Abstract
Ceftaroline is a new cephalosporin with broad-spectrum activity against Gram-positive and -negative organisms. The prodrug of ceftaroline, ceftaroline fosamil, combined with the β-lactamase inhibitor avibactam (formerly NXL104), was tested against Enterobacteriaceae strains producing Ambler class A, B, C, and D enzymes, including strains producing multiple enzymes, as well as Pseudomonas aeruginosa, Acinetobacter spp., and methicillin-susceptible and methicillin-resistant Staphylococcus aureus (MRSA) strains. Isolates were collected from 1999 to 2008 from global surveillance programs, and susceptibility testing was performed by reference broth microdilution methods. Ceftaroline-avibactam exhibited potent activity against Enterobacteriaceae producing various β-lactamase types (MIC90, 0.25 to 2 μg/ml, except for metalloenzymes), including 99 strains carrying multiple enzymes (2 to 4 β-lactamases; MIC90, 2 μg/ml). All isolates were inhibited by ceftaroline-avibactam at ≤4 μg/ml. Ceftaroline-avibactam (MIC90, 0.5 to 1 μg/ml) was more active than meropenem (MIC90, >8 μg/ml) and other comparators when tested against KPC-producing strains. S. aureus strains, including MRSA with four staphylococcal cassette chromosome mec (SCCmec) types, were dominantly (99.1%) inhibited by ceftaroline-avibactam at ≤2 μg/ml, and the ceftaroline MIC was not adversely affected by the addition of the β-lactamase inhibitor (MIC50/90, 1 and 2 μg/ml for ceftaroline with and without avibactam). Ceftaroline-avibactam demonstrated limited activity against Acinetobacter spp. and P. aeruginosa (MIC50s, 32 and 16 μg/ml, respectively). These results document that ceftaroline-avibactam has potent activity against Enterobacteriaceae that produce KPC, various ESBL types (CTX-M types), and AmpC (chromosomally derepressed or plasmid-mediated enzymes), as well as against those producing more than one of these β-lactamase types, and its development as a therapeutic option for the treatment of infections caused by multidrug-resistant Enterobacteriaceae as well as MRSA is warranted.
INTRODUCTION
The evolution of antimicrobial resistance among nosocomial and, more recently, community-acquired pathogens is of great concern, and it has compromised the treatment of serious bacterial infections (15). Among the emerging multidrug-resistant (MDR) organisms, Staphylococcus aureus, Streptococcus pneumoniae, Enterococcus faecium, and Gram-negative bacilli (Enterobacteriaceae species, Pseudomonas aeruginosa, and Acinetobacter baumannii) are particularly threatening our ability to manage these infections (10, 15). The advent of vaccines, the development of new compounds active against MDR S. aureus and S. pneumoniae, and the successful implementation of infection control measures are diminishing the burden of infections with Gram-positive organisms (10); however, fewer options are available for MDR Gram-negative organisms. Among those resistance mechanisms, β-lactamases are especially worrisome because (i) they act against an important class of antimicrobial agents that is widely used in clinical practice; (ii) they can potentially acquire mutations that broaden their spectrum of hydrolysis against different β-lactams; and (iii) they have the ability to disseminate as they are carried by plasmids and/or other mobile genetic structures (3).
Ceftaroline fosamil is the parenterally administered prodrug form of ceftaroline, a new cephalosporin with potent activity against Gram-positive organisms, including methicillin-resistant S. aureus (MRSA) and MDR S. pneumoniae (1, 8; D. J. Farrell et al., submitted for publication; R. K. Flamm et al., submitted for publication; H. S. Sader et al., submitted for publication). In phase III trials, ceftaroline fosamil was shown to be noninferior to ceftriaxone for the treatment of patients with acute bacterial skin and skin structure infection (ABSSSI) or community-acquired bacterial pneumonia (CABP) requiring hospitalization (1, 7, 17). Ceftaroline fosamil has been approved by the U.S. Food and Drug Administration (FDA) for ABSSSI and CABP (1). Ceftaroline is also active against most species of Enterobacteriaceae but, like other cephalosporins, has limited activity against isolates producing extended-spectrum β-lactamases (ESBL), cephalosporinases, and carbapenemases (8). However, when ceftaroline is combined with avibactam (formerly NXL104), a new non-β-lactam β-lactamase inhibitor with a prolonged deacylation rate that inhibits Ambler classes A (e.g., ESBL, KPC), C (AmpC), and D (OXA-like) enzymes (16), its spectrum of activity is significantly expanded (13).
We evaluated the spectrum of activity and potency of ceftaroline combined with avibactam, as well as those of comparator antimicrobial agents, against a collection of Gram-negative isolates, including strains with well-characterized β-lactamase-encoding genes, and methicillin-susceptible S. aureus and MRSA strains carrying four different staphylococcal cassette chromosome mec (SCCmec) types.
MATERIALS AND METHODS
Bacterial isolates.
A total of 421 Gram-negative (321 strains) (Enterobacteriaceae, P. aeruginosa, and Acinetobacter spp.) and Gram-positive (110 S. aureus strains) isolates were tested. These strains were identified during the 1999–2008 period (82.2% from 2006 to 2008) in two global surveillance studies (the SENTRY Antimicrobial Surveillance Program and the MYSTIC Program) and were collected from patients with bloodstream, respiratory tract, and skin and skin structure infections. Species identification was confirmed by standard biochemical tests, the Vitek2 System (bioMérieux, Hazelwood, MO), or sequencing-based methods, when necessary.
Different PCR approaches were used to detect ESBL, plasmid AmpC, and carbapenemase-encoding genes among Gram-negative bacteria. Generic primers were used to detect genes encoding PER, GES, VEB, CTX-M, OXY (K1), oxacillinases (OXA-ESBL), TEM, and SHV (2). Plasmid AmpC genes were detected as described elsewhere (14). Isolates showing reduced susceptibility to imipenem or meropenem (MIC, ≥2 μg/ml) were tested for the presence of carbapenemase genes, including blaIMP, blaVIM, blaKPC, blaSME, and blaGES variants, and for blaIMI, blaNMC-A, and blaOXA-48, combined in two amplification reactions (4). Acinetobacter spp. were screened for the presence of carbapenem-hydrolyzing oxacillinases according to he methods described by Woodford et al. (18). PCR amplicons were sequenced on both strands, and the nucleotide sequences and deduced amino acid sequences were analyzed using the Lasergene software package (DNAStar, Madison, WI). Sequences were compared with others available via NCBI/BLAST. Citrobacter spp., Enterobacter spp., and Serratia spp. that putatively produce stably derepressed chromosomal AmpC were selected according to ceftazidime resistance (MIC, ≥16 μg/ml) and susceptibility to cefepime (MIC, ≤2 μg/ml).
Staphylococcal cassette chromosome mec (SCCmec) typing was performed using a multiplex PCR strategy as described previously (12). Isolates were compared to MRSA clonal types (USA100, USA300, USA400, USA700, and Hungarian/Brazilian) by pulsed-field gel electrophoresis (PFGE), single-locus (spa) typing, and multilocus sequence typing (MLST) methods as described by Mendes et al. (11).
Antimicrobial susceptibility testing.
All strains were tested for antimicrobial susceptibility by using the reference broth microdilution method as described by the Clinical and Laboratory Standards Institute (CLSI) (5). Ceftaroline was combined with avibactam at a fixed concentration of 4 μg/ml. Quality control (QC) was performed using Escherichia coli ATCC 25922, S. aureus ATCC 29213, and P. aeruginosa ATCC 27853. All QC results were within specified ranges as published in CLSI document M100–S22 (6).
RESULTS AND DISCUSSION
A total of 272 β-lactamase-producing Enterobacteriaceae strains were tested, including isolates producing ESBLs (33 strains), plasmid-mediated AmpC (36 strains), carbapenemases (69 strains), and a combination of two to four enzymes (99 strains). These isolates belonged to 15 bacterial species, including Klebsiella pneumoniae (76 strains), Enterobacter spp. (64 strains), Escherichia coli (62 strains), Citrobacter freundii (21 strains), Serratia marcescens (20 strains), Klebsiella oxytoca (17 strains), Salmonella spp. (6 strains), Proteus mirabilis (5 strains), and Providencia stuartii (1 strain).
ESBL-producing Enterobacteriaceae carried genes encoding five CTX-M variants (CTX-M-15 [12 strains], CTX-M-14 [6 strains], CTX-M-3 and CTX-M-5 [2 strains each], and CTX-M-2 [1 strain]), four SHV variants (one strain each for SHV-5, SHV-7, SHV-12, and SHV-31), OXY-like enzymes, or TEM-10. Ceftaroline-avibactam MICs for these ESBL producers ranged from ≤0.015 to only 1 μg/ml. The majority (16 of 23 [69.6%]) of CTX-M-producing strains exhibited a ceftaroline-avibactam MIC of ≤0.12 μg/ml (MICs ranged from 0.03 to 1 μg/ml). One CTX-M-2-producing S. marcescens strain and one Enterobacter sp. strain carrying the CTX-M-15-encoding gene had ceftaroline-avibactam MIC results at 1 μg/ml. SHV-producing Enterobacteriaceae exhibited a broad range of ceftaroline-avibactam MIC values (≤0.015 to 1 μg/ml), and the highest MIC values (0.5 to 1 μg/ml) were observed among Enterobacter sp. isolates. All SHV-producing Klebsiella sp. strains were inhibited at ≤0.25 μg/ml, and the only SHV-12-positive E. coli strain tested had a ceftaroline-avibactam MIC of 0.03 μg/ml.
Isolates carrying plasmid-mediated AmpC enzymes harbored blaCMY-2 or blaFOX-5 (24 and 12 strains, respectively) and were represented by E. coli (24 strains), Klebsiella spp. (6 strains), Salmonella spp. (4 strains), and P. mirabilis (2 strains). Ceftaroline-avibactam was very potent (MIC90, 0.25 μg/ml) against these strains, inhibiting all isolates at ≤0.5 μg/ml (Table 1). Additionally, for a group of ceftazidime-resistant (MIC, ≥16 μg/ml) Citrobacter spp., Enterobacter spp., and Serratia spp. that putatively have stably derepressed chromosomal AmpC, the ceftaroline-avibactam MIC50 was 0.12 and the MIC90 was 0.5 μg/ml (see Table 3).
Table 1.
Frequency distributions for ceftaroline-avibactam tested against β-lactamase-producing Enterobacteriaceae grouped by enzyme type, nonfermentative Gram-negative bacilli, and S. aureus strains, including MRSA with defined SCCmec types
| Organism and type of enzyme or SCCmec (no. of strains tested) | No. of strains (cumulative % inhibited) inhibited at a ceftaroline-avibactama MIC (μg/ml) of: |
|||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| ≤0.015 | 0.03 | 0.06 | 0.12 | 0.25 | 0.5 | 1 | 2 | 4 | 8 | 16 | ≥32 | |
| Enterobacteriaceae (272) | ||||||||||||
| ESBL (33) | 1 (3.0) | 3 (12.1) | 11 (45.4) | 11 (79.8) | 4 (90.9) | 0 (90.9) | 3 (100.0) | |||||
| Plasmid-mediated AmpC (36) | 4 (11.1) | 8 (33.3) | 14 (72.2) | 8 (94.4) | 2 (100.0) | |||||||
| Ceftazidime-resistant AmpC-producing species (27) | 3 (11.1) | 13 (59.3) | 7 (85.2) | 3 (96.3) | 1 (100.0) | |||||||
| Serine-carbapenemase (32) | 1 (3.1) | 3 (12.5) | 3 (21.9) | 15 (68.7) | 6 (87.5) | 2 (93.7) | 2 (100.0) | |||||
| Serine-carbapenemase in AmpC-producing species (37) | 1 (2.7) | 2 (5.4) | 6 (24.3) | 8 (45.9) | 13 (81.1) | 6 (97.3) | 1 (100.0) | |||||
| Metallo-β-lactamase (8) | 8 (100.0) | |||||||||||
| Multiple β-lactamases (57) | 1 (1.7) | 1 (3.5) | 24 (45.6) | 12 (66.7) | 12 (87.7) | 3 (98.2) | 1 (100.0) | |||||
| Multiple β-lactamases including KPC (15) | 3 (20.0) | 7 (66.7) | 3 (86.7) | 1 (93.3) | 1 (100.0) | |||||||
| Multiple β-lactamases in AmpC-producing species (27) | 4 (14.8) | 6 (37.0) | 5 (55.6) | 5 (74.1) | 6 (96.3) | 1 (100.0) | ||||||
| P. aeruginosa (25) | 3 (12.0) | 3 (24.0) | 1 (28.0) | 7 (56.0) | 11 (100.0) | |||||||
| Acinetobacter spp. (24) | 1 (4.2) | 1 (8.3) | 2 (16.7) | 3 (29.2) | 0 (29.2) | 4 (45.8) | 13 (100.0) | |||||
| S. aureus (110) | 10 (9.1) | 28 (34.5) | 45 (75.4) | 26 (99.1) | 1 (100) | - | ||||||
| SCCmec type I (19) | 1 (5.3) | 17 (94.7) | 1 (100.0) | |||||||||
| SCCmec type II (20) | 2 (10.0) | 15 (85.0) | 3 (100.0) | |||||||||
| SCCmec type III (20) | 2 (10.0) | 12 (70.0) | 6 (100.0) | |||||||||
| SCCmec type IV (41) | 24 (58.5) | 17 (100.0) | ||||||||||
Ceftaroline combined with avibactam at a fixed concentration of 4 μg/ml.
Table 3.
In vitro activities of ceftaroline-avibactam and selected antimicrobial agents tested against a well-characterized collection of β-lactamase-producing Enterobacteriaceae, non-fermentative Gram-negative bacilli, and S. aureus strains, including MRSA strains
| Organism or group, enzyme(s) (no. of strains tested), and antimicrobial agent | MIC (μg/ml) |
% Susceptible/% resistanta by: |
|||
|---|---|---|---|---|---|
| 50% | 90% | Range | CLSI | EUCAST | |
| Enterobacteriaceae | |||||
| ESBL (33) | |||||
| Ceftaroline-avibactam | 0.12 | 0.25 | ≤0.015–1 | NA | NA |
| Ceftaroline | >64 | >64 | 0.25–>64 | 3.0/97.0b | NA |
| Ceftriaxone | >32 | >32 | ≤0.25–>32 | 3.0/93.9 | 3.0/93.9 |
| Ceftazidime | >16 | >16 | ≤16–>16 | 30.3/63.6 | 27.3/69.7 |
| Meropenem | ≤0.12 | ≤0.12 | ≤0.12–0.25 | 100.0/0.0 | 100.0/0.0 |
| Piperacillin-tazobactam | 16 | >64 | ≤8–>64 | 54.5/27.3 | 30.3/45.5 |
| Gentamicin | ≤4 | >8 | ≤4–>8 | 75.8/18.2 | 69.7/24.2 |
| Levofloxacin | >4 | >4 | ≤0.5–>4 | 42.4/51.5 | 39.4/57.6 |
| Plasmid-mediated AmpC (36) | |||||
| Ceftaroline-avibactam | 0.12 | 0.25 | 0.06–1 | NA | NA |
| Ceftaroline | 16 | 64 | 4–>64 | 2.8/97.2b | NA |
| Ceftriaxone | 16 | >32 | 8–>32 | 0.0/88.9 | 0.0/88.9 |
| Ceftazidime | >16 | >16 | 16–>16 | 5.6/94.4 | 0.0/94.4 |
| Meropenem | ≤0.12 | 0.12 | ≤0.06–0.25 | 100.0/0.0 | 100.0/0.0 |
| Piperacillin-tazobactam | 16 | >64 | 2–>64 | 63.9/25.0 | 44.4/36.1 |
| Gentamicin | ≤4 | >8 | ≤2–>8 | 72.2/13.9 | 44.4/27.8 |
| Levofloxacin | >4 | >4 | ≤0.5–>4 | 41.7/52.8 | 41.7/58.3 |
| Ceftazidime-resistant AmpC-producing speciesb (27) | |||||
| Ceftaroline-avibactam | 0.12 | 0.5 | 0.03–0.5 | NA | NA |
| Ceftaroline | 64 | >64 | 0.25–>128 | 0.0/100.0b | NA |
| Ceftriaxone | 32 | >32 | ≤8–>32 | 0.0/100.0 | 0.0/100.0 |
| Ceftazidime | >16 | >16 | ≤16–>16 | 0.0/100.0 | 0.0/100.0 |
| Meropenem | ≤0.06 | 0.12 | ≤0.12–0.12 | 100.0/0.0 | 100.0/0.0 |
| Piperacillin-tazobactam | 64 | >64 | ≤8–>64 | 14.8/44.4 | 14.8/85.2 |
| Gentamicin | ≤2 | >8 | ≤4–>8 | 74.1/18.5 | 74.1/25.9 |
| Levofloxacin | ≤0.5 | >4 | ≤0.5–>4 | 77.8/18.5 | 70.4/22.2 |
| Carbapenemase (32) | |||||
| Ceftaroline-avibactam | 0.5 | 2 | 0.06–4 | NA | NA |
| Ceftaroline | >64 | >64 | 2–>64 | 0.0/100.0b | NA |
| Ceftriaxone | >32 | >32 | 0.5–>32 | 6.3/93.8 | 6.3/93.8 |
| Ceftazidime | >16 | >16 | ≤16–>16 | 6.3/87.5 | 3.1/93.8 |
| Meropenem | >8 | >8 | ≤0.06–>8 | 9.4/84.4 | 15.6/53.1 |
| Piperacillin-tazobactam | >64 | >64 | 32–>64 | 0.0/93.8 | 0.0/100.0 |
| Gentamicin | 8 | >8 | ≤2–>8 | 48.4/45.2 | 35.5/51.6 |
| Levofloxacin | >4 | >4 | ≤0.5–>4 | 37.0/59.3 | 22.2/63.0 |
| Carbapenemase in AmpC-producing speciesb (37) | |||||
| Ceftaroline-avibactam | 1 | 2 | 0.06–4 | NA | NA |
| Ceftaroline | >64 | >64 | ≤0.12–>64 | 2.7/86.5b | NA |
| Ceftriaxone | >32 | >32 | ≤8–>32 | 18.9/81.1 | 18.9/81.1 |
| Ceftazidime | >16 | >16 | ≤16–>16 | 18.9/78.4 | 18.9/81.1 |
| Meropenem | 8 | >8 | 0.12–>8 | 2.7/83.8 | 16.2/40.5 |
| Piperacillin-tazobactam | >64 | >64 | 1–>64 | 24.3/70.3 | 24.3/75.7 |
| Gentamicin | 8 | >8 | ≤2–>8 | 38.9/44.4 | 36.1/61.1 |
| Levofloxacin | 4 | >4 | ≤0.5–>4 | 39.4/48.5 | 27.3/60.6 |
| Metallo-β-lactamase (8) | |||||
| Ceftaroline-avibactam | >32 | – | >32 | NA | NA |
| Ceftaroline | >128 | – | 32–>128 | 0.0/100.0b | NA |
| Ceftriaxone | >32 | – | 16–>32 | 0.0/100.0 | 0.0/100.0 |
| Ceftazidime | >32 | – | 32–>32 | 0.0/100.0 | 0.0/100.0 |
| Meropenem | 8 | – | 1–>8 | 12.5/75.0 | 25.0/37.5 |
| Piperacillin-tazobactam | 64 | – | 32–>64 | 0.0/50.0 | 0.0/100.0 |
| Gentamicin | ≤2 | – | ≤2–>8 | 75.0/12.5 | 75.0/25.0 |
| Levofloxacin | 1 | – | ≤0.5–>4 | 62.5/37.5 | 50.0/37.5 |
| Multiple β-lactamases (57) | |||||
| Ceftaroline-avibactam | 0.12 | 0.5 | ≤0.015–2 | NA | NA |
| Ceftaroline | >64 | >64 | 4–>64 | 0.0/100.0b | NA |
| Ceftriaxone | >32 | >32 | ≤0.25–>32 | 3.5/94.7 | 3.5/94.7 |
| Ceftazidime | >16 | >16 | ≤16–>16 | 29.8/70.2 | 12.3/70.2 |
| Meropenem | ≤0.12 | ≤0.12 | ≤0.12–4 | 98.2/1.8 | 98.2/0.0 |
| Piperacillin-tazobactam | 16 | >64 | ≤8–>64 | 52.6/35.1 | 43.9/47.4 |
| Gentamicin | >8 | >8 | ≤4–>8 | 38.6/50.9 | 35.1/61.4 |
| Levofloxacin | 4 | >4 | ≤0.5–>4 | 47.4/47.4 | 42.1/52.6 |
| Multiple β-lactamases, including KPC (15) | |||||
| Ceftaroline-avibactam | 0.5 | 2 | 0.25–4 | NA | NA |
| Ceftaroline | >64 | >64 | 64–>64 | 0.0/100.0b | NA |
| Ceftriaxone | >32 | >32 | 16–>32 | 0.0/100.0 | 0.0/100.0 |
| Ceftazidime | >16 | >16 | 16–>16 | 0.0/100.0 | 0.0/100.0 |
| Meropenem | >8 | >8 | 4–>8 | 0.0/100.0 | 0.0/66.7 |
| Piperacillin-tazobactam | >64 | >64 | >64 | 0.0/100.0 | 0.0/100.0 |
| Gentamicin | 4 | >8 | ≤2–>8 | 53.3/20.0 | 33.3/46.7 |
| Levofloxacin | >4 | >4 | ≤0.5–>4 | 20.0/80.0 | 13.3/80.0 |
| Multiple β-lactamases in AmpC-producing speciesb (27) | |||||
| Ceftaroline-avibactam | 0.5 | 2 | 0.12–4 | NA | NA |
| Ceftaroline | 256 | >256 | 1–>256 | 0.0/96.3b | NA |
| Ceftriaxone | >32 | >32 | 16–>32 | 0.0/100.0 | 0.0/100.0 |
| Ceftazidime | 256 | >256 | 1–>256 | 14.8/81.5 | 3.7/85.2 |
| Meropenem | ≤0.12 | 0.5 | ≤0.12–>8 | 96.3/3.7 | 96.3/3.7 |
| Piperacillin-tazobactam | 32 | >64 | ≤8–>64 | 37.0/25.9 | 33.3/63.0 |
| Gentamicin | >8 | >8 | ≤2–>8 | 25.9/59.3 | 14.8/74.1 |
| Levofloxacin | 4 | >4 | ≤0.5–>4 | 44.4/48.1 | 40.7/55.6 |
| P. aeruginosa (25) | |||||
| Ceftaroline-avibactam | 16 | >32 | 2–>32 | NA | NA |
| Ceftaroline | >64 | >64 | 8–>64 | NA | NA |
| Ceftriaxone | >32 | >32 | 16–>32 | 0.0/86.4b | NA |
| Ceftazidime | >16 | >16 | ≤16–>16 | 8.0/56.0 | 8.0/92.0 |
| Meropenem | >8 | >8 | 0.25–>8 | 28.0/68.0 | 28.0/56.0 |
| Piperacillin-tazobactam | 64 | >64 | 4–>64 | 36.0/44.0 | 36.0/64.0 |
| Gentamicin | >8 | >8 | ≤2–>8 | 41.2/52.9 | 41.2/58.8 |
| Levofloxacin | 4 | >4 | ≤0.5–>4 | 41.2/47.1 | 41.2/58.8 |
| Acinetobacter spp. (24) | |||||
| Ceftaroline-avibactam | 32 | >32 | 0.5–>32 | NA | NA |
| Ceftaroline | >64 | >64 | 1–>64 | NA | NA |
| Ceftriaxone | >32 | >32 | ≤8–>32 | 25.0/66.7 | NA |
| Ceftazidime | >16 | >16 | ≤16–>16 | 8.3/62.5 | NA |
| Meropenem | >8 | >8 | ≤0.12–>8 | 29.2/62.5 | 29.2/62.5 |
| Piperacillin-tazobactam | >64 | >64 | ≤0.5–>64 | 29.2/70.8 | NA |
| Gentamicin | >8 | >8 | ≤2–>8 | 41.7/58.3 | 41.7/58.3 |
| Levofloxacin | >4 | >4 | ≤0.5–>4 | 29.2/66.7 | 29.2/70.8 |
| S. aureus (110) | |||||
| Ceftaroline-avibactam | 1 | 2 | 0.25–4 | NA | NA |
| Ceftaroline | 1 | 2 | 0.25–4 | 75.4b,c,d | NA |
| Ceftriaxone | >32 | >32 | ≤8–>32 | 0.0/90.9 | 9.1/90.9 |
| Ceftazidime | >16 | >16 | ≤16–>16 | 0.0/90.9 | 9.1/90.9 |
| Meropenem | 8 | >8 | ≤0.12–>8 | 9.1/90.9 | 9.1/90.9 |
| Piperacillin-tazobactam | 64 | >64 | ≤0.5–>64 | 9.1/90.9 | 9.1/90.9 |
| Imipenem | 8 | >8 | ≤0.12–>8 | 9.1/90.9 | 9.1/90.9 |
| Vancomycin | 1 | 1 | 0.25–2 | NA | NA |
Results obtained by the criteria published by the CLSI (2012) (6) or EUCAST (2011) (9a). β-Lactam susceptibility should be guided by the oxacillin test results for S. aureus. NA, not available.
U.S. FDA breakpoints were applied (Rocephin package insert, 2010; Genentech, Inc.) for comparison purposes.
All S. aureus isolates with SCCmec type IV were susceptible to ceftaroline.
Percentage of susceptible isolates.
Isolates producing serine carbapenemases carried genes encoding KPC-2 (32 strains), KPC-3 (23 strains), SME-1 or -2 (7 strains), OXA-48 (5 strains), and KPC-4 and NMC-A (1 strain each), among 32 Klebsiella spp. and E. coli strains. In addition, 37 of those enzymes were found in bacterial species that putatively hyperproduce chromosomal AmpC enzymes (listed above). In general, carbapenemase-producing Enterobacteriaceae strains (n = 69) exhibited ceftaroline-avibactam MIC values slightly higher than those of ESBL- or AmpC-producing strains (MIC50, 0.5 to 1 μg/ml compared to 0.12 μg/ml). This novel β-lactam–β-lactamase combination inhibited 58 (84.0%) strains at ≤1 μg/ml, and only 3 strains exhibited ceftaroline-avibactam MIC values of >4 μg/ml (1 Enterobacter cloacae strain and 2 K. pneumoniae strains producing KPC-4, KPC-2, and OXA-48, respectively). Serine carbapenemase-producing isolates from species that might hyperproduce AmpC enzymes (Citrobacter spp., Enterobacter spp., and Serratia spp.) were modestly less susceptible to ceftaroline-avibactam than Klebsiella spp. and E. coli strains also carrying serine carbapenemases (MIC50, 1 and 0.5 μg/ml, respectively). As expected, SME-producing S. marcescens and NMC-A-producing E. cloacae displayed low MIC values for ceftaroline-avibactam and other cephalosporins (all ≤2 μg/ml) compared to meropenem MIC results (>8 μg/ml). Ceftaroline-avibactam showed limited activity against isolates carrying metallo-β-lactamase (MBL) genes encoding VIM variants and IMP variants (MIC50, >32 μg/ml) (Table 1).
A total of 99 Enterobacteriaceae carrying two or more β-lactamases were tested against ceftaroline-avibactam and comparator agents (Table 2). These strains were grouped as follows: (i) Klebsiella spp. and E. coli, (ii) species that might hyperexpress chromosomal AmpC (Citrobacter spp., Enterobacter spp., and Serratia spp.), and (iii) isolates producing serine carbapenemases (KPC) in combination with other enzymes (Table 1). The description of the enzymes carried by these isolates is found in Table 2, and more than 70 enzyme arrays/combinations (>1 β-lactamase) were selected. Ceftaroline-avibactam inhibited 90.0% of strains producing multiple enzymes at ≤2 μg/ml. When strains were grouped according to (i) Klebsiella spp. and E. coli, (ii) AmpC producers, and (iii) strains producing multiple β-lactamases including KPC, 100.0, 96.3, and 93.3% of the isolates were inhibited at the same MIC value (≤2 μg/ml) (Table 1), respectively. It is noteworthy that MIC reductions of 4- to 8,192-fold were noted when avibactam (4 μg/ml) combined with ceftaroline was tested against strains producing multiple β-lactamases, including serine carbapenemases (Table 2).
Table 2.
Enterobacteriaceae strains carrying more than one β-lactamase gene
| No. of enzymes | Enzymea |
No. of strains | Fold (log2) reduction in the ceftaroline MIC in the presence of 4 μg/ml avibactamb | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| ESBL |
Non-ESBL |
Plasmid AmpC | KPC | |||||||||
| CTX-M | SHV | GES | OXA | OXY | SHV | TEM | OXA | |||||
| 2 | X | X | 3 | 256–2,048 | ||||||||
| 2 | X | X | 5 | 512–8,192 | ||||||||
| 2 | X | X | 7 | 512–8,192 | ||||||||
| 2 | X | X | 3 | 32–8,192 | ||||||||
| 2 | X | X | 2 | 64–2,048 | ||||||||
| 2 | X | X | 1 | 2,048 | ||||||||
| 2 | X | X | 1 | 4,096 | ||||||||
| 2 | X | X | 1 | 512 | ||||||||
| 2 | X | X | 2 | 32–8,192 | ||||||||
| 2c | X | 2 | 4–128 | |||||||||
| 2c | X | 15 | 16–1,024 | |||||||||
| 2c | X | 1 | 512 | |||||||||
| 2c | X | 1 | 512 | |||||||||
| 3 | X | X | X | 1 | 8,192 | |||||||
| 3 | X | X | X | 1 | 4,096 | |||||||
| 3 | X | X | X | 1 | 8,192 | |||||||
| 3 | X | X | X | 4 | 512–4,096 | |||||||
| 3 | X | X | X | 1 | 512 | |||||||
| 3 | X | X | X | 1 | 256 | |||||||
| 3 | X | X | X | 2 | 2,048–8,192 | |||||||
| 3 | X | X | X | 1 | 512 | |||||||
| 3c | X | X | 1 | 1,024 | ||||||||
| 3c | X | X | 5 | 32–2,048 | ||||||||
| 4c | X | X | X | 2 | 128–512 | |||||||
| 2 | X | X | 1 | 512 | ||||||||
| 3 | X | X | X | 1 | 1,024 | |||||||
| 3 | X | X | X | 1 | 512 | |||||||
| 3 | X | X | X | 1 | 256 | |||||||
| 3 | X | X | X | 3 | 512–1,024 | |||||||
The presence of a particular enzyme is indicated by the letter X.
Off-scale results were represented by the immediately higher dilution concentration.
Including species that putatively have derepressed AmpC.
Among comparators, meropenem displayed the greatest activity against ESBL- and AmpC-producing Enterobacteriaceae (Table 3); however, this carbapenem had limited or no activity against carbapenemase-producing strains. Ceftaroline-avibactam (MIC50/90, ≤1/2 μg/ml) displayed the greatest activity and potency of all antimicrobial agents when tested against strains producing serine carbapenemases with or without the coproduction of other enzymes. This subset of strains exhibited very high resistance rates for comparator agents (MICs for susceptible strains ranged from 0.0 to 9.4% for meropenem and 0.0 to 18.9% for ceftazidime) (Table 3). Troublesome β-lactamase-producing strains also displayed low rates of susceptibility to comparator agents (including other β-lactams) gentamicin and levofloxacin (Table 3). Ceftazidime and piperacillin-tazobactam had limited activity against all organism/enzyme categories within the Enterobacteriaceae (Table 3).
Among tested P. aeruginosa strains, ceftaroline-avibactam MICs ranged from 2 to >32 μg/ml. Wild-type strains (ceftazidime MIC, <16 μg/ml) (n = 10) showed the lowest ceftaroline-avibactam MIC results (4 and 16 μg/ml), and the highest MIC values for this compound were observed among the MBL-producing strains (>32 μg/ml) (n = 15). Similarly, wild-type Acinetobacter strains (ceftazidime MIC, ≤16 μg/ml) (n = 7) exhibited ceftaroline-avibactam values of 1 or 2 μg/ml (data not shown); however, all strains producing carbapenem-hydrolyzing oxacillinases (OXA-23, OXA-24/-40, or OXA-58) (n = 17) showed ceftaroline-avibactam MIC values of ≥32 μg/ml. These strains were often resistant to all comparator agents tested (8.3 to 41.7% susceptible to all compounds) (Table 3).
A collection of 110 S. aureus strains was included in the study; among these, 10 were methicillin susceptible (MSSA) and 100 were MRSA, including strains carrying SCCmec types I, II, II, IV, and IVa. Strains of types IV and IVa were combined for analysis, and representatives of the USA300 clone were also tested. All MRSA strains, except one (SCCmec type I), were inhibited at ≤2 μg/ml of ceftaroline-avibactam (MIC50, 1 μg/ml) (Tables 1 and 3). Furthermore, the ceftaroline MIC was not adversely affected by the addition of avibactam; MIC values for ceftaroline with and without avibactam were essentially identical or only varied within ±1 doubling dilution step for MSSA and MRSA strains (data not shown). Among MRSA strains (MICs, 0.25 μg/ml), the lowest ceftaroline-avibactam MIC values were observed for SCCmec type IV (MIC mode, 0.5 μg/ml), while the highest MIC values were observed among MRSA strains with SCCmec type I (MIC mode, 2 μg/ml) (Table 1).
Our results showed that, as demonstrated by Mushtaq et al. (13) with a more limited subset of β-lactamase-producing organisms, ceftaroline-avibactam is active against a wide variety of clinical serine-β-lactamase-carrying strains, including combinations of β-lactamase enzymes and KPC producers that are often highly resistant to other antimicrobial agents. Additionally, it has been shown that avibactam provided good coverage of K. pneumoniae KPC-producing isolates when combined with several β-lactam agents (9), and we expand this knowledge to include the ceftaroline-avibactam combination. The activity of this β-lactamase–β-lactamase inhibitor combination was limited against Gram-negative bacilli producing metallo-β-lactamases, OXA-producing MDR Acinetobacter spp., and P. aeruginosa strains.
Strains producing β-lactamases of various classes have been highlighted by the epidemiologic impact and antimicrobial therapy limitations that their dissemination can cause (17). Ceftaroline-avibactam appears to present broad antimicrobial coverage and greater activity than the β-lactams currently available for clinical use against organisms carrying β-lactamases such as serine carbapenemases (KPC). Furthermore, against MRSA, avibactam did not alter ceftaroline MIC values, and ceftaroline-avibactam was active against the more common strains in the community (e.g., CA-MRSA USA300 possessing SCCmec type IV).
ACKNOWLEDGMENTS
We express our appreciation to S. Benning and M. Janecheck for the preparation of the manuscript and to the JMI staff members for scientific assistance in performing this study.
This study was funded by educational/research grants from Cerexa, Inc. (Oakland, CA), a wholly owned subsidiary of Forest Laboratories, Inc. (New York, NY). Cerexa, Inc., was involved in the study design and the decision to present these results. Cerexa, Inc., had no involvement in the collection, analysis, or interpretation of data. Scientific Therapeutics Information, Inc., provided editorial assistance, which was funded by Forest Research Institute, Inc. JMI Laboratories, Inc., has received research and educational grants in 2009–2011 from Achaogen, Aires, the American Proficiency Institute (API), Anacor, Astellas, AstraZeneca, Bayer, bioMérieux, Cempra, Cerexa, Cosmo Technologies, Contrafect, Cubist, Daiichi, Dipexium, Enanta, Furiex, GlaxoSmithKline, Johnson & Johnson (Ortho McNeil), LegoChem Biosciences Inc., Meiji Seika Kaisha, Merck, Nabriva, Novartis, Paratek, Pfizer (Wyeth), PPD Therapeutics, Premier Research Group, Rempex, Rib-X Pharmaceuticals, Seachaid, Shionogi, Shionogi USA, The Medicines Co., Theravance, ThermoFisher, TREK Diagnostics, Vertex Pharmaceuticals, and some other corporations.
R.N.J. and H.S.S. are advisors/consultants for Astellas, Cubist, Pfizer, Cempra, Cerexa-Forest, J&J, and Theravance. We have no activity to declare with regard to speakers' bureaus and stock options.
Footnotes
Published ahead of print 25 June 2012
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