Ceftazidime-avibactam activity when tested against

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Jun 17, 2015 - expressed and the known β-lactamase inhibition profile of avibactam. (CLSI, 2015b; Huband et al., 2015). Ceftazidime-avibactam breakpoints.
Diagnostic Microbiology and Infectious Disease 83 (2015) 389–394

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Ceftazidime-avibactam activity when tested against ceftazidime-nonsusceptible Citrobacter spp., Enterobacter spp., Serratia marcescens, and Pseudomonas aeruginosa from Unites States medical centers (2011–2014) Helio S. Sader ⁎, Mariana Castanheira, David J. Farrell, Robert K. Flamm, Ronald N. Jones JMI Laboratories, North Liberty, IA, USA

a r t i c l e

i n f o

Article history: Received 3 April 2015 Received in revised form 9 June 2015 Accepted 13 June 2015 Available online 17 June 2015 Keywords: Derepressed AmpC Class C beta-lactamases cephalosporinases Gram-negative bacilli

a b s t r a c t A total of 6910 Enterobacteriaceae with inducible AmpC β-lactamases and 5328 Pseudomonas aeruginosa were collected from 71 US hospitals in 2011–2014 and were susceptibility tested using the reference broth microdilution method. Ceftazidime-avibactam demonstrated potent in vitro activity against all 3 Enterobacteriaceae genus groups evaluated, with MIC50/MIC90 values of 0.12/0.5 μg/mL for Enterobacter spp. and Serratia marcescens and 0.12/0.25 μg/mL for Citrobacter spp. (99.8–99.9% susceptibility rates at ≤8 μg/mL). Furthermore, 99.3% of ceftazidime-nonsusceptible Enterobacteriaceae isolates (1015 of 1022) were susceptible to ceftazidime-avibactam (MIC50/MIC90 of 0.25–0.5/1–2 μg/mL), whereas susceptibility rates for cefepime ranged from 45.9% (Citrobacter spp.) to 80.0% (S. marcescens). Ceftazidime-avibactam was also very active against P. aeruginosa (MIC50/MIC90, 2/4 μg/mL; 96.8% inhibited at ≤8 μg/mL). P. aeruginosa susceptibility rates to other antipseudomonal β-lactams ranged from 79.6% for piperacillin-tazobactam to 84.5% for cefepime. Ceftazidimeavibactam inhibited 67.4% of isolates at ≤8 μg/mL (MIC50/MIC90, 8/32 μg/mL) that were nonsusceptible to ceftazidime, cefepime, piperacillin-tazobactam, and meropenem. © 2015 Elsevier Inc. All rights reserved.

1. Introduction Enterobacteriaceae with inducible cephalosporinases, such as Citrobacter spp., Enterobacter spp., and Serratia spp., may become resistant to oxyimino-cephalosporins (cefotaxime and ceftazidime), 7-αmethoxy-cephalosporins (cephamycins such as cefoxitin), and monobactams by hyperproducing their chromosomal AmpC βlactamases (Goldstein, 2002; Jacoby, 2009). This phenomenon has led to a controversy about the optimal antimicrobial therapy for severe infections caused by these organisms (Livermore et al., 2004). Because of reports of clinical failure due to the selection of derepressed mutants during cephalosporin treatment (Chow et al., 1991), the Clinical and Laboratory Standards Institute (CLSI) recommends reporting β-lactam susceptibilities of these organisms with a footnote suggesting that resistance to third-generation cephalosporins may develop during therapy (CLSI, 2015a). Pseudomonas aeruginosa also carries an inducible AmpC cephalosporinase, and when the production of this β-lactamase is significantly elevated, P. aeruginosa exhibits resistance to most β-lactams currently available for clinical use, with the exception of the carbapenems. Moreover, carbapenem resistance can arise due to up-regulation

⁎ Corresponding author. Tel.: +1-319-665-3370; fax: +1-319-665-3371. E-mail address: [email protected] (H.S. Sader). http://dx.doi.org/10.1016/j.diagmicrobio.2015.06.008 0732-8893/© 2015 Elsevier Inc. All rights reserved.

of MexA-MexB-OprM and loss of OprD, which are considered the most prevalent mechanisms of carbapenem resistance in P. aeruginosa, and these mechanisms are usually associated with AmpC hyperproduction (Lister et al., 2009). Ceftazidime-avibactam is a combination agent consisting of the β-lactamase inhibitor avibactam and the broad-spectrum cephalosporin, ceftazidime (Zhanel et al., 2013). Avibactam acts as a reversible, covalent inhibitor and is a member of a novel class of non–β-lactam β-lactamase inhibitors, the diazabicyclooctanes (Coleman, 2011). Avibactam is more potent and has a broader spectrum of enzyme inhibition when compared to current clinically available β-lactamase inhibitors. Importantly, avibactam effectively inactivates class C β-lactamases, protecting companion β-lactams from hydrolysis by AmpC hyperproducing strains of Enterobacteriaceae and P. aeruginosa (Lahiri et al., 2014; Levasseur et al., 2014; Li et al., 2014). In addition to class C, avibactam inhibits class A (including KPC) and some D βlactamases, with low IC50 (concentration resulting in 50% inhibition) values and low turnover numbers. Thus, avibactam restores ceftazidime in vitro activity against Gram-negative organisms producing a variety of clinically relevant enzymes (Castanheira et al., 2014a). Ceftazidime-avibactam combination has been approved by the US Food and Drug Administration (FDA) for treatment of complicated intraabdominal infections and complicated urinary tract infections, including pyelonephritis, in patients with limited or no alternative treatment options (AVYCAZ, 2015). Ceftazidime-avibactam is also

0.5 0.25 1 0.5 0.5 2 0.25 0.25 1 4 32 1 (100.0) 105 (98.8) 77 (86.9) 0 (99.4) 308 (96.8) 120 (67.4)

1 (93.2)a 0 (99.9)a

0 (99.6) 1 (99.8)a

10 (99.6) 1 (99.7) 1 (100.0) 0 (90.9) 1 (99.9)

1 (99.4) 758 (91.0) 102 (37.1) 6 (98.8) 1681 (76.8) 42 (11.4)

3 (100.0) 1 (100.0)

31 (99.4) 25 (93.2)

1 (100.0) 2 (99.9) 3 (100.0)

1 (100.0)

17 (99.7) 1 (100.0) 16 (98.4) 8 (99.7) 4 (99.9) 4 (90.9) 6 (99.9)

111 (99.2) 14 (100.0) 97 (96.4) 23 (99.2) 16 (99.7) 7 (81.8) 24 (99.4) 4 (100.0) 20 (95.1) 2067 (45.3) 2 (0.8) 405 (96.4) 112 (99.5) 293 (84.5) 171 (97.7) 156 (98.6) 15 (65.9) 75 (97.7) 25 (99.7) 50 (82.9) 266 (6.5) 1 (0.3) 1023 (86.2) 766 (96.0) 257 (48.5) 544 (86.6) 536 (88.2) 8 (31.8) 220 (92.4) 165 (97.7) 55 (52.4) 48 (1.5) 1836 (60.5) 1727 (71.7) 109 (17.0) 691 (51.3) 685 (52.4) 6 (13.6) 538 (76.6) 511 (84.3) 27 (18.9) 21 (0.6) 534 (38.2) 530 (42.9) 4 (2.4) 10 (0.2)

565 (14.2) 536 (17.0) 29 (3.6) 99 (6.4) 99 (6.6)

Enterobacter spp. (3970) CAZ-S (3156) CAZ-NS (814) S. marcescens (1541) CAZ-S (1497) CAZ-NS (44) Citrobacter spp. (1399) CAZ-S (1235) CAZ-NS (164) P. aeruginosa (5328) CAZ, CPM, P/T and MER-NS (396)b

CAZ-S = ceftazidime susceptible (≤4 μg/mL (CLSI, 2015a)); CAZ-NS = ceftazidime nonsusceptible (≥8 μg/mL (CLSI, 2015a)); CAZ = ceftazidime; CPM = cefepime; P/T = piperacillin-tazobactam; MER = meropenem. a Underlined values indicate percentage inhibited at ceftazidime-avibactam susceptible breakpoint of ≤8 μg/mL (AVYCAZ, 2015). b Isolates nonsusceptible to ceftazidime (MIC, ≥16 μg/mL), cefepime (MIC, ≥16 μg/mL), piperacillin-tazobactam (MIC, ≥32 μg/mL), and meropenem (MIC, ≥4 μg/mL) according to the CLSI breakpoint criteria (CLSI, 2015a).

33 (100.0) 27 (100.0)

0.12 0.12 0.5 0.12 0.12 0.5 0.12 0.12 0.25 2 8 a

0 (99.9) 10 (99.9)

8 4 2 1 0.5 0.25 0.12 ≤0.06

No. of isolates (cumulative %) inhibited at ceftazidime-avibactam MIC (μg/mL) of

Organism

Table 1 Summary of ceftazidime-avibactam activity tested against Enterobacter spp., S. marcescens, Citrobacter spp., and P. aeruginosa isolates collected from US hospitals (2011–2014).

a

16

2 (100.0)

32

N32

50%

90%

H.S. Sader et al. / Diagnostic Microbiology and Infectious Disease 83 (2015) 389–394 MIC (μg/mL)

390

under clinical development for treatment of nosocomial pneumonia (http://clinicaltrials.gov; NCT01808092). Previous reports from the International Network for Optimal Resistance Monitoring (INFORM) program have provided analyses of ceftazidime-avibactam activity against collections of bacterial isolates collected during the initial years of the program and stratified by site of infection and year of isolation (Flamm et al., 2014; Sader et al., 2014, 2015). In this report, we evaluated the activity of ceftazidime-avibactam and comparator agents against a large collection of Enterobacteriaceae isolates from species that produce inducible cephalosporinases (AmpC β-lactamase) and P. aeruginosa collected over 4 years (2011–2014) of the INFORM Program in the United States. 2. Materials and methods 2.1. Bacterial isolates A total of 12,238 Gram-negative organisms, including 6910 Enterobacteriaceae and 5328 P. aeruginosa, were collected from 71 US hospitals from January 2011 to October 2014 as part of the INFORM program. These isolates were collected from bloodstream, respiratory tract, and skin and skin structure infections, according to defined protocols (Sader et al., 2014). Only clinically significant isolates were included in the study (1 per patient episode). Species identification was confirmed when necessary by matrix-assisted laser desorption ionization time-of-flight mass spectrometry using the Bruker Daltonics MALDI Biotyper (Billerica, MA, USA) by following manufacturer instructions. 2.2. Antimicrobial susceptibility testing All isolates were susceptibility tested using the reference broth microdilution method as described by the CLSI (2015b). Ceftazidime was combined with avibactam at a fixed concentration of 4 μg/mL. The combination of ceftazidime plus a constant 4 μg/mL of avibactam was chosen for susceptibility testing over alternatives and standardized by CLSI and EUCAST, based on the pharmacokinetic of avibactam and the ability of this combination to separate isolates that have been predefined as “susceptible” or “resistant” based on the β-lactamases expressed and the known β-lactamase inhibition profile of avibactam (CLSI, 2015b; Huband et al., 2015). Ceftazidime-avibactam breakpoints approved by the US-FDA (≤8 μg/mL for susceptible and ≥16 μg/mL for resistance) were applied for all Enterobacteriaceae species and P. aeruginosa (AVYCAZ, 2015). All Enterobacteriaceae isolates displaying ceftazidime-avibactam MIC of N8 μg/mL were screened for the presence of metallo-β-lactamase and serine-carbapenemase encoding gene families blaIMP, blaVIM, blaNDM, blaKPC, blaOXA-48, blaGES, blaIMI, blaNMC-A, and blaSME by PCR as previously described (Kaiser et al., 2013). Categorical interpretations for all comparator agents were those found in CLSI document M100-S25 (CLSI, 2015a) and EUCAST breakpoint tables (EUCAST, 2015), except for tigecycline where the US-FDA breakpoints were applied (Tygacil®, 2012). Quality control (QC) was performed using Escherichia coli ATCC 25922 and 35218, Klebsiella pneumoniae 700603, and P. aeruginosa ATCC 27853. All QC MIC results were within acceptable ranges as published in CLSI documents (CLSI, 2015a). 3. Results Ceftazidime-avibactam demonstrated potent in vitro activity against all 3 Enterobacteriaceae genus groups evaluated, with MIC50 results at 0.12 μg/mL, MIC90 values at 0.25–0.5 μg/mL, and ≥99.8% susceptible at ≤8 μg/mL (AVYCAZ, 2015) (Table 1). When tested against ceftazidime alone, the susceptibility (CLSI criteria) rate was lowest for Enterobacter spp. (79.5%), followed by Citrobacter spp. (88.3%) and Serratia spp. (97.1%; Table 2). Almost 4000 Enterobacter spp. isolates were tested against ceftazidime-avibactam (MIC50/MIC90, 0.12/0.5 μg/mL), and only 3

H.S. Sader et al. / Diagnostic Microbiology and Infectious Disease 83 (2015) 389–394

391

Table 2 Activity of ceftazidime-avibactam and comparator antimicrobial agents when tested against Enterobacter spp, S. marcescens, Citrobacter spp., and P. aeruginosa from US hospitals (2011–2014). Antimicrobial agent

MIC (μg/mL)

%S/%I/%R

50%

90%

Range

CLSIa

EUCASTa

0.12 0.25 ≤0.5 0.25 16 2 ≤0.06 ≤0.12 ≤1 0.25 0.5

0.5 N32 2 N8 N32 64 ≤0.06 0.5 ≤1 0.5 N8

≤0.03 to 32 0.03 to N32 ≤0.5 to N16 ≤0.06 to N8 ≤0.25 to N32 ≤0.5 to N64 ≤0.06 to N8 ≤0.12 to N4 ≤1 to N8 ≤0.015 to 4 0.12 to N8

99.9/0.0/0.1c 79.5/1.5/19.0 93.3/4.2/2.5 75.6/2.0/22.4 31.3/24.2/44.5 84.0/9.4/6.6 99.4/0.1/0.5 95.7/1.2/3.1 96.4/0.6/3.0 99.1/0.9/0.0d -/-/-

-/-/77.1/2.4/20.5 89.5/6.9/3.6 75.6/2.0/22.4 31.3/0.0/68.7 80.0/4.0/16.0 99.5/0.4/0.1 94.7/1.0/4.3 96.0/0.4/3.6 95.7/3.4/0.9 84.7/0.0/15.3

0.25 N32 2 N8 N32 64 ≤0.06 0.5 ≤1 0.25 0.5

1 N32 16 N8 N32 N64 0.12 N4 N8 1 N8

≤0.015 to 16 8 to N32 ≤0.5 to N16 2 to N8 4 to N32 1 to N64 ≤0.06 to N8 ≤0.12 to N4 ≤1 to N8 0.06 to 4 0.12 to N8

99.6/0.0/0.4c 0.0/3.0/97.0 68.3/19.6/12.1 0.0/1.8/98.2 1.8/1.2/97.0 27.0/35.0/38.0 95.7/1.2/3.1 71.3/9.8/18.9 74.4/1.2/24.4 99.4/0.6/0.0d -/-/-

-/-/0.0/0.0/100.0 49.8/32.7/17.6 0.0/1.8/98.2 1.8/0.0/98.2 12.9/14.1/73.0 96.9/1.9/1.2 61.0/10.3/28.7 72.0/2.4/25.6 93.9/5.5/0.6 83.0/0.0/17.0

0.12 0.25 ≤0.5 0.25 N8 32 2 ≤0.06 ≤0.12 ≤1 0.5 N8

0.5 0.5 ≤0.5 2 N8 N32 8 ≤0.06 1 ≤1 1 N8

≤0.03 to 16 0.03 to N32 ≤0.5 to N16 ≤0.06 to N8 2 to N8 1 to N32 ≤0.5 to N64 ≤0.06 to N8 ≤0.12 to N4 ≤1 to N8 0.12 to N4 0.25 to N8

99.8/0.0/0.2c 97.1/0.5/2.4 97.7/1.7/0.6 89.6/2.5/7.9 3.3/0.0/96.7 7.7/15.4/76.9 96.4/2.4/1.2 99.2/0.2/0.6 96.0/2.0/2.0 97.6/0.6/1.8 99.1/0.7/0.2d -/-/-

-/-/96.2/0.9/2.9 97.2/1.4/1.4 89.6/2.5/7.9 3.3/0.0/96.7 7.7/0.0/92.3 94.7/1.7/3.6 99.4/0.2/0.4 93.0/3.0/4.0 96.7/0.9/2.4 95.5/3.6/0.9 6.0/0.0/94.0

0.5 32 4 N8 N32 16 ≤0.06 1 2 1 N8

2 N32 16 N8 N32 N64 2 N4 N8 2 N8

0.12–16 8 to N32 ≤0.5 to N16 0.25 to N8 16 to N32 1 to N64 ≤0.06 to N8 ≤0.12 to N4 ≤1 to N8 0.12 to 4 1 to N8

93.2/0.0/6.8c 0.0/15.9/84.1 45.9/35.1/18.9 4.7/0.0/95.3 0.0/4.5/95.5 52.3/13.6/34.1 86.4/4.5/9.1 68.2/13.6/18.2 54.5/13.7/31.8 97.7/2.3/0.0d -/-/-

-/-/0.0/0.0/100.0 37.8/21.6/40.5 4.7/0.0/95.3 0.0/0.0/100.0 47.7/4.6/47.7 90.9/6.8/2.3 50.0/18.2/31.8 52.3/2.2/45.5 86.4/11.3/2.3 27.8/0.0/72.2

0.12 0.25 ≤0.5 0.12 4 2 ≤0.06 ≤0.12 ≤1 0.12 0.5

0.25 16 ≤0.5 N8 N32 16 ≤0.06 1 ≤1 0.5 1

≤0.03 to 16 0.03 to N32 ≤0.5 - N16 ≤0.06 to N8 ≤0.25 to N32 ≤0.5 to N64 ≤0.06 to N8 ≤0.12 to N4 ≤1 to N8 0.03–4 0.12 to N8

99.9/0.0/0.1c 88.3/0.3/11.4 97.3/1.0/1.6 87.7/0.3/12.0 76.3/7.8/15.9 90.5/4.7/4.8 99.4/0.2/0.4 94.8/1.8/3.4 95.0/0.1/4.9 99.7/0.3/0.0d -/-/-

-/-/86.7/1.6/11.7 95.3/2.8/1.9 87.7/0.3/12.0 76.3/0.0/23.7 85.6/4.9/9.5 99.6/0.3/0.1 92.1/2.7/5.2 94.6/0.4/5.0 98.2/1.5/0.3 99.8/0.0/0.2

0.25 N32 1 N8 N32 64 ≤0.06 0.5 ≤1 0.25

1 N32 16 N8 N32 N64 0.12 N4 N8 1

≤0.015 to 16 8 to N32 ≤0.5 to N16 2 to N8 4 to N32 1 to N64 ≤0.06 to N8 ≤0.12 to N4 ≤1 to N8 0.06–4

99.4/0.0/0.6c 0.0/3.0/97.0 80.0/7.2/12.8 0.0/1.8/98.2 1.8/1.2/97.0 27.0/35.0/38.0 95.7/1.2/3.1 71.3/9.8/18.9 74.4/1.2/24.4 99.4/0.6/0.0d

-/-/0.0/0.0/100.0 64.0/21.6/14.4 0.0/1.8/98.2 1.8/0.0/98.2 12.9/14.1/73.0 96.9/1.9/1.2 61.0/10.3/28.7 72.0/2.4/25.6 93.9/5.5/0.6

b

Enterobacter spp. (3970) Ceftazidime-avibactam Ceftazidime Cefepime Ceftriaxone Ampicillin/sulbactam Piperacillin-tazobactam Meropenem Levofloxacin Gentamicin Tigecycline Colistin Ceftazidime nonsusceptiblee (814) Ceftazidime-avibactam Ceftazidime Cefepime Ceftriaxone Ampicillin/sulbactam Piperacillin-tazobactam Meropenem Levofloxacin Gentamicin Tigecycline Colistin S. marcescens (1541) Ceftazidime-avibactam Ceftazidime Cefepime Ceftriaxone Ampicillin Ampicillin/sulbactam Piperacillin-tazobactam Meropenem Levofloxacin Gentamicin Tigecycline Colistin Ceftazidime nonsusceptible (44) Ceftazidime-avibactam Ceftazidime Cefepime Ceftriaxone Ampicillin/sulbactam Piperacillin-tazobactam Meropenem Levofloxacin Gentamicin Tigecycline Colistin Citrobacter spp. (1399)f Ceftazidime-avibactam c Ceftazidime Cefepime Ceftriaxone Ampicillin/sulbactam Piperacillin-tazobactam Meropenem Levofloxacin Gentamicin Tigecycline Colistin Ceftazidime nonsusceptibleg (164) Ceftazidime-avibactam Ceftazidime Cefepime Ceftriaxone Ampicillin/sulbactam Piperacillin-tazobactam Meropenem Levofloxacin Gentamicin Tigecycline

(continued on next page)

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Table 2 (continued) Antimicrobial agent

Colistin P. aeruginosa (5328) Ceftazidime-avibactam Ceftazidime Cefepime Piperacillin-tazobactam Meropenem Levofloxacin Gentamicin Amikacin Tobramycin Colistin CAZ, CPM, P/T, and MER nonsusceptible (396)h Ceftazidime/avibactam Levofloxacin Gentamicin Amikacin Tobramycin Colistin

MIC (μg/mL)

%S/%I/%R

50%

90%

Range

CLSIa

0.5

1

0.25–1

-/-/-

EUCASTa 100.0/0.0/0.0 c

2 2 2 8 0.5 0.5 ≤1 2 0.5 1

4 32 16 N64 8 N4 8 8 4 2

0.03 to N32 0.06 to N32 ≤0.5 to N16 ≤0.5 to N64 ≤0.06 to N8 ≤0.12 to N4 ≤1 to N8 ≤0.25 to N32 ≤0.12 - N16 ≤0.5 to N8

96.8/0.0/3.2 83.9/3.8/12.3 84.5/8.2/7.3 79.6/9.0/11.4 82.5/5.6/11.9 75.0/6.4/18.6 89.1/3.2/7.7 97.4/1.1/1.5 91.7/0.2/8.1 N99.9/0.0/b0.1

-/-/83.9/0.0/16.1 84.5/0.0/15.5 79.6/0.0/20.4 82.5/11.5/6.0 66.3/8.7/25.0 89.1/0.0/10.9 94.2/3.2/2.6 91.7/0.0/8.3 N99.9/0.0/b0.1

8 N4 4 4 1 1

32 N4 N8 32 N16 2

0.5 to N32 ≤0.12 to N4 ≤1 to N8 ≤0.25 to N32 ≤0.12 to N16 ≤0.5 to 2

67.4/0.0/32.6c 17.9/8.4/73.7 53.3/7.8/38.9 87.4/5.3/7.3 62.6/3.8/33.6 100.0/0.0/0.0

-/-/11.9/6.0/82.1 53.3/0.0/46.7 77.8/9.6/12.6 62.6/0.0/37.4 100.0/0.0/0.0

a

Criteria as published by the CLSI (CLSI, 2015a) or US-FDA when CLSI breakpoints are not available and EUCAST (EUCAST, 2015). Includes: E. aerogenes (1050 strains), E. cloacae (2517 strains), and E. cloacae species complex (403 strains). Ceftazidime-avibactam breakpoints approved by the US-FDA (≤8 μg/mL for susceptible and ≥16 μg/mL for resistance) were applied (AVYCAZ, 2015). d US-FDA breakpoints were applied when available (Tygacil®, 2012). e Includes E. aerogenes (215 strains), E. cloacae (526 strains), and E. cloacae species complex (73 strains). f Includes C. freundii (145 strains), C. freundii species complex (10 strains), and Citrobacter koseri (9 strains). g Includes C. freundii (692 strains), C. freundii species complex (59 strains), and C. koseri (648 strains). h Isolates nonsusceptible to ceftazidime (MIC, ≥16 μg/mL), cefepime (MIC, ≥16 μg/mL), piperacillin-tazobactam (MIC, ≥32 μg/mL), and meropenem (MIC, ≥4 μg/mL) according to the CLSI breakpoint criteria (CLSI, 2015a). b c

isolates exhibited an MIC at N8 μg/mL (0.08%), 1 Enterobacter aerogenes with ceftazidime-avibactam MIC of 16 μg/mL isolated in 2012 and 2 Enterobacter cloacae isolates from 2013 and 2014 (Table 3). PCR screening for carbapenemases was negative for all 3 strains. Meropenem was also very active against Enterobacter spp. (MIC50/MIC90, ≤0.06/ ≤0.06 μg/mL; 99.4% susceptible), whereas cefepime (MIC50/MIC90, ≤0.5/2 μg/mL) was active against 93.3% of isolates at the CLSI susceptible breakpoint of ≤2 μg/mL. Other compounds with good in vitro activity (N90% susceptibility) against Enterobacter spp. include levofloxacin (MIC50/MIC90, ≤0.12/0.5 μg/mL; 95.7% susceptible), gentamicin (MIC50/ MIC90, ≤1/≤1 μg/mL; 96.4% susceptible), and tigecycline (MIC50/MIC90, 0.25/0.5 μg/mL; 99.1% susceptible; Table 2). Ceftazidime-avibactam (MIC50/MIC90, 0.25/1 μg/mL; 99.6% susceptible), meropenem (MIC50/MIC90, ≤0.06/0.12 μg/mL; 95.7% susceptible), and tigecycline (MIC50/MIC90, 0.25/1 μg/mL; 99.4% susceptible) were the most active compounds tested against ceftazidime-nonsusceptible Enterobacter spp. (n = 814), and all other compounds exhibited susceptibility rates at b75.0% (Table 2). Cefepime (MIC50/MIC90, 2/16 μg/mL) was 8- to 16-fold less active than ceftazidime-avibactam against these organisms and inhibited only 68.3% of isolates at the CLSI susceptible breakpoint (Table 2). Ceftazidime-avibactam was also very active against S. marcescens (MIC50/MIC90, 0.12/0.5 μg/mL; 99.8% susceptible), with only 3 nonsusceptible isolates, all with ceftazidime-avibactam MIC of 16 μg/mL, which is only 1 doubling dilution above the susceptible breakpoint (Table 3). The ceftazidime-avibactam–nonsusceptible strains again exhibited negative PCR results for the carbapenemase families tested and were isolated from patients with pneumonia in 2012 (Hackensack, NJ), 2013 (New York, NY) and 2014 (Hackensack, NJ) (Table 3). Other β-lactams that demonstrated good activity against S. marcescens include meropenem (MIC50/MIC90, ≤0.06/≤0.06 μg/mL; 99.2% susceptible), cefepime (MIC50/MIC90, ≤0.5/≤0.5 μg/mL; 97.7% susceptible), ceftazidime (MIC50/MIC90, 0.25/0.5 μg/mL; 97.1% susceptible), and piperacillin-tazobactam (MIC50/MIC90, 2/8 μg/mL; 96.4% susceptible; Table 2). In contrast, ceftazidime-avibactam (MIC50/MIC90, 0.5/ 2 μg/mL; 93.2% susceptible) was the only β-lactam compound active

against N90.0% of ceftazidime-nonsusceptible S. marcescens isolates (Table 2). Only 1 Citrobacter spp. isolate (0.07%) exhibited a ceftazidimeavibactam MIC at N8 μg/mL, a Citrobacter freundii isolated in 2012 from a patient with pneumonia (Table 3). Meropenem (MIC50/MIC90, ≤0.06/ ≤0.06 μg/mL; 99.4% susceptible) and cefepime (MIC50/MIC90, ≤0.5/ ≤0.5 μg/mL; 97.3% susceptible) were also very active against Citrobacter spp. isolates. Furthermore, ceftazidime-avibactam (MIC50/MIC90, 0.25/ 1 μg/mL; 99.4% susceptible) remained very active against ceftazidimenonsusceptible Citrobacter spp. isolates (Table 2). Ceftazidime-avibactam (MIC50/MIC90, 2/4 μg/mL; 96.8% susceptible at ≤8 μg/mL; Table 1) showed greater in vitro activity than ceftazidime tested alone (MIC50/MIC90, 2/32 μg/mL; 83.9% susceptible at ≤8 μg/mL) when tested against the entire collection of P. aeruginosa (n = 5328; Table 2). Overall susceptibility (by CLSI criteria; Table 2) rates for cefepime (84.5%) and meropenem (82.5%) were similar to that of ceftazidime (83.9%), much lower than that of ceftazidime-avibactam at ≤8 μg/mL (96.8%) and slightly higher than that of piperacillintazobactam (79.6%; Table 2). Among the non–β-lactam comparator agents, amikacin (MIC50/MIC90, 2/8 μg/mL, 97.4% susceptible at ≤16 μg/mL [CLSI criteria]) and colistin (MIC50/MIC90, 1/2 μg/mL, N99.9% susceptible at ≤2 μg/mL [CLSI]) were the most active compounds (Table 2). Moreover, ceftazidime-avibactam inhibited 67.4% of P. aeruginosa isolates nonsusceptible to ceftazidime (MIC, ≥16 μg/mL), cefepime (MIC, ≥16 μg/mL), piperacillin-tazobactam (MIC, ≥32 μg/mL), and meropenem (MIC, ≥4 μg/mL) at ≤8 μg/mL (Tables 1 and 2). 4. Discussion Bacterial organisms expressing derepressed AmpC β-lactamases are often resistant to multiple agents, making the selection of an effective antimicrobial treatment very difficult. These organisms are generally resistant to most cephalosporins and piperacillin-tazobactam, and whether cefepime can be used remains the subject of some debate. Cefepime is a poor inducer of AmpC β-lactamase, rapidly penetrates through the outer cell membrane, and is weakly hydrolyzed by the enzyme

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393

Table 3 Antimicrobial susceptibility and demographic information of ceftazidime-avibactam–nonsusceptible isolates. MIC (μg/mL) Organism

CAZ-AVI

CAZ

MER

P/T

AZT

GEN

LEV

C. freundii E. aerogenes E. cloacae E. cloacae S. marcescens S. marcescens S. marcescens

16 16 32 16 16 16 16

N32 N32 N32 N32 32 32 32

0.5 ≤0.06 1 4 1 1 1

N64 N64 N64 N64 N64 N64 N64

N16 N16 N16 N16 8 4 16

N8 ≤1 ≤1 ≤1 ≤1 ≤1 ≤1

4.1 0.119 4.1 4.1 4 4 2

Infection type

Year of isolation

Pneumonia BSI UTI IAI Pneumonia Pneumonia Pneumonia

2012 2012 2013 2014 2012 2013 2014

City, state Birmingham, AL Little Rock, AR New York, NY Indianapolis, IN Hackensack, NJ New York, NY Hackensack, NJ

Abbreviations: CAZ-AVI = ceftazidime-avibactam (avibactam at fixed 4 μg/mL); CAZ = ceftazidime; MER = meropenem; P/T = piperacillin-tazobactam; AZT = aztreonam; GEN = gentamicin; LEV = levofloxacin; BSI = bloodstream infection; UTI = urinary tract infection; and IAI = intraabdominal infection.

(Endimiani et al., 2008); thus, AmpC-producing organisms may test susceptible to cefepime (Pfaller et al., 2006). However, in the present study, cefepime exhibited limited activity against ceftazidime-nonsusceptible Enterobacteriaceae, especially Enterobacter spp. (MIC50/MIC90, 2/ 16 μg/mL; 68.3% susceptible [CLSI]) and S. marcescens (MIC50/MIC90, 4/ 16 μg/mL; 45.9% susceptible [CLSI]) isolates. In contrast, ceftazidimeavibactam retained good activity against these organisms (MIC50/ MIC90, 0.12–0.5/0.25–2 μg/mL) and inhibited 99.3% (1015 of 1022) of ceftazidime-nonsusceptible Enterobacteriaceae at the susceptible breakpoint of MIC of ≤8 μg/mL. Of note, all 7 ceftazidime-avibactam– nonsusceptible Enterobacteriaceae isolates had negative PCR results for the following carbapenemase encoding gene families: blaIMP, blaVIM, blaNDM, blaKPC, blaOXA-48, blaGES, blaIMI, blaNMC-A, and blaSME. β-Lactam compounds, including cephalosporins, carbapenems, and antipseudomonal penicillins, have become key agents for the treatment of infections caused by P. aeruginosa. However, the overall prevalence of resistance to these first-line agents has continuously increased in recent years, and infections caused by multidrug-resistant P. aeruginosa strains are associated with significant morbidity and mortality (Lister et al., 2009). While the acquisition of potent β-lactamases, such as the metallo-β-lactamases, represents an important threat in some geographic regions, β-lactam resistance among P. aeruginosa in the United States is still much more commonly associated with the selection of a complex range of chromosomal mutations, including those causing hyperproduction of the chromosomal AmpC, repression or inactivation of the porin OprD, and up-regulation of one of the several efflux pumps (Caille et al., 2014; Castanheira et al., 2014b; Lister et al., 2009). Furthermore, two or more of these mechanisms are frequently combined, leading to the emergence of resistance to all currently available β-lactams (Castanheira et al., 2014b). In the present study, nonsusceptibility to the key antipseudomonal β-lactams (i.e., ceftazidime, cefepime, piperacillin-tazobactam, and meropenem) ranged from 15.5% to 20.4%. Importantly, approximately two-thirds (67.4%) of the isolates expressing decreased susceptibility to all these 4 agents remained susceptible to ceftazidime-avibactam. Inhibition of class C β-lactamases is one of the most significant and unique attributes of avibactam (Lahiri et al., 2014). The results of the present study indicate that ceftazidime-avibactam is highly active against Enterobacteriaceae organisms producing derepressed AmpC and remained active against a large proportion of P. aeruginosa strains resistant to multiple antipseudomonal β-lactams. It is important to note that the in vitro activity of ceftazidime-avibactam against AmpCproducing/ceftazidime-resistant Enterobacteriaceae and P. aeruginosa translates into good efficacy in animal infection models (Housman et al., 2014; Levasseur et al., 2014). Results from phase 2 and 3 clinical trials also indicates that ceftazidime-avibactam is as effective as carbapenem comparator therapy for complicated intraabdominal infection and complicated urinary tract infection, including infection caused by ceftazidime-resistant Gram-negative bacilli (Zhanel et al., 2013). Thus, ceftazidime-avibactam could become a very valuable addition to the shrinking group of antimicrobial agents currently available for the

treatment of serious infections caused by emerging multidrugresistant Gram-negative bacilli. Disclosures This study was supported by Cerexa, a wholly owned subsidiary of Forest Laboratories. Forest Laboratories was involved in the design and decision to present these results. Forest Laboratories had no involvement in the collection, analysis, and interpretation of data. JMI Laboratories has also received research and educational grants in 2012–2014 from Achaogen, Actelion, Affinium, American Proficiency Institute, AmpliPhi Bio, Anacor, Astellas, AstraZeneca, Basilea, BioVersys, Cardeas, Cempra, Cubist, Daiichi, Dipexium, Durata, Exela, Fedora, Furiex, Genentech, GlaxoSmithKline, Janssen, Johnson & Johnson, Medpace, Meiji Seika Kaisha, Melinta, Merck, Methylgene, Nabriva, Nanosphere, Novartis, Pfizer, Polyphor, Rempex, Roche, Seachaid, Shionogi, Synthes, The Medicines Co., Theravance, Thermo Fisher, Venatorx, Vertex, Waterloo, and Wockhardt. Some JMI employees are advisors/consultants for Astellas, Cubist, Pfizer, Cempra, Cerexa-Forest, and Theravance. In regard to speakers' bureaus and stock options, none to declare. Acknowledgments The authors would like to thank all participants of the INFORM program for providing bacterial isolates. References AVYCAZ. AVYCAZ Prescribing Information. Forest Pharmaceuticals, Cincinnati, OH. Available at http://www.avycaz.com, 2015. (Accessed March 23, 2015). Caille O, Zincke D, Merighi M, Balasubramanian D, Kumari H, Kong KF, et al. Structural and functional characterization of Pseudomonas aeruginosa global regulator AmpR. J Bacteriol 2014;196:3890–902. Castanheira M, Farrell SE, Krause KM, Jones RN, Sader HS. Contemporary diversity of βlactamases among Enterobacteriaceae in the nine United States census regions and ceftazidime-avibactam activity tested against isolates producing the most prevalent β-lactamase groups. Antimicrob Agents Chemother 2014a;58:833–8. Castanheira M, Mills JC, Farrell DJ, Jones RN. Mutation-driven β-lactam resistance mechanisms among contemporary ceftazidime non-susceptible Pseudomonas aeruginosa isolates from USA hospitals. Antimicrob Agents Chemother 2014b;58:6844–50. Chow JW, Fine MJ, Shlaes DM, Quinn JP, Hooper DC, Johnson MP, et al. Enterobacter bacteremia: clinical features and emergence of antibiotic resistance during therapy. Ann Intern Med 1991;115:585–90. Clinical and Laboratory Standards Institute (CLSI). . M100-S25 Performance standards for antimicrobial susceptibility testing: 25th informational supplement. Wayne, PA: CLSI; 2015a. Clinical and Laboratory Standards Institute (CLSI). Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard-tenth edition. M07-A10 Wayne, PA: CLSI; 2015b. Coleman K. Diazabicyclooctanes: a potent new class of non-β-lactam β-lactamase inhibitors. Curr Opin Microbiol 2011;14:550–5. Endimiani A, Perez F, Bonomo RA. Cefepime: a reappraisal in an era of increasing antimicrobial resistance. Expert Rev Anti Infect Ther 2008;6:805–24. EUCAST. Breakpoint tables for interpretation of MICs and zone diameters. Version 5.0, January 2015. Available at http://www.eucast.org/clinical_breakpoints/, 2015. (Accessed January 2015).

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