecycline), a folate synthesis inhibitor (trimethoprim-sulfamethoxazole), and polymyxins (colistin and polymyxin B) (7). Isolate MICs were interpreted as susceptible, intermediate, or resistant for each of the antimicrobial agents based on Clinical and Laboratory Standards Institute (CLSI) breakpoints, where applicable (8). Isolates for which MIC values were ≤2 μg/ml for tigecycline (the FDA susceptible breakpoint when testing Enterobacteriaceae), colistin, and polymyxin B were considered susceptible to those agents. The CLSI interpretive criteria for imipenem were applied to imipenem-relebactam. The percentages of isolates that tested susceptible, intermediate, or resistant, as well as the MIC50 and MIC90, were calculated for each of the antimicrobial agents.
The 106 CRE bloodstream isolates represented six unique species, namely, Klebsiella pneumoniae (n = 97), Enterobacter cloacae (n = 3), Escherichia coli (n = 3), Morganella morganii (n = 1), Pluralibacter (formerly Enterobacter) gergoviae (n = 1), and Klebsiella (formerly Enterobacter) aerogenes (n = 1) (see Table S1 in the supplemental material) (9). Carbapenemases were identified in 93.4% of the isolates (99/106 isolates). The 7 isolates without carbapenemases included K. pneumoniae (n = 4), P. gergoviae (n = 1), and E. coli (n = 2) (Table S2). The number of β-lactamases present per isolate was 3 ± 0.5 β-lactamases (median ± standard deviation).
Of the 99 carbapenemase-producing CRE isolates, KPC-2 and KPC-3 were present in 47 and 51 isolates, respectively. The remaining carbapenemase-producing isolate was an OXA-48-producing K. pneumoniae strain. No isolates with multiple carbapenemases were identified. Ninety-two of the carbapenem-resistant K. pneumoniae isolates expressed KPC, of which 84% (77/92 isolates) were sequence type 258 (ST258). In addition, 37% of isolates (34/92 isolates) harbored an extended-spectrum β-lactamase (ESBL). The most common wzi (a gene that encodes an outer membrane protein that correlates with capsular type [10]) alleles were wzi154 (43/92 isolates), wzi29 (19/92 isolates), wzi50 (8/92 isolates), and wzi83 (7/92 isolates). Forty-five KPC-expressing K. pneumoniae isolates (49% [45/92 isolates]) had mutations in the gene that encodes OmpK35, an outer membrane porin that allows for passage of carbapenems, and 31 (34% [31/92 isolates]) had cooccurrences of ompK35 and ompK36 mutations. Of note, no isolate had an ompK36 mutation without an ompK35 mutation.
The OXA-48-like CRE isolate collection was composed of five unique species (Table S3), namely, K. pneumoniae (n = 14), Klebsiella (formerly Enterobacter) aerogenes (n = 2), Escherichia coli (n = 2), Proteus mirabilis (n = 1), and Klebsiella ozaenae (n = 1). Eighty percent of those isolates (16/20 isolates) coharbored ESBLs. OXA-48 (n = 8) and OXA-181 (n = 8) enzymes were the most common, followed by OXA-232 (n = 3) and OXA-163 (n = 1). Porin mutations were present in 86% of K. pneumoniae isolates (12/14 isolates), including ompK35 mutations alone (n = 9), ompK36 mutations alone (n = 2), and cooccurrence of ompK35 and ompK36 mutations (n = 1).
Of the 19 antimicrobial agents evaluated for the CRE bloodstream isolates, only 8/19 agents (imipenem-relebactam, ceftazidime-avibactam, amikacin, gentamicin, minocycline, tigecycline, colistin, and polymyxin B) from 5/9 antimicrobial classes demonstrated in vitro activity against >50% of the strains tested, and only 2 (ceftazidime-avibactam and imipenem-relebactam) exhibited activity against >90% (Table S4). The imipenem-relebactam MIC50 and MIC90 were ≤0.25/4 μg/ml and 0.5/4 μg/ml, respectively. Notably, all isolates for which the MIC exceeded the mode for imipenem-relebactam harbored genes for multiple mechanisms of β-lactam resistance.
Of the 19 antimicrobial agents evaluated for activity against the OXA-48-like CRE isolates, only 4/19 agents (ceftazidime-avibactam, tigecycline, colistin, and polymyxin B) from 3/9 antimicrobial classes were active against >50% of the isolates, and only 3 (ceftazidime-avibactam, colistin, and polymyxin B) were active against >90% (Table S5). The addition of avibactam restored in vitro susceptibility to ceftazidime in 14/16 OXA-48-like strains that were resistant to ceftazidime alone. In contrast, the addition of relebactam to imipenem did not restore in vitro susceptibility to imipenem in any of the 17 strains that were resistant to imipenem alone. Of note, the MIC50 and MIC90 of ceftazidime-avibactam were unchanged in the OXA-48-like CRE isolates, compared to the CRE bloodstream isolates.
All 92 KPC-expressing K. pneumoniae bloodstream isolates were susceptible to imipenem-relebactam, whereas 90 (98%) KPC-expressing K. pneumoniae isolates were susceptible to ceftazidime-avibactam. Of note, the 2 K. pneumoniae isolates that were resistant to ceftazidime-avibactam were both KPC-3 expressing, with coexpression of 2 additional β-lactamases (wild-type TEM and wild-type SHV). In addition, the ceftazidime-avibactam-resistant isolates included 1 isolate with an ompK35 mutation alone and another with both ompK35 and ompK36 mutations.
The in vitro activities of imipenem-relebactam and ceftazidime-avibactam against KPC-expressing K. pneumoniae isolates were further stratified by KPC allele, ESBL status, ST258 status, wzi allele, and mutation status for ompK35 and ompK36 outer membrane porin genes (Tables 1 and 2; also see Tables S6 to S9). There was no difference in imipenem-relebactam MIC values between KPC-2-expressing K. pneumoniae isolates and KPC-3-expressing K. pneumoniae isolates (MIC90 of 0.5/4 μg/ml for both; P = 0.58). Coharboring an ESBL gene did not increase imipenem-relebactam MICs. Additionally, the in vitro activity of imipenem-relebactam was not affected by wzi allele status or ST. However, outer membrane porin mutations had an impact on imipenem-relebactam MIC values (Table 1; also see Table S8), with strains harboring mutations in ompK35 and ompK36 having increased MIC values, compared to wild-type isolates (MIC90 values of 1/4 μg/ml versus ≤0.25/4 μg/ml; P = 0.023). No statistically significant increases in MIC values were observed for isolates with ompK35 mutations only (P = 0.14). Unlike findings for ceftazidime-avibactam (Table S6), the KPC allelic variant had minimal impact on the effect of outer membrane porin mutations on imipenem-relebactam MIC values.
TABLE 1.
Imipenem-relebactam MIC values for KPC-expressing K. pneumoniae bloodstream isolates stratified by ESBL status, ST258 status, wzi allele, and mutations in ompK35 and ompK36 outer membrane porin genesa
| Isolate type | MIC50 (μg/ml) | MIC90 (μg/ml) | MIC range (μg/ml) |
|---|---|---|---|
| KPC-expressing K. pneumoniae (n = 92) | ≤0.25/4 | 0.5/4 | ≤0.25/4 to 1/4 |
| ESBL (n = 34) | ≤0.25/4 | 0.5/4 | ≤0.25/4 to 1/4 |
| ST258 (n = 77) | ≤0.25/4 | 0.5/4 | ≤0.25/4 to 1/4 |
| Non-ST258 (n = 15) | ≤0.25/4 | ≤0.25/4 | ≤0.25/4 to ≤0.25/4 |
| wzi29 (n = 19) | ≤0.25/4 | 0.5/4 | ≤0.25/4 to 1/4 |
| wzi50 (n = 8) | ≤0.25/4 | 0.5/4 | ≤0.25/4 to 0.5/4 |
| wzi83 (n = 7) | ≤0.25/4 | 1/4 | ≤0.25/4 to 1/4 |
| wzi154 (n = 43) | ≤0.25/4 | 0.5/4 | ≤0.25/4 to 1/4 |
| Wild-type ompK35 and ompK36 (n = 15) | ≤0.25/4 | ≤0.25/4 | ≤0.25/4 to ≤0.25/4 |
| ompK35 mutation only (n = 45) | ≤0.25/4 | 0.5/4 | ≤0.25/4 to 1/4 |
| ompK35 and ompK36 mutations (n = 31) | ≤0.25/4 | 1/4 | ≤0.25/4 to 1/4 |
| KPC-2-expressing K. pneumoniae (n = 44) | ≤0.25/4 | 0.5/4 | ≤0.25/4 to 1/4 |
| ESBL (n = 19) | ≤0.25/4 | 0.5/4 | ≤0.25/4 to 1/4 |
| ST258 (n = 35) | ≤0.25/4 | 0.5/4 | ≤0.25/4 to 1/4 |
| Non-ST258 (n = 9) | ≤0.25/4 | ≤0.25/4 | ≤0.25/4 to ≤0.25/4 |
| wzi29 (n = 18) | ≤0.25/4 | 0.5/4 | ≤0.25/4 to 1/4 |
| wzi50 (n = 7) | ≤0.25/4 | 0.5/4 | ≤0.25/4 to 0.5/4 |
| wzi83 (n = 6) | ≤0.25/4 | 1/4 | ≤0.25/4 to 1/4 |
| wzi154 (n = 4) | ≤0.25/4 | 1/4 | ≤0.25/4 to 1/4 |
| Wild-type ompK35 and ompK36 (n = 9) | ≤0.25/4 | ≤0.25/4 | ≤0.25/4 to ≤0.25/4 |
| ompK35 mutation only (n = 25) | ≤0.25/4 | 0.5/4 | ≤0.25/4 to 1/4 |
| ompK35 and ompK36 mutations (n = 10) | ≤0.25/4 | 1/4 | ≤0.25/4 to 1/4 |
| KPC-3-expressing K. pneumoniae (n = 48) | ≤0.25/4 | 0.5/4 | ≤0.25/4 to 1/4 |
| ESBL (n = 15) | ≤0.25/4 | 0.5/4 | ≤0.25/4 to 0.5/4 |
| ST258 (n = 42) | ≤0.25/4 | 0.5/4 | ≤0.25/4 to 1/4 |
| Non-ST258 (n = 6) | ≤0.25/4 | 4/4 | ≤0.25/4 to ≤0.25/4 |
| wzi29 (n = 1) | 0.5/4 | 0.5/4 | 0.5/4 to 0.5/4 |
| wzi50 (n = 1) | ≤0.25/4 | ≤0.25/4 | ≤0.25/4 to ≤0.25/4 |
| wzi83 (n = 1) | 0.5/4 | 0.5/4 | 0.5/4 to 0.5/4 |
| wzi154 (n = 39) | ≤0.25/4 | 0.5/4 | ≤0.25/4 to 1/4 |
| Wild-type ompK35 and ompK36 (n = 6) | ≤0.25/4 | ≤0.25/4 | ≤0.25/4 to ≤0.25/4 |
| ompK35 mutation only (n = 20) | ≤0.25/4 | 0.5/4 | ≤0.25/4 to 0.5/4 |
| ompK35 and ompK36 mutations (n = 21) | ≤0.25/4 | 0.5/4 | ≤0.25/4 to 1/4 |
The CLSI interpretive criteria for imipenem were applied to imipenem-relebactam.
TABLE 2.
Imipenem and imipenem-relebactam MIC values for OXA-48-like isolates stratified by ESBL status, OXA-like enzyme, and mutations in ompK35 and ompK36 outer membrane porin genesa
| Drug and isolate type | MIC50 (μg/ml) | MIC90 (μg/ml) | MIC range (μg/ml) |
|---|---|---|---|
| Imipenem | |||
| OXA-48-like (n = 20) | 8 | ≥32 | 8 to ≥32 |
| ESBL (n = 16) | 8 | ≥32 | 0.5/4 to ≥32 |
| OXA-48 (n = 8) | 16 | ≥32 | ≤0.25/4 to ≥32 |
| OXA-163 (n = 1) | ≤0.25 | ≤0.25 | ≤0.25 to ≤0.25 |
| OXA-181 (n = 8) | 4 | ≥32 | 4 to ≥32 |
| OXA-232 (n = 3) | ≥32 | ≥32 | 16 to ≥32 |
| OXA-48-like-expressing K. pneumoniae (n = 14) | 16 | ≥32 | ≤0.25 to ≥32 |
| Wild-type ompK35 and ompK36 (n = 2) | 16 | ≥32 | 16 to ≥32 |
| ompK35 mutation only (n = 9) | 8 | ≥32 | ≤0.25 to ≥32 |
| ompK36 mutation only (n = 2) | 4 | ≥32 | 4 to ≥32 |
| ompK35 and ompK36 mutations (n = 1) | 16 | 16 | 16 to 16 |
| Imipenem-relebactam | |||
| OXA-48-like (n = 20) | 4/4 | ≥32/4 | ≤0.25/4 to ≥32/4 |
| ESBL (n = 16) | 4/4 | ≥32/4 | 0.5/4 to ≥32/4 |
| OXA-48 (n = 8) | 4/4 | ≥32/4 | ≤0.25/4 to ≥32/4 |
| OXA-163 (n = 1) | 0.5/4 | 0.5/4 | 0.5/4 to 0.5/4 |
| OXA-181 (n = 8) | 4/4 | ≥32/4 | 2/4 to ≥32/4 |
| OXA-232 (n = 3) | 8/4 | 16/4 | 8/4 to 16/4 |
| OXA-48-like-expressing K. pneumoniae (n = 14) | 8/4 | ≥32/4 | 0.5/4 to ≥32/4 |
| Wild-type ompK35 and ompK36 (n = 2) | 8/4 | ≥32/4 | ≤0.25/4 to ≥32/4 |
| ompK35 mutation only (n = 9) | 4/4 | ≥32/4 | 0.5/4 to ≥32/4 |
| ompK36 mutation only (n = 2) | 4/4 | ≥32/4 | 4/4 to ≥32/4 |
| ompK35 and ompK36 mutations (n = 1) | 4/4 | 4/4 | 4/4 to 4/4 |
The CLSI interpretive criteria for imipenem were applied to imipenem-relebactam.
In summary, both imipenem-relebactam and ceftazidime-avibactam were effective against KPC-expressing K. pneumoniae CRE isolates, while only ceftazidime-avibactam was effective against OXA-48-like CRE isolates. Elevation of imipenem-relebactam MIC values was observed only among KPC-expressing K. pneumoniae isolates harboring both ompK35 and ompK36 mutations, although isolates with elevated MIC values were generally still considered susceptible when CLSI interpretive criteria for imipenem were applied.
Supplementary Material
ACKNOWLEDGMENTS
This work was supported by an investigator-initiated grant provided by Merck and Co., Inc.
M.J.S. has received research grant funding from Merck and Co., Inc., and Allergan and has received consulting fees from Shionogi, Inc., and Achaogen. S.G.J. has received research grant funding from Merck and Co., Inc., and has received consulting fees from Shionogi, Inc. All other authors have no competing financial interests.
Footnotes
Supplemental material for this article may be found at https://doi.org/10.1128/AAC.00672-19.
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