Abstract
Among 177 carbapenemase-producing Gram-negative bacilli (108 KPC, 32 NDM, 11 IMP, 8 OXA-48, 4 OXA-181, 2 OXA-232, 5 IMI, 4 VIM, and 3 SME producers), aztreonam-avibactam was active against all isolates except two NDM producers with elevated MICs of 8/4 and 16/4 mg/liter; ceftazidime-avibactam was active against all KPC-, IMI-, SME-, and most OXA-48 group-producing isolates (93%) but not metallo-β-lactamase producers. Among older and contemporary antimicrobials, the most active were colistin, tigecycline, and fosfomycin, with overall susceptibilities of 88%, 79%, and 78%, respectively.
TEXT
The recent emergence and global dissemination of carbapenemase-producing Gram-negative bacilli (CP-GNB) pose a significant therapeutic challenge. Avibactam is a diazabicyclooctane non-β-lactam β-lactamase inhibitor with broad activity against Ambler class A and C β-lactamases and certain class D β-lactamases by covalent acylation of the β-lactamase active site serine residue. It restores susceptibility of Enterobacteriaceae harboring extended-spectrum β-lactamases (ESBLs), AmpC cephalosporinases, and class A carbapenemases to ceftazidime or ceftaroline (1). In vitro studies of avibactam in combination with aztreonam have also demonstrated activity against Enterobacteriaceae harboring NDM (a class B metallo-β-lactamase); however, there are scant data for the other less commonly encountered carbapenemases (2–4).
The aim of this study was to examine the activities of ceftazidime and aztreonam with and without avibactam against a large, contemporary, international collection of CP-GNB with diverse resistance mechanisms, with MICs determined using agar dilution as recommended by the Clinical and Laboratory Standards Institute (CLSI) (5, 6). A secondary aim was to evaluate the activity of antimicrobials commonly used to treat CP-GNB infections, including the “legacy antibiotics” colistin, amdinocillin (mecillinam), and fosfomycin. A total of 177 CP-GNB were studied (Table 1), comprising 122 and 53 clinical isolates from the United States and Singapore, respectively, and 2 NCTC (National Collection of Type Cultures, United Kingdom) reference isolates. These consisted of 172 Enterobacteriaceae isolates (107 KPC, 32 NDM, 8 OXA-48, 4 OXA-181, 2 OXA-232, 5 IMI, 3 SME, and 11 IMP producers) and 5 Pseudomonas aeruginosa isolates (4 VIM producers and 1 KPC producer). Genotypic characterization was performed using PCR/sequencing as previously described (7–15). All CP-GNB isolates tested positive by the CarbaNP test (16), except for one isolate each of OXA-181 and OXA-232, which were CarbaNP negative, and one OXA-48-producing isolate, which was CarbaNP indeterminate. In addition, as a control/comparator group, we studied 29 Enterobacteriaceae (11 Klebsiella pneumoniae and 18 Escherichia coli isolates), including 18 ESBL producers (10 with porin loss), 6 plasmid-mediated AmpC producers (1 with porin loss and another coproducing an ESBL), and 5 derepressed AmpC mutants (2 with porin loss).
TABLE 1.
CP-GNB isolates studied
| Origin of CP-GNB isolates | Ambler class | Resistance mechanism (n) | Species (n) |
|---|---|---|---|
| Singapore | A | IMI-1 (4) | Enterobacter cloacae complex (4) |
| IMI (unspecified) (1) | E. cloacae complex (1) | ||
| B | NDM-1 (19) | Klebsiella pneumoniae complex (9), Escherichia coli (5), E. cloacae complex (4), Citrobacter sedlakii (1) | |
| NDM-7 (1) | E. coli (1) | ||
| NDM (unspecified) (6) | K. pneumoniae complex (4), E. cloacae complex (1), Citrobacter freundii (1) | ||
| IMP-1 (7) | K. pneumoniae complex (4), E. cloacae complex (2), Citrobacter freundii (1) | ||
| IMP-4 (1) | K. pneumoniae complex (1) | ||
| IMP (unspecified) (3) | E. cloacae complex (2), E. coli (1) | ||
| D | OXA-48 (5) | K. pneumoniae complex (3), E. coli (1), Citrobacter koseri (1) | |
| OXA-181a (4) | K. pneumoniae complex (4) | ||
| OXA-232a (2) | K. pneumoniae complex (2) | ||
| U.S. | A | KPC, unspecified (108) | K. pneumoniae complex (84), E. cloacae (6), E. coli (5), Enterobacter aerogenes (3), C. freundii (2), C. koseri (2), Proteus stuartii (2), Serratia marcescens (2), Proteus mirabilis (1), Pseudomonas aeruginosa (1) |
| SME (unspecified) (3) | S. marcescens (3) | ||
| B | NDM-1 (1) | E. coli (1) | |
| NDM-7 (2) | E. coli (1), K. pneumoniae complex (1) | ||
| NDM (unspecified) (2) | K. pneumoniae complex (2) | ||
| VIM-2 (4) | P. aeruginosa (4) | ||
| D | OXA-48 (2) | K. pneumoniae complex (2) | |
| Reference NCTCb isolates | B | NDM-1 (NCTC 13443) (1) | K. pneumoniae complex (1) |
| D | OXA-48 (NCTC 13442) (1) | K. pneumoniae complex (1) |
OXA-181 and OXA-232 are part of the OXA-48 group.
NCTC, National Collection of Type Cultures, Central Public Health Laboratory, London, United Kingdom.
(This work was presented in part at the 54th Interscience Conference on Antimicrobial Agents and Chemotherapy, 5 to 9 September 2014, Washington, DC.)
Antimicrobial susceptibility testing was performed by agar dilution for ceftazidime and aztreonam (with or without avibactam at a fixed concentration of 4 mg/liter), cefepime, meropenem, piperacillin-tazobactam, levofloxacin, ciprofloxacin, colistin, gentamicin, tobramycin, amikacin, and, for non-Pseudomonas aeruginosa isolates, ceftriaxone, ertapenem, fosfomycin, amdinocillin, nitrofurantoin, and trimethoprim-sulfamethoxazole (6). Due to intrinsic resistance, nitrofurantoin and colistin were not evaluated against Serratia, Proteus, or Providencia species. Tigecycline MICs were determined by gradient diffusion (bioMérieux, France, or Liofiochem, Italy) on cation-adjusted Mueller-Hinton agar (BBL, Becton, Dickinson and Company, Franklin Lakes, NJ) for all isolates, except those with intrinsic resistance (P. aeruginosa, Proteus species, and Providencia species). CLSI interpretive breakpoints were applied with the following exceptions: aztreonam-avibactam activity was extrapolated from the aztreonam FDA breakpoints (≤4 mg/liter or ≤8 mg/liter for Enterobacteriaceae and P. aeruginosa isolates, respectively) as there are currently no interpretive criteria. FDA breakpoints were used for ceftazidime-avibactam (susceptible, ≤8/4 mg/liter) and tigecycline (susceptible, ≤2 mg/liter), and EUCAST breakpoints for colistin were used for Enterobacteriaceae (susceptible, ≤2 mg/liter). Finally, susceptibility to amdinocillin (MIC of ≤8 mg/liter) for non-E. coli Enterobacteriaceae was extrapolated from the EUCAST urinary breakpoint for E. coli. American Type Culture Collection (ATCC) strains E. coli 25922 and 35218, P. aeruginosa 27853, Staphylococcus aureus 29213, and K. pneumoniae 700603 were used as quality control (QC) organisms.
Aztreonam-avibactam was highly active against CP-GNB of all resistance types tested, all with MICs of ≤4/4 mg/liter (Enterobacteriaceae) and ≤8/4 mg/liter (P. aeruginosa), except for 2 NDM-positive E. coli isolates from Singapore: these were NDM-1 and NDM-7 positive, with MICs of 8/4 and 16/4 mg/liter, respectively. Ceftazidime-avibactam was active against all KPC-, IMI-, and SME-producing isolates and the majority of OXA-48 group CP-GNB (93%), but not against class B CP-GNB, which is not unexpected given the mechanism of action of avibactam (Table 2). In comparison, the overall susceptibilities of CP-GNB to the other antimicrobials tested were as follows: colistin, 88%; tigecycline, 79%; fosfomycin, 78%; amikacin, 51%; gentamicin, 48%; tobramycin, 15%; trimethoprim-sulfamethoxazole, 23%; nitrofurantoin, 16%; amdinocillin, 11%; levofloxacin, 17%; and ciprofloxacin, 11%. Colistin, which is often resorted to for treatment of serious CP-GNB infections, had only 91% and 87% susceptibilities for NDM- and KPC-positive isolates, respectively (Table 3). All ESBL- and AmpC-producing GNB tested were susceptible to aztreonam-avibactam and ceftazidime-avibactam, except for one ESBL (SHV)- and plasmid-mediated AmpC (DHA)-positive E. coli isolate with a MIC of 8/4 mg/liter to aztreonam-avibactam; the ceftazidime-avibactam MIC was 2/4 mg/liter.
TABLE 2.
Susceptibility of CP-GNB to ceftazidime and aztreonam with or without avibactam
| CP-GNB resistance genotype (n) | Antibiotica | No. (cumulative %) of isolates inhibited at ceftazidime or aztreonam concn (mg/liter) shown with or without 4 mg/liter avibactam |
MIC(μg/ml)b |
% S | |||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| ≤0.06 | 0.12 | 0.25 | 0.5 | 1 | 2 | 4 | 8 | 16 | 32 | 64 | 128 | 256 | 512 | >512 | 50% | 90% | |||
| KPC (108)c | ATM | 1 (1) | 1 (2) | 1 (3) | 2 (5) | 7 (11) | 11 (21) | 17 (37) | 28 (63) | 40 (100) | 512 | >512 | 2 | ||||||
| AZA | 20 (19) | 35 (51) | 38 (86) | 9 (94) | 3 (97) | 2 (99) | 1 (100) | 0.12/4 | 0.5/4 | 100 | |||||||||
| CAZ | 1 (1) | 3 (4) | 3 (7) | 6 (12) | 12 (23) | 23 (44) | 26 (69) | 31 (97) | 3 (100) | 256 | 512 | 2 | |||||||
| CZA | 7 (6) | 3 (9) | 9 (18) | 22 (38) | 44 (79) | 15 (92) | 5 (97) | 3 (100) | 1/4 | 2/4 | 100 | ||||||||
| NDM (32) | ATM | 2 (6) | 1 (9) | 1 (13) | 2 (19) | 6 (38) | 1 (41) | 7 (63) | 3 (72) | 9 (100) | 256 | >512 | 9 | ||||||
| AZA | 6 (19) | 11 (53) | 4 (66) | 4 (78) | 4 (91) | 1 (94) | 1 (97) | 1 (100) | 0.12/4 | 1/4 | 94 | ||||||||
| CAZ | 32 (100) | >512 | >512 | 0 | |||||||||||||||
| CZA | 32 (100) | >512/4 | >512/4 | 0 | |||||||||||||||
| OXA-48 group (14) | ATM | 1 (7) | 1 (14) | 1 (21) | 3 (43) | 4 (71) | 1 (79) | 3 (100) | 128 | >512 | 14 | ||||||||
| AZA | 4 (29) | 4 (57) | 2 (71) | 2 (86) | 2 (100) | 0.12/4 | 1/4 | 100 | |||||||||||
| CAZ | 1 (7) | 0 (7) | 1 (14) | 5 (50) | 1 (57) | 2 (71) | 1 (79) | 2 (93) | 1 (100) | 32 | 512 | 14 | |||||||
| CZA | 1 (7) | 1 (14) | 2 (28) | 3 (50) | 5 (86) | 1 (93) | 1 (100) | 0.5/4 | 4/4 | 93 | |||||||||
| IMP (11) | ATM | 1 (9) | 1 (18) | 1 (27) | 1 (36) | 2 (55) | 2 (72.7) | 1 (82) | 2 (100) | 128 | >512 | 18 | |||||||
| AZA | 2 (18) | 5 (46) | 2 (82) | 2 (100) | 0.25/4 | 1/4 | 100 | ||||||||||||
| CAZ | 2 (18) | 3 (46) | 3 (73) | 3 (100) | >512 | >512 | 0 | ||||||||||||
| CZA | 1 (9) | 1 (18) | 3 (46) | 2 (64) | 4 (100) | 256/4 | >512/4 | 9 | |||||||||||
| IMI (5) | ATM | 1 (25) | 4 (100) | 100 | |||||||||||||||
| AZA | 3 (60) | 2 (100) | 100 | ||||||||||||||||
| CAZ | 1 (20) | 1 (40) | 1 (60) | 1 (80) | 1 (100) | 80 | |||||||||||||
| CZA | 1 (20) | 4 (100) | 100 | ||||||||||||||||
| VIM (4)c | ATM | 5 (100) | 100 | ||||||||||||||||
| AZA | 1 (25) | 4 (100) | 100 | ||||||||||||||||
| CAZ | 2 (50) | 2 (100) | 0 | ||||||||||||||||
| CZA | 2 (50) | 2 (100) | 0 | ||||||||||||||||
| SME (3) | ATM | 2 (67) | 1 (100) | 0 | |||||||||||||||
| AZA | 1 (33) | 1 (67) | 1 (100) | 100 | |||||||||||||||
| CAZ | 1 (33) | 1 (67) | 1 (100) | 67 | |||||||||||||||
| CZA | 1 (33) | 1 (67) | 1 (100) | 100 | |||||||||||||||
ATM, aztreonam; AZA, aztreonam-avibactam; CAZ, ceftazidime; CZA, ceftazidime-avibactam. Because no CLSI breakpoints exist for AZA, Enterobacteriaceae and Pseudomonas aeruginosa isolates with MICs of ≤4 mg/liter and ≤8 mg/liter, respectively, to the aztreonam component were considered susceptible (S). For CAZ and CZA, FDA breakpoints were used: MICs of ≤8 mg/liter and ≤8/4 mg/liter, respectively, were considered susceptible.
50% and 90%, MIC50 and MIC90, respectively.
All KPC-producing CP-GNB were Enterobacteriaceae with MICs to AZA of ≤4/4 mg/liter, except for one P. aeruginosa isolate with a MIC of 8/4 mg/liter (susceptible). All VIM CP-GNB were P. aeruginosa.
TABLE 3.
Comparative susceptibilities of carbapenemase-producing Gram-negative bacilli to various antimicrobials
| Antimicrobiala | % susceptibility with resistance mechanism shown (total no. of isolates) |
||||||
|---|---|---|---|---|---|---|---|
| KPC (108) | NDM (32) | OXA-48 group (14) | IMP (11) | IMI (5) | VIM (4) | SME (3) | |
| β-Lactams and β-lactam–β-lactamase inhibitor combinations | |||||||
| ATM | 2 | 9 | 14 | 18 | 100 | 100 | 0 |
| AZA | 100 | 94 | 100 | 100 | 100 | 100 | 100 |
| CAZ | 2 | 0 | 14 | 0 | 80 | 0 | 67 |
| CZA | 100 | 0 | 93 | 9 | 100 | 0 | 100 |
| CROb | 1 | 0 | 14 | 0 | 100 | 67 | |
| FEPc | 2 | 0 | 14 | 0 | 80 | 0 | 33 |
| ETPb | 2 | 0 | 7 | 0 | 0 | 0 | |
| MEM | 5 | 0 | 43 | 36 | 20 | 0 | 0 |
| TZP | 1 | 0 | 0 | 27 | 100 | 0 | 67 |
| MECb | 0 | 16 | 43 | 18 | 100 | 33 | |
| Aminoglycosides | |||||||
| GEN | 63 | 16 | 36 | 0 | 80 | 0 | 100 |
| TOB | 9 | 9 | 36 | 9 | 80 | 0 | 100 |
| AMK | 51 | 41 | 43 | 82 | 100 | 0 | 100 |
| Fluoroquinolones | |||||||
| LVX | 11 | 13 | 14 | 36 | 100 | 0 | 100 |
| CIP | 10 | 0 | 7 | 18 | 80 | 0 | 67 |
| Other antimicrobial agents | |||||||
| SXTb | 21 | 9 | 14 | 45 | 80 | 100 | |
| NITb | 14 | 25 | 7 | 27 | 20 | ||
| FOFb | 78 | 78 | 71 | 73 | 100 | 100 | |
| CSTd | 87 | 91 | 100 | 91 | 40 | 75 | |
| TGCb,d | 81 | 69 | 93 | 55 | 100 | 100 | |
Antimicrobial abbreviations: ATM, aztreonam; AZA, aztreonam-avibactam; CAZ, ceftazidime; CZA, ceftazidime-avibactam; CRO, ceftriaxone; FEP, cefepime; ETP, ertapenem; MEM, meropenem; TZP, piperacillin-tazobactam; MEC, amdinocillin; GEN, gentamicin; TOB, tobramycin; AMK, amikacin; LVX, levofloxacin; CIP, ciprofloxacin; SXT, trimethoprim-sulfamethoxazole; NIT, nitrofurantoin; FOF, fosfomycin; CST, colistin; TGC, tigecycline.
Excluding Pseudomonas aeruginosa isolates for the antimicrobial agents with no inherent antipseudomonal activity and/or without CLSI breakpoints (CRO, ETP, MEC, SXT, NIT, FOF, and TGC). Additionally, for NIT, Proteus, Providencia, and Serratia species were excluded due to intrinsic resistance. An amdinocillin (MEC) breakpoint of ≤8 mg/liter was applied to Enterobacteriaceae.
Susceptibility to FEP was defined as ≤2 mg/liter, per CLSI M100-S25 (6). Susceptible dose-dependent isolates (MICs of 4 to 8 mg/liter) were not included in this category.
EUCAST breakpoints were used for CST for Enterobacteriaceae and FDA breakpoints for TGC. Due to intrinsic resistance, for CST, Proteus, Providencia, and Serratia species were excluded, and for TGC, Pseudomonas, Proteus, and Providencia species were excluded.
Our findings are consistent with the reported and expected activities of aztreonam-avibactam and ceftazidime-avibactam, except for a few isolates with elevated MICs to aztreonam-avibactam. While breakpoints have not been established for aztreonam-avibactam, a single carbapenemase-negative, but SHV- and DHA-positive, E. coli isolate and one NDM-positive isolate had an aztreonam-avibactam MIC of 8/4 mg/liter, and a single NDM-positive isolate had an aztreonam-avibactam MIC of 16/4 mg/liter. Aztreonam has a specific affinity to penicillin-binding protein-3 (PBP3); we postulate that in the isolates with elevated aztreonam-avibactam MICs, there may have been altered PBP3 affinity, as has been recently described in NDM-positive E. coli isolates (17). Resistance to avibactam may also result from changes in its β-lactamase binding site—e.g., from single amino acid substitutions or Ω-loop mutations in coproduced class A and C β-lactamases (18–20). Also, porin loss and overexpression of efflux pumps may be contributory, although we did not assess our isolates for these. As β-lactam–avibactam combinations are introduced into clinical use, it will be important to monitor for and further characterize nonsusceptible isolates and correlate clinical outcomes with the various resistance genotypes.
Interestingly, among the β-lactams, amdinocillin, an old extended-spectrum penicillin antibiotic, demonstrated the most activity against NDM (16%) and OXA-48 (43%) CP-GNB, after ceftazidime-avibactam (0% and 93% activities against NDM and the OXA-48 group, respectively) and aztreonam-avibactam (94% and 100% activities against NDM and the OXA-48 group, respectively). The interpretive criteria used, however, were for urinary isolates as this agent is currently mostly used in an oral formulation (pivmecillinam) in Nordic countries for the treatment of urinary tract infections. Although an inoculum effect has been demonstrated (21), amdinocillin achieves high urinary concentrations and is relatively stable to ESBLs and AmpCs. An intravenous formulation is available in Denmark (22). A previous study found that amdinocillin with avibactam showed a modest reduction in MICs in ESBL and AmpC producers (23). While we did not specifically study this combination, further studies of avibactam in combination with older penicillins such as amdinocillin are warranted.
Overall, we found that aztreonam-avibactam showed excellent activity against CP-GNB from all Ambler classes tested (A, B, and D). The ceftazidime-avibactam combination showed activity against class A CP-GNB and the majority of OXA-48 group CP-GNB tested, but not against class B CP-GNB. The evaluated β-lactam–avibactam combinations may provide an important advance in the treatment of CP-GNB infections.
ACKNOWLEDGMENTS
Cerexa, Inc., a wholly owned subsidiary of Forest Laboratories, LLC, and Forest Laboratories, LLC, kindly provided avibactam powder for this study. We thank Mary Hayden and Karen Lolans for sharing reference isolates and leftover rectal surveillance swabs from which CP-GNB isolates were isolated and Paul Schreckenberger, Patricia Simner, George Zhanel, and Daryl Hoban for sharing reference isolates.
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