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In Vitro Activity of Aztreonam-Avibactam against Enterobacteriaceae and Pseudomonas aeruginosa Isolated by Clinical Laboratories in 40 Countries from 2012 to 2015.

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  • 1. Department of Medical Microbiology, College of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada.
      Authors
    • Karlowsky JA 1
    (1 author)
  • 2. International Health Management Associates, Inc., Schaumburg, Illinois, USA
      Authors
    • Kazmierczak KM 2
    (1 author)
  • 3. AstraZeneca Pharmaceuticals, Waltham, Massachusetts, USA.
      Authors
    • de Jonge BLM 3
    • Bradford PA 3
    (2 authors)
  • 4. International Health Management Associates, Inc., Schaumburg, Illinois, USA.
      Authors
    • Hackel MA 4
    • Sahm DF 4
    (2 authors)

ORCIDs linked to this article

Antimicrobial Agents and Chemotherapy, 24 Aug 2017, 61(9):e00472-17
https://doi.org/10.1128/aac.00472-17 PMID: 28630192 PMCID: PMC5571336

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Abstract 

The combination of the monobactam aztreonam and the non-β-lactam β-lactamase inhibitor avibactam is currently in clinical development for the treatment of serious infections caused by metallo-β-lactamase (MBL)-producing Enterobacteriaceae, a difficult-to-treat subtype of carbapenem-resistant Enterobacteriaceae for which therapeutic options are currently very limited. The present study tested clinically significant isolates of Enterobacteriaceae (n = 51,352) and Pseudomonas aeruginosa (n = 11,842) collected from hospitalized patients in 208 medical center laboratories from 40 countries from 2012 to 2015 for in vitro susceptibility to aztreonam-avibactam, aztreonam, and comparator antimicrobial agents using a standard broth microdilution methodology. Avibactam was tested at a fixed concentration of 4 μg/ml in combination with 2-fold dilutions of aztreonam. The MIC90s of aztreonam-avibactam and aztreonam were 0.12 and 64 μg/ml, respectively, for all Enterobacteriaceae isolates; >99.9% of all isolates and 99.8% of meropenem-nonsusceptible isolates (n = 1,498) were inhibited by aztreonam-avibactam at a concentration of ≤8 μg/ml. PCR and DNA sequencing identified 267 Enterobacteriaceae isolates positive for MBL genes (NDM, VIM, IMP); all Enterobacteriaceae carrying MBLs demonstrated aztreonam-avibactam MICs of ≤8 μg/ml and a MIC90 of 1 μg/ml. Against all P. aeruginosa isolates tested, the MIC90 of both aztreonam-avibactam and aztreonam was 32 μg/ml; against MBL-positive P. aeruginosa isolates (n = 452), MIC90 values for aztreonam-avibactam and aztreonam were 32 and 64 μg/ml, respectively. The current study demonstrated that aztreonam-avibactam possesses potent in vitro activity against a recent, sizeable global collection of Enterobacteriaceae clinical isolates, including isolates that were meropenem nonsusceptible, and against MBL-positive isolates of Enterobacteriaceae, for which there are few treatment options.

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Antimicrob Agents Chemother. 2017 Sep; 61(9): e00472-17.
Published online 2017 Aug 24. Prepublished online 2017 Jun 19. https://doi.org/10.1128/AAC.00472-17
PMCID: PMC5571336
PMID: 28630192

In Vitro Activity of Aztreonam-Avibactam against Enterobacteriaceae and Pseudomonas aeruginosa Isolated by Clinical Laboratories in 40 Countries from 2012 to 2015

James A. Karlowsky,a Krystyna M. Kazmierczak,corresponding authorb Boudewijn L. M. de Jonge,c,* Meredith A. Hackel,b Daniel F. Sahm,b and Patricia A. Bradfordc,*
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ABSTRACT

The combination of the monobactam aztreonam and the non-β-lactam β-lactamase inhibitor avibactam is currently in clinical development for the treatment of serious infections caused by metallo-β-lactamase (MBL)-producing Enterobacteriaceae, a difficult-to-treat subtype of carbapenem-resistant Enterobacteriaceae for which therapeutic options are currently very limited. The present study tested clinically significant isolates of Enterobacteriaceae (n = 51,352) and Pseudomonas aeruginosa (n = 11,842) collected from hospitalized patients in 208 medical center laboratories from 40 countries from 2012 to 2015 for in vitro susceptibility to aztreonam-avibactam, aztreonam, and comparator antimicrobial agents using a standard broth microdilution methodology. Avibactam was tested at a fixed concentration of 4 μg/ml in combination with 2-fold dilutions of aztreonam. The MIC90s of aztreonam-avibactam and aztreonam were 0.12 and 64 μg/ml, respectively, for all Enterobacteriaceae isolates; >99.9% of all isolates and 99.8% of meropenem-nonsusceptible isolates (n = 1,498) were inhibited by aztreonam-avibactam at a concentration of ≤8 μg/ml. PCR and DNA sequencing identified 267 Enterobacteriaceae isolates positive for MBL genes (NDM, VIM, IMP); all Enterobacteriaceae carrying MBLs demonstrated aztreonam-avibactam MICs of ≤8 μg/ml and a MIC90 of 1 μg/ml. Against all P. aeruginosa isolates tested, the MIC90 of both aztreonam-avibactam and aztreonam was 32 μg/ml; against MBL-positive P. aeruginosa isolates (n = 452), MIC90 values for aztreonam-avibactam and aztreonam were 32 and 64 μg/ml, respectively. The current study demonstrated that aztreonam-avibactam possesses potent in vitro activity against a recent, sizeable global collection of Enterobacteriaceae clinical isolates, including isolates that were meropenem nonsusceptible, and against MBL-positive isolates of Enterobacteriaceae, for which there are few treatment options.

KEYWORDS: aztreonam-avibactam, Gram-negative bacteria, metallo-β-lactamase, surveillance studies

INTRODUCTION

Carbapenem resistance in Enterobacteriaceae has been reported to arise from one or a combination of the following mechanisms: carbapenemase production; reduced outer membrane permeability, often in the milieu of an extended-spectrum β-lactamase (ESBL), AmpC, or multiple β-lactamases; efflux across the outer membrane; or mutations in penicillin binding proteins (PBP) (1,–5). Of these mechanisms, carbapenemase production is regarded as the most worrisome because many of the genes encoding carbapenemases reside on plasmids, creating the potential for horizontal spread. Carbapenemases are a diverse collection of β-lactamases and include members of Ambler class A β-lactamases (e.g., Klebsiella pneumoniae carbapenemase [KPC], GES), class B metallo-β-lactamases (MBLs; e.g., NDM, VIM, IMP, SPM), and class D β-lactamases (e.g., OXA) (6). MBLs in particular are problematic because they hydrolyze all classes of β-lactams except monobactams (aztreonam) and are not inhibited by classic serine β-lactamase inhibitors (clavulanic acid, tazobactam, sulbactam).

Aztreonam, a monobactam, is a unique agent among currently marketed β-lactams, in that it is stable to hydrolysis by MBLs. Unfortunately, aztreonam is easily inactivated by Ambler class A β-lactamases, including ESBLs and KPCs, and plasmid-encoded or stably derepressed chromosomally encoded class C (AmpC) β-lactamases (6,–8). Gram-negative bacilli (GNB) carrying an MBL also commonly carry additional β-lactamases, including ESBLs, AmpC, or serine carbapenemases (i.e., KPCs), that inactivate aztreonam, negating the activity of aztreonam against these isolates (9). Over time, the susceptibility of Enterobacteriaceae to aztreonam has been reduced (6, 7).

Avibactam is a bridged diazabicyclooctanone, non-β-lactam β-lactamase inhibitor of a wide range of serine β-lactamases that offers a broader β-lactamase inhibition profile than current β-lactamase inhibitors (clavulanic acid, tazobactam, and sulbactam), which inactivate only specific class A enzymes (3, 10). Avibactam protects β-lactams from hydrolysis by class A enzymes (including TEM, CTX-M ESBLs, and KPCs), class C enzymes (e.g., CMY, ACT, FOX), and some class D enzymes (e.g., OXA-48, OXA-139) (2, 10,–13). When combined with avibactam, aztreonam is able to inhibit cell wall synthesis in MBL-producing bacteria, despite the presence of cocarried serine β-lactamases (2, 9, 14).

To better define the inhibitory profile of aztreonam-avibactam, a recent (2012 to 2015) global collection of clinically significant isolates of Enterobacteriaceae and Pseudomonas aeruginosa from hospitalized patients was generated. The current study determined the profiles of in vitro susceptibility to aztreonam-avibactam, aztreonam, and comparator antimicrobial agents of these isolates using the Clinical and Laboratory Standards Institute (CLSI) broth microdilution method (15, 16). The study also identified carbapenem-nonsusceptible and MBL-positive isolates of Enterobacteriaceae in the collection and focused attention on the activity of aztreonam-avibactam against isolates with these important emerging resistant phenotypes and genotypes. This study is an expansion of one previously published that examined isolates from 2012 and 2013 only (9).

RESULTS

Table 1 displays the in vitro activities of aztreonam-avibactam, aztreonam, and the comparator antimicrobial agents tested against all Enterobacteriaceae isolates collected in the five geographic regions from 2012 to 2015. For all regions, aztreonam-avibactam was the most potent agent tested, with a MIC90 of 0.12 μg/ml for all Enterobacteriaceae isolates in four (Asia/South Pacific, Europe, Middle East/Africa, and North America) of the five geographic regions (Table 1). The MIC90 of aztreonam-avibactam for Enterobacteriaceae isolates collected in Latin America was 1 dilution higher at 0.25 μg/ml. In comparison, the MIC90 of (and percent susceptibility to) aztreonam ranged from 16 μg/ml (87.6% susceptible) for isolates from North America to 128 μg/ml (68.0% susceptible) for isolates from Latin America. Only meropenem showed activity comparable to that of aztreonam-avibactam against these isolates (MIC90 = 0.12 μg/ml). Other comparator agents showed much higher MIC90 values (≥2 μg/ml), resulting from the presence of a subpopulation of isolates with intrinsic resistance (Proteeae for tigecycline and colistin) or acquired resistance (the remainder of the compounds).

TABLE 1

Regional activities of aztreonam-avibactam, aztreonam, and comparator antimicrobial agents tested against 51,352 clinical isolates of Enterobacteriaceae categorized by MBL status

Region and category (ng)DrugMIC (μg/ml)
% of isolates with MIC interpretation of susceptiblea
50%90%Range
Global (51,352)Aztreonam-avibactam0.060.12≤0.015 to >128NAb
Aztreonam0.1264≤0.015 to >12876.0
Meropenem0.030.12≤0.004 to >897.1
Cefepime≤0.12>16≤0.12 to >1679.0
Ceftazidime0.2564≤0.015 to >12877.0
Piperacillin-tazobactam264≤0.25 to >12884.6
Amikacin28≤0.25 to >3296.7
Tigecycline0.52≤0.015 to >893.5
Levofloxacin0.06>4≤0.03 to >476.1
Colistin (n = 27,836)c1>4≤0.12 to >483.2
MBL negatived (51,085)Aztreonam-avibactam0.060.12≤0.015 to >128NA
Aztreonam0.1264≤0.015 to >12876.3
Meropenem0.030.12≤0.004 to >897.6
Cefepime≤0.12>16≤0.12 to >1679.4
Ceftazidime0.2564≤0.015 to >12877.4
Piperacillin-tazobactam264≤0.25 to >12885.0
Amikacin28≤0.25 to >3296.9
Tigecycline0.52≤0.015 to >893.6
Levofloxacin0.06>4≤0.03 to >476.3
Colistin (n = 27,664)1>4≤0.12 to >483.2
MBL positivee (267)Aztreonam-avibactam0.121≤0.015 to 8NA
Aztreonam64>128≤0.015 to >12829.2
Meropenem>8>80.25 to >86.4
Cefepime>16>16≤0.12 to >164.9
Ceftazidime>128>1280.25 to >1281.5
Piperacillin-tazobactam>128>1280.5 to >1286.0
Amikacin16>320.5 to >3257.7
Tigecycline140.06 to 889.1
Levofloxacin>4>4≤0.03 to >428.8
Colistin (n = 172)1>40.25 to >487.8
Asia/South Pacific (9,149)Aztreonam-avibactam0.060.12≤0.015 to 32NA
Aztreonam0.1264≤0.015 to >12873.9
Meropenem0.030.12≤0.004 to >898.4
Cefepime≤0.12>16≤0.12 to >1677.8
Ceftazidime0.2564≤0.015 to >12875.1
Piperacillin-tazobactam264≤0.25 to >12886.9
Amikacin28≤0.25 to >3297.5
Tigecycline0.52≤0.015 to >893.9
Levofloxacin0.12>4≤0.03 to >474.6
Colistin (n = 4,140)0.5>4≤0.12 to >483.0
MBL negative (9,075)Aztreonam-avibactam0.060.12≤0.015 to 32NA
Aztreonam0.1264≤0.015 to >12874.3
Meropenem0.030.12≤0.004 to >899.1
Cefepime≤0.12>16≤0.12 to >1678.4
Ceftazidime0.2564≤0.015 to >12875.7
Piperacillin-tazobactam232≤0.25 to >12887.5
Amikacin28≤0.25 to >3297.8
Tigecycline0.52≤0.015 to >894.0
Levofloxacin0.12>4≤0.03 to >474.9
Colistin (n = 4,103)0.5>4≤0.12 to >482.9
MBL positive (74)Aztreonam-avibactam0.120.5≤0.015 to 8NA
Aztreonam64>128≤0.015 to >12825.7
Meropenem>8>80.5 to >88.1
Cefepime>16>161 to >162.7
Ceftazidime>128>12832 to >1280
Piperacillin-tazobactam>128>1280.5 to >12816.2
Amikacin4>320.5 to >3266.2
Tigecycline140.06 to 889.2
Levofloxacin>4>40.06 to >436.5
Colistin (n = 37)1>40.25 to >486.5
Europe (24,826)Aztreonam-avibactam0.060.12≤0.015 to >128NA
Aztreonam0.1264≤0.015 to >12877.4
Meropenem0.030.12≤0.004 to >896.8
Cefepime≤0.12>16≤0.12 to >1680.4
Ceftazidime0.2564≤0.015 to >12878.1
Piperacillin-tazobactam2128≤0.25 to >12883.5
Amikacin28≤0.25 to >3296.3
Tigecycline0.52≤0.015 to >893.1
Levofloxacin0.06>4≤0.03 to >478.3
Colistin (n = 13,902)1>4≤0.12 to >482.8
MBL negative (24,695)Aztreonam-avibactam0.060.12≤0.015 to >128NA
Aztreonam0.1264≤0.015 to >12877.7
Meropenem0.030.12≤0.004 to >897.3
Cefepime≤0.12>16≤0.12 to >1680.8
Ceftazidime0.2564≤0.015 to >12878.5
Piperacillin-tazobactam2128≤0.25 to >12883.9
Amikacin28≤0.25 to >3296.6
Tigecycline0.52≤0.015 to >893.1
Levofloxacin0.06>4≤0.03 to >478.7
Colistin (n = 13,809)1>4≤0.12 to >482.8
MBL positive (131)Aztreonam-avibactam0.251≤0.015 to 2NA
Aztreonam64>1280.06 to >12829.8
Meropenem>8>80.25 to >87.6
Cefepime>16>16≤0.12 to >165.3
Ceftazidime>128>12816 to >1280
Piperacillin-tazobactam>128>12816 to >1282.3
Amikacin16>321 to >3251.1
Tigecycline140.12 to >888.6
Levofloxacin>4>4≤0.03 to >413.7
Colistin (n = 93)0.520.25 to >490.3
Latin America (7,665)Aztreonam-avibactam0.060.25≤0.015 to 16NA
Aztreonam0.12128≤0.015 to >12868.0
Meropenem0.030.12≤0.004 to >894.9
Cefepime≤0.12>16≤0.12 to >1670.4
Ceftazidime0.2564≤0.015 to >12869.9
Piperacillin-tazobactam4>128≤0.25 to >12881.1
Amikacin28≤0.25 to >3294.9
Tigecycline0.52≤0.015 to >893.7
Levofloxacin0.25>4≤0.03 to >467.5
Colistin (n = 4,516)0.5>4≤0.12 to >483.0
MBL negative (7,648)Aztreonam-avibactam0.060.25≤0.015 to 16NA
Aztreonam0.12128≤0.015 to >12868.0
Meropenem0.030.12≤0.004 to >895.1
Cefepime≤0.12>16≤0.12 to >1670.5
Ceftazidime0.2564≤0.015 to >12870.0
Piperacillin-tazobactam4128≤0.25 to >12881.3
Amikacin28≤0.25 to >3295.0
Tigecycline0.52≤0.015 to >893.8
Levofloxacin0.25>4≤0.03 to >467.6
Colistin (n = 4,500)0.5>4≤0.12 to >483.1
MBL positive (17)Aztreonam-avibactam0.120.25≤0.015 to 1NA
Aztreonam161280.03 to >12847.1
Meropenem8>81 to >85.9
Cefepime>16>161 to >165.9
Ceftazidime>128>1280.25 to >1285.9
Piperacillin-tazobactam>128>1281 to >1285.9
Amikacin16>322 to >3264.7
Tigecycline140.5 to 482.4
Levofloxacin1>40.25 to >458.8
Colistin (n = 16)1>40.25 to >468.8
Middle East/Africa (4,232)Aztreonam-avibactam0.030.12≤0.015 to >128NA
Aztreonam0.1264≤0.015 to >12872.3
Meropenem0.030.12≤0.004 to >898.0
Cefepime≤0.12>16≤0.12 to >1672.6
Ceftazidime0.2564≤0.015 to >12873.4
Piperacillin-tazobactam464≤0.25 to >12885.1
Amikacin28≤0.25 to >3297.1
Tigecycline0.52≤0.015 to >894.5
Levofloxacin0.12>4≤0.03 to >474.8
Colistin (n = 2,429)1>4≤0.12 to >483.7
MBL negative (4,191)Aztreonam-avibactam0.030.12≤0.015 to >128NA
Aztreonam0.1264≤0.015 to >12872.8
Meropenem0.030.12≤0.004 to >899.0
Cefepime≤0.12>16≤0.12 to >1673.3
Ceftazidime0.2564≤0.015 to >12874.0
Piperacillin-tazobactam264≤0.25 to >12886.0
Amikacin28≤0.25 to >3297.4
Tigecycline0.52≤0.015 to >894.5
Levofloxacin0.12>4≤0.03 to >475.0
Colistin (n = 2,404)1>4≤0.12 to >483.6
MBL positive (41)Aztreonam-avibactam0.120.25≤0.015 to 8NA
Aztreonam64128≤0.015 to >12824.4
Meropenem>8>82 to >80
Cefepime>16>16≤0.12 to >167.3
Ceftazidime>128>1280.5 to >1287.3
Piperacillin-tazobactam>128>12832 to >1280
Amikacin8>321 to >3263.4
Tigecycline120.25 to 892.7
Levofloxacin2>40.06 to >451.2
Colistin (n = 25)120.25 to >492.0
North America (5,480)Aztreonam-avibactam0.030.12≤0.015 to 16NA
Aztreonam0.1216≤0.015 to >12887.6
Meropenem0.030.12≤0.004 to >898.5
Cefepime≤0.121≤0.12 to >1691.5
Ceftazidime0.2516≤0.015 to >12887.6
Piperacillin-tazobactam216≤0.25 to >12890.6
Amikacin24≤0.25 to >3298.8
Tigecycline0.52≤0.015 to >893.7
Levofloxacin0.06>4≤0.03 to >481.5
Colistin (n = 2,849)1>4≤0.12 to >485.2
MBL negative (5,476)Aztreonam-avibactam0.030.12≤0.015 to 16NA
Aztreonam0.1216≤0.015 to >12887.6
Meropenem0.030.12≤0.004 to >898.6
Cefepime≤0.121≤0.12 to >1691.6
Ceftazidime0.2516≤0.015 to >12887.7
Piperacillin-tazobactam216≤0.25 to >12890.7
Amikacin24≤0.25 to >3298.9
Tigecycline0.52≤0.015 to >893.7
Levofloxacin0.06>4≤0.03 to >481.6
Colistin (n = 2,848)1>4≤0.12 to >485.2
MBL positive (4)Aztreonam-avibactamNDfND0.06 to 0.5NA
AztreonamNDND0.12 to >12850.0
MeropenemNDND8 to >80
CefepimeNDND>160
CeftazidimeNDND>1280
Piperacillin-tazobactamNDND>1280
AmikacinNDND16 to >3225.0
TigecyclineNDND0.5 to 2100
LevofloxacinNDND0.5 to >425.0
Colistin (n = 1)NDND1100
aMICs were interpreted according to CLSI breakpoints (16), with the exception of those for aztreonam-avibactam, for which clinical breakpoints have not yet been assigned; tigecycline, for which MIC interpretative criteria published by the U.S. FDA were used (33); and colistin, for which EUCAST breakpoints were applied (34).
bNA, not available (no breakpoint criteria has been defined for aztreonam-avibactam).
cColistin was tested without added surfactant; data are for isolates collected in 2014 and 2015 only.
dMBL negative, no gene encoding a metallo-β-lactamase included in the screening algorithm was detected by PCR. This isolate type includes carbapenem-sensitive and carbapenem-nonsusceptible metallo-β-lactamase-negative isolates.
eMBL positive, a gene encoding a metallo-β-lactamase was detected by PCR. This isolate type can also carry one or more additional serine β-lactamases.
fND, not determined; i.e., the MIC50s and MIC90s were not calculated when there were <10 isolates.
gn, number of isolates tested, unless indicated otherwise.

Table 1 also summarizes regional data for MBL-negative and MBL-positive Enterobacteriaceae isolates from each region. MBL-positive isolates constituted 1.0% (41/4,232) of isolates from the Middle East/Africa region, 0.8% (74/9,149) of isolates from Asia/South Pacific, 0.5% (131/24,826) of isolates from Europe, 0.2% (17/7,665) of isolates from Latin America, and 0.1% (4/5,480) of isolates from North America. Against MBL-positive isolates of Enterobacteriaceae, the MIC90 of aztreonam-avibactam ranged from 0.25 μg/ml (Latin America and Middle East/Africa) to 1 μg/ml (Europe). The MIC of aztreonam-avibactam was ≤8 μg/ml for all MBL-positive Enterobacteriaceae (Table 2).

TABLE 2

MIC cumulative frequency distribution for aztreonam-avibactam and aztreonam tested against clinical isolates of Enterobacteriaceae and P. aeruginosa collected worldwide from 2012 to 2015

Organism (nd) and drug% frequency distribution by MIC (μg/ml)a:
≤0.0150.030.060.120.250.51248163264128>128
Enterobacteriaceae
All (51,352)
Aztreonam-avibactam20.948.779.191.496.398.399.299.799.9>99.9>99.9>99.9>99.9>99.9100
Aztreonam9.116.041.262.669.872.173.474.476.077.880.786.092.096.1100
Meropenem nonsusceptible (1,498)
Aztreonam-avibactam2.55.915.239.072.488.294.097.599.299.899.9100
Aztreonam0.50.61.32.55.36.57.17.78.59.110.914.824.542.1100
Meropenem nonsusceptible, MBL negativeb (1,248)
Aztreonam-avibactam2.25.013.136.170.687.993.697.499.299.899.9100
Aztreonam0.20.30.61.42.63.23.43.84.55.06.49.517.933.8100
Meropenem nonsusceptible, MBL positivec (250)
Aztreonam-avibactam4.010.425.653.681.689.696.098.499.2100
Aztreonam2.02.05.28.018.822.825.627.628.429.633.241.257.283.2100
All MBL-positive isolates (267)
Aztreonam-avibactam4.912.027.754.782.089.595.998.199.3100
Aztreonam2.22.66.09.019.523.226.228.529.230.333.742.359.684.3100
P. aeruginosa
All (11,842)
Aztreonam-avibactam0.60.72.23.74.78.742.073.486.996.399.299.7100
Aztreonam0.20.31.32.73.66.734.461.777.490.695.897.7100
MBL positive (452)
Aztreonam-avibactam0.20.40.41.113.938.170.690.797.398.7100
Aztreonam0.20.40.40.910.424.859.783.890.592.9100
aFor each MIC distribution, MIC90 values are in bold.
bMBL negative, no gene encoding a metallo-β-lactamase included in the screening algorithm was detected by PCR. This isolate type includes carbapenem-sensitive and carbapenem-nonsusceptible metallo-β-lactamase-negative isolates.
cMBL positive, a gene encoding a metallo-β-lactamase was detected by PCR. This isolate type can also carry one or more additional serine β-lactamases.
dn, number of isolates tested.

Aztreonam-avibactam and meropenem were the two most potent antimicrobial agents tested (i.e., they had the lowest MIC90 values) against MBL-negative isolates of Enterobacteriaceae, with their MIC90s being equal to or within 1 2-fold dilution. However, against MBL-positive Enterobacteriaceae, aztreonam-avibactam was >8- to >32-fold more potent (by comparison of MIC90 values) than meropenem against isolates from Asia/South Pacific, Europe, Latin America, and the Middle East/Africa (Table 1). The percentages of isolates susceptible to antimicrobial agents other than aztreonam-avibactam were markedly lower for MBL-positive Enterobacteriaceae isolates than for MBL-negative isolates for agents other than tigecycline and colistin (Table 1).

Avibactam lowered the MIC90 of aztreonam for all Enterobacteriaceae isolates tested by 512-fold, from 64 μg/ml to 0.12 μg/ml (Table 2). A concentration of aztreonam-avibactam of ≤8 μg/ml inhibited >99.9% (51,327/51,352) of all Enterobacteriaceae isolates (Table 2). Aztreonam-avibactam remained very potent against meropenem-nonsusceptible Enterobacteriaceae isolates (MIC90 of 1 μg/ml), regardless of the presence or absence of an MBL. Twenty-five Enterobacteriaceae isolates tested with aztreonam-avibactam MICs ranging from 16 to >128 μg/ml, and all were MBL negative. These included isolates of Escherichia coli (n = 8; 5 of these isolates carried CMY-type AmpC β-lactamases), Enterobacter cloacae (n = 5), Klebsiella pneumoniae (n = 4), Proteus mirabilis (n = 2), and one isolate each of Enterobacter aerogenes, Morganella morganii, Proteus vulgaris, Providencia rettgeri, Raoultella ornithinolytica, and Serratia marcescens, collected in 21 hospitals in 14 countries (Argentina, Belgium, China, Denmark, Germany, Italy, Kenya, Kuwait, Portugal, Russia, Spain, Taiwan, Thailand, Venezuela). The mechanism(s) responsible for the increased MICs of aztreonam-avibactam in these 25 isolates remains undefined; however, 22 and 16 of the 25 isolates were susceptible to meropenem and ceftazidime-avibactam, respectively (data not shown).

Table 3 provides a summary of the in vitro activities of aztreonam-avibactam, aztreonam, and the other antimicrobial agents tested against Enterobacteriaceae isolates collected in 2012 and 2013 compared with the activities against isolates collected in 2014 and 2015. The MIC90 of aztreonam-avibactam was consistent across both the 2012 and 2013 and the 2014 and 2015 data sets, with a MIC90 of 0.12 μg/ml for MBL-negative isolates of Enterobacteriaceae and with a MIC90 of 0.5 to 1 μg/ml for MBL-positive isolates.

TABLE 3

Longitudinal analysis of in vitro activities of aztreonam-avibactam, aztreonam, and comparative antimicrobial agents tested against clinical isolates of Enterobacteriaceae collected worldwide in 2012 and 2013 and in 2014 and 2015 and categorized by MBL status

Time period and category (nf)DrugMIC (μg/ml)
% of isolates with MIC interpretation of susceptiblea
50%90%Range
2012–2013
All (23,516)Aztreonam-avibactam0.060.12≤0.015 to >128NAb
Aztreonam0.1264≤0.015 to >12876.2
Meropenem0.030.12≤0.004 to >897.6
Cefepime≤0.12>16≤0.12 to >1679.4
Ceftazidime0.2564≤0.015 to >12877.2
Piperacillin-tazobactam264≤0.25 to >12884.2
Amikacin28≤0.25 to >3296.6
Tigecycline0.52≤0.015 to >891.8
Levofloxacin0.06>4≤0.03 to >475.8
MBL negativec (23,421)Aztreonam-avibactam0.060.12≤0.015 to >128NA
Aztreonam0.1264≤0.015 to >12876.3
Meropenem0.030.12≤0.004 to >897.9
Cefepime≤0.12>16≤0.12 to >1679.7
Ceftazidime0.2564≤0.015 to >12877.5
Piperacillin-tazobactam264≤0.25 to >12884.5
Amikacin28≤0.25 to >3296.7
Tigecycline0.52≤0.015 to >891.8
Levofloxacin0.06>4≤0.03 to >476.0
MBL positived (95)Aztreonam-avibactam0.121≤0.015 to 4NA
Aztreonam32>128≤0.015 to >12836.8
Meropenem>8>80.25 to >810.5
Cefepime>16>16≤0.12 to >164.2
Ceftazidime>128>1280.25 to >1284.2
Piperacillin-tazobactam>128>1280.5 to >12812.6
Amikacin16>321 to >3261.1
Tigecycline140.06 to >888.4
Levofloxacin>4>40.06 to >436.8
2014–2015
All (27,836)Aztreonam-avibactam0.060.12≤0.015 to 64NA
Aztreonam0.1264≤0.015 to >12875.9
Meropenem0.060.12≤0.004 to >896.7
Cefepime≤0.12>16≤0.12 to >1678.7
Ceftazidime0.2564≤0.015 to >12876.8
Piperacillin-tazobactam264≤0.25 to >12885.0
Amikacin28≤0.25 to >3296.7
Tigecycline0.52≤0.015 to >895.0
Levofloxacin0.12>4≤0.03 to >476.3
Colistine1>4≤0.12 to >483.2
MBL negative (27,664)Aztreonam-avibactam0.060.12≤0.015 to 64NA
Aztreonam0.1264≤0.015 to >12876.3
Meropenem0.060.12≤0.004 to >897.3
Cefepime≤0.12>16≤0.12 to >1679.1
Ceftazidime0.2564≤0.015 to >12877.3
Piperacillin-tazobactam264≤0.25 to >12885.5
Amikacin28≤0.25 to >3297.0
Tigecycline0.52≤0.015 to >895.1
Levofloxacin0.12>4≤0.03 to >476.6
Colistin1>4≤0.12 to >483.2
MBL positive (172)Aztreonam-avibactam0.120.5≤0.015 to 8NA
Aztreonam64>128≤0.015 to >12825.0
Meropenem>8>80.25 to >84.1
Cefepime>16>160.5 to >165.2
Ceftazidime>128>12832 to >1280
Piperacillin-tazobactam>128>1284 to >1282.3
Amikacin16>320.5 to >3255.8
Tigecycline140.12 to 889.5
Levofloxacin>4>4≤0.03 to >424.4
Colistin1>40.25 to >487.8
aMICs were interpreted according to CLSI breakpoints (16), with the exception of those for aztreonam-avibactam, for which clinical breakpoints have not yet been assigned; tigecycline, for which MIC interpretative criteria published by the U.S. FDA were used (33); and colistin, for which EUCAST breakpoints were applied (34).
bNA, not available (no breakpoint criteria have been defined for aztreonam-avibactam).
cMBL negative, no gene encoding a metallo-β-lactamase included in the screening algorithm was detected by PCR. This isolate type includes carbapenem-sensitive and carbapenem-nonsusceptible metallo-β-lactamase-negative isolates.
dMBL positive, a gene encoding a metallo-β-lactamase was detected by PCR. This isolate type can also carry one or more additional serine β-lactamases.
eColistin was tested without added surfactant; data for colistin are for isolates collected in 2014 and 2015 only.
fn, number of isolates tested.

Table 4 describes the comparative MICs of aztreonam-avibactam and aztreonam alone against the 267 isolates of MBL-positive Enterobacteriaceae (with or without additional serine β-lactamase enzymes) identified in this study. The 267 isolates with an MBL (139 K. pneumoniae, 59 E. cloacae, 18 Citrobacter freundii, 12 E. coli, 10 Klebsiella oxytoca, 10 P. mirabilis, 5 Providencia stuartii, 5 S. marcescens, 4 Enterobacter asburiae, 3 P. rettgeri, and 2 E. aerogenes isolates) were composed of 142 isolates with NDM, 96 isolates with VIM, and 29 isolates with IMP. The majority of the characterized isolates with an MBL also produced ESBLs, class C enzymes, and/or serine carbapenemases. For isolates carrying only an MBL (n = 6), aztreonam alone inhibited all isolates at a concentration of 1 μg/ml. However, for isolates with an MBL plus one or more serine β-lactamases, the majority of isolates produced aztreonam MICs of >128 μg/ml. Aztreonam-avibactam was much more potent against these isolates, with all isolates being inhibited by a concentration of 8 μg/ml and with MIC90s, where calculable, ranging from 0.5 to 2 μg/ml, emphasizing the benefit of combining avibactam with aztreonam against isolates harboring serine β-lactamases in addition to an MBL. The 237 isolates that coproduced an MBL and an ESBL, AmpC, and/or serine carbapenemase produced aztreonam-avibactam and aztreonam MIC90 values of 1 μg/ml (MIC range, ≤0.015 to 8 μg/ml) and >128 μg/ml (MIC range, ≤0.015 to >128 μg/ml), respectively.

TABLE 4

Comparative MICs of aztreonam-avibactam and aztreonam against 267 isolates of Enterobacteriaceae positive for an MBL gene alone or positive for an MBL gene and one or more additional β-lactamase genes

Group (n)aMICb (μg/ml) for:
Aztreonam-avibactam
Aztreonam
50%90%Range50%90%Range
All MBL producers (267)0.121≤0.015 to 864>128≤0.015 to >128
MBL only (6)c−−≤0.015 to 0.06−−≤0.015 to 1
MBL + OSBL (24)0.120.250.03 to 0.50.2520.06 to 64
MBL + ESBL (26)0.250.250.03 to 0.2564>1280.06 to >128
MBL + ESBL + OSBL (69)0.120.25≤0.015 to 0.5128>1282 to >128
MBL + AmpC (33)0.122≤0.015 to 80.2564≤0.015 to 128
MBL + AmpC + OSBL (28)0.52≤0.015 to 816128≤0.015 to >128
MBL + ESBL + AmpC (13)0.250.250.03 to 164>1281 to >128
MBL + ESBL + AmpC + OSBL (30)0.120.5≤0.015 to 2128>1280.5 to >128
MBL + KPC (1)−−0.5−−>128
MBL + KPC + ESBL (2)−−0.5 to 1−−>128
MBL + KPC + ESBL + OSBL (2)−−0.5 to 2−−>128
MBL + KPC + AmpC + OSBL (1)−−0.5−−>128
MBL + KPC + ESBL + AmpC + OSBL (2)−−0.5−−>128
MBL + OXA-48-like + OSBL (5)d−−0.12 to 2−−0.25 to 1
MBL + OXA-48 + ESBL (3)−−0.12 to 0.25−−>128
MBL + OXA-48-like + ESBL + OSBL (13)d0.250.50.12 to 1>128>128128 to >128
MBL + OXA-48 + AmpC (3)−−0.25 to 1−−0.25 to 16
MBL + OXA-48 + AmpC + OSBL (5)−−0.25−−32
MBL + OXA-48 + ESBL + AmpC + OSBL (1)−−0.25−−128
aThe MBLs included NDM (142 isolates), VIM (96 isolates), and IMP (29 isolates); the ESBLs included SHV, CTX-M, VEB, and the endogenous ESBL common to K. oxytoca. OSBL, original-spectrum β-lactamase. Original-spectrum β-lactamases are enzymes that do not hydrolyze expanded-spectrum cephalosporins or carbapenems and include TEM-1, SHV-1, and SHV-11. n, number of isolates tested.
b−, MIC50s and MIC90s were not calculated when there were <10 isolates.
cDoes not include species with endogenous AmpC or ESBL enzymes.
dIncludes isolates carrying OXA-48 and OXA-232.

Aztreonam alone consistently demonstrated low potency against most Enterobacteriaceae isolates that produced class A or class C enzymes alone or in combination with an MBL. Of particular note were isolates that expressed a serine carbapenemase in combination with an MBL. There were eight isolates that produced both an MBL and a KPC enzyme (six K. pneumoniae isolates cocarrying KPC-2 and VIM-1 [n = 4] or KPC-2 and VIM-26 [n = 2] were collected in two hospitals in Greece, and two K. oxytoca isolates cocarrying KPC-2 and IMP-4 were collected in one hospital in China). Thirty additional isolates produced an MBL and an OXA-48-like enzyme: 21 K. pneumoniae isolates cocarried NDM-1 and OXA-48 (n = 14, two hospitals in Romania in 2014 and 2015) or NDM-1 and OXA-232 (one hospital each in Thailand [n = 5; 2015], Belgium [n = 1; 2013], and Kuwait [n = 1; 2015]), 5 isolates of C. freundii cocarried VIM-31 and OXA-48 (Turkey, one hospital in 2015), and 4 E. cloacae isolates cocarried VIM-4 and OXA-48 (n = 2, 1 hospital in Kuwait), VIM-31 and OXA-48 (n = 1, Turkey), or NDM-1 and OXA-48 (n = 1, Romania). All of these isolates were inhibited by aztreonam-avibactam at a concentration of 0.12 to 2 μg/ml.

Against 11,842 clinical isolates of P. aeruginosa collected worldwide from 2012 to 2015, the MIC90s for both aztreonam-avibactam and aztreonam alone were 32 μg/ml (Table 2). Against MBL-positive P. aeruginosa isolates, the MIC90 of aztreonam-avibactam (32 μg/ml) was 1 2-fold dilution lower than that of aztreonam alone (64 μg/ml); the MIC distributions for aztreonam-avibactam and aztreonam alone were similar for all P. aeruginosa isolates and MBL-positive isolates. In comparison, the MIC90 values of meropenem and colistin were >8 μg/ml and 2 μg/ml, respectively, against all and MBL-positive P. aeruginosa (data not shown).

DISCUSSION

The monobactam–β-lactamase inhibitor combination aztreonam-avibactam is in development to treat serious infections caused by MBL-producing Enterobacteriaceae, a difficult-to-treat subtype of carbapenem-resistant Enterobacteriaceae for which therapeutic options are currently very limited (17, 18). A phase 2 study to determine the pharmacokinetics, safety, and tolerability of aztreonam-avibactam for the treatment of complicated intra-abdominal infections in hospitalized patients is under way (19). Use of the combination of aztreonam-avibactam presents a novel approach to the treatment of infections caused by pathogens containing multiple β-lactamases, including isolates carrying MBLs, and may serve to address the need for new antimicrobial agents with activity against these carbapenem-nonsusceptible isolates of Gram-negative bacilli (17, 20, 21).

In the current study, aztreonam-avibactam demonstrated potent in vitro activity (MIC90, 0.12 μg/ml) against >50,000 clinical isolates of Enterobacteriaceae collected in 40 countries worldwide between 2012 and 2015. Compared with the potency of aztreonam alone, the addition of avibactam resulted in a ≥128-fold improvement in potency for aztreonam-avibactam against isolates that were meropenem nonsusceptible due to the presence of an MBL or other mechanisms of carbapenem resistance (e.g., the production of a KPC). No regional differences in the activity of aztreonam-avibactam were observed, and the activity was maintained against isolates collected over the 4 years surveyed. These data demonstrate the ability of avibactam to restore the activity of aztreonam against Enterobacteriaceae.

Previous studies have also demonstrated that avibactam improved the potency of aztreonam against Enterobacteriaceae isolates that produced MBLs as well as class A (KPC), class C (AmpC), and class D (OXA-48) β-lactamases (2, 3, 9, 12, 14, 22,–26). The current data set extended the observations made previously from studies using smaller collections of isolates (14, 27). A study performed by investigators in China demonstrated that avibactam potentiated the activity of aztreonam against 193 Enterobacteriaceae isolates, including isolates with ESBLs and stably derepressed AmpC β-lactamases, and generated a MIC90 of 0.25 μg/ml, with MICs ranging from ≤0.015 to ≥128 μg/ml (22). In the same study, 26 isolates of meropenem-nonsusceptible Enterobacteriaceae, including isolates with NDM and IMP MBLs, were tested, and they generated an aztreonam-avibactam MIC90 of 2 μg/ml and a MIC range of ≤0.06 to 2 μg/ml (22). A European study reported that all 339 Enterobacteriaceae isolates tested had aztreonam-avibactam MICs of ≤4 μg/ml (23). In another study, for 65 Enterobacteriaceae isolates that were carbapenem resistant by serine carbapenemase production, MBL production, or porin loss plus AmpC or ESBL production, the MIC range for aztreonam-avibactam was ≤0.03 to 4 μg/ml (3). Avibactam was reported to lower the aztreonam MICs of AmpC-, ESBL-, and KPC-producing Enterobacteriaceae by up to 2,048-fold, to a maximum MIC of 4 μg/ml (2). Aztreonam-avibactam also demonstrated potent in vitro activity (MIC90, 0.5 μg/ml; MIC range, ≤0.06 to 4 μg/ml) against 133 porin-deficient, carbapenem-nonsusceptible Enterobacteriaceae isolates from northern France (24). Lastly, aztreonam-avibactam inhibited all isolates in a collection of 177 (172 Enterobacteriaceae isolates and 5 P. aeruginosa isolates) 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) from the United States and Singapore at a concentration of 16 μg/ml (MIC90, 1 μg/ml) (25).

In this study, 25 isolates of MBL-negative Enterobacteriaceae with aztreonam-avibactam MICs of ≥16 μg/ml and meropenem MICs ranging from 0.03 to >8 μg/ml were detected (9 isolates in 2012, 4 in 2013, 5 in 2014, and 7 in 2015). The mechanism responsible for the increased MICs of aztreonam-avibactam in these 25 isolates remains unclear and may represent a combination of factors. For 16 isolates, the mechanism(s) appears to be aztreonam related rather than avibactam related, because those isolates remained susceptible to ceftazidime-avibactam. It is possible that some of the isolates may harbor mutations in PBP3, such as the 4-amino-acid insertion shown to reduce the activity of aztreonam-avibactam (28). Resistance may also arise from single amino acid substitutions or Ω-loop mutations in coresident class A and C serine β-lactamases that can lead to a reduced affinity of avibactam for the β-lactamase binding site, resulting in poor inhibition of β-lactamase activity (13, 29, 30). Porin deficits and overexpression of efflux pumps may also be contributory. Other investigators have shown that variants of KPC-2 can be resistant to avibactam by mutations in residue S130, as well as residues K234 and R220, which are important residues for avibactam binding to and inactivation of KPC-2 (31).

The in vitro activities of aztreonam-avibactam and aztreonam alone were less potent (MIC90, 32 μg/ml) against P. aeruginosa than against Enterobacteriaceae, and the addition of avibactam to aztreonam did not improve the potency of aztreonam, suggesting that resistance to aztreonam in P. aeruginosa is, at least in part, a function of mechanisms other than β-lactamases, such as upregulation of the MexAB-OprM efflux transport system (32). Other investigators have also observed that the addition of avibactam to aztreonam did not generate an improvement in the potency of aztreonam against clinical isolates of P. aeruginosa (22). The distributions of the MICs of aztreonam-avibactam for MBL-negative and MBL-positive P. aeruginosa isolates have been shown to be similar to those for meropenem-susceptible and meropenem-nonsusceptible populations, respectively (9). The treatment of patients with infections due to carbapenem-resistant nonfermentative Gram-negative bacilli remains difficult because of the MDR profiles of many of these isolates.

The clinical development of aztreonam-avibactam appears to be well justified, as it may be a valuable alternative for the treatment of infections caused by isolates of the Enterobacteriaceae that are carbapenem nonsusceptible, especially infections caused by MBL-producing pathogens, as infections caused by these pathogens continue to emerge worldwide. Infection control and antimicrobial stewardship practices will remain important to maintaining the low prevalence of mobile carbapenemase genes and to preserving the activities of all antimicrobial agents. As β-lactam–avibactam combinations are introduced into clinical use, ongoing surveillance for β-lactamase-mediated resistance and other mechanisms of resistance is imperative to ensure the long-term viability and effectiveness of these and other antimicrobial agents. It will also be important to monitor for and characterize nonsusceptible isolates and to correlate clinical outcomes with identified resistance genotypes.

MATERIALS AND METHODS

Bacterial isolates.

Isolates were collected from 2012 to 2015 from multiple sites around the world, including 208 medical center laboratories in 40 countries. The sites were requested each year to collect predefined quotas of clinical isolates of selected bacterial species (limited to one isolate per patient per annum) from hospitalized patients with specific infection types (intra-abdominal, urinary tract, skin and soft tissue, lower respiratory, and bloodstream infections) and to submit them to a central laboratory (International Health Management Associates, Inc. [IHMA], Schaumburg, IL, USA) for confirmatory identification and reference antimicrobial susceptibility testing. In total, from 2012 to 2015, 51,352 Enterobacteriaceae isolates and 11,842 P. aeruginosa isolates were collected, their identities were confirmed, and reference antimicrobial susceptibility testing was performed. Because quotas of selected bacterial species were used for the collection of isolates, this study was not structured to evaluate the prevalence of organisms causing specific infections or resistance phenotypes. The identities of all isolates were confirmed using matrix-assisted laser desorption ionization–time of flight spectrometry (Bruker Daltonics, Billerica, MA, USA). All components of this study were coordinated by IHMA, including the development and management of the central study database.

Isolates submitted by medical center laboratories were grouped by country into five major geographical regions: Asia/South Pacific (Australia, China, Hong Kong, Japan, Malaysia, Philippines, South Korea, Taiwan, Thailand), Europe (Austria, Belgium, Czech Republic, Denmark, France, Germany, Greece, Hungary, Ireland, Italy, Netherlands, Poland, Portugal, Romania, Russia, Spain, Sweden, Turkey, United Kingdom), Latin America (Argentina, Brazil, Chile, Colombia, Mexico, Venezuela), Middle East/Africa (Israel, Kenya, Kuwait, Nigeria, South Africa), and North America (United States). Of the 51,352 Enterobacteriaceae isolates included in this study, 17.8% (n = 9,149) were from the Asia/South Pacific region, 48.3% (n = 24,826) were from Europe, 14.9% (n = 7,665) were from Latin America, 8.2% (n = 4,232) were from the Middle East/Africa region, and 10.7% (n = 5,480) were from North America. The composition of the Enterobacteriaceae isolates collected in each region, by species, is shown in the Table S1 in the supplemental material. Of the 11,842 P. aeruginosa isolates included in this study, 17.2% (n = 2,038) were from the Asia/South Pacific region, 48.4% (n = 5,728) were from Europe, 15.1% (n = 1,794) were from Latin America, 8.1% (n = 964) were from the Middle East/Africa region, and 11.1% (n = 1,318) were from North America.

The composition of the Enterobacteriaceae isolates by specimen source was as follows: intra-abdominal, n = 10,025 (19.5% of all isolates tested); urinary tract, n = 14,604 (28.4%); skin and soft tissue, n = 12,698 (24.7%); lower respiratory tract, n = 11,426 (22.3%); bloodstream, n = 2,458 (4.8%); and other, n = 141 (0.3%). The composition of the P. aeruginosa isolates by specimen source was as follows: intra-abdominal, n = 1,171 (9.9% of all isolates tested); urinary tract, n = 1,696 (14.3%); skin and soft tissue, n = 3,521 (29.7%); lower respiratory tract, n = 5,052 (42.7%); bloodstream, n = 387 (3.3%); and other, n = 15 (0.1%).

Antimicrobial susceptibility testing.

All antimicrobial susceptibility testing was performed at IHMA using in-house-prepared broth microdilution panels produced, inoculated, incubated, interpreted, and quality controlled by standardized CLSI methods (15, 16). Avibactam was tested at a fixed concentration of 4 μg/ml in combination with 2-fold dilutions of aztreonam. MICs were interpreted using CLSI breakpoints (16) for all antimicrobial agents except for those for which CLSI breakpoints are not available: aztreonam-avibactam, for which clinical breakpoints have not yet been assigned; tigecycline, for which U.S. FDA MIC breakpoints were used (33); and colistin, for which EUCAST MIC breakpoints (34) were applied.

Isolates of E. coli, K. pneumoniae, K. oxytoca, and P. mirabilis were phenotypically screened for possible ESBL production using ceftazidime and aztreonam MICs (>1 μg/ml) and confirmed to be ESBL producers by testing with clavulanate in combination with ceftazidime and cefotaxime, following CLSI guidelines (16). If the MICs of the screening β-lactams exceeded the highest dilution on the broth microdilution panel, confirmation of ESBL production was determined using disk diffusion testing (16).

Data analysis included grouping of the Enterobacteriaceae isolates into MBL-negative and MBL-positive isolates. An MBL-negative isolate was defined as an isolate for which molecular testing (i.e., PCR [described below]) did not detect the presence of an MBL gene. MBL-negative isolates included isolates that were susceptible to all carbapenems tested, isolates whose genomes encoded serine carbapenemases or other serine β-lactamases, and isolates that were resistant to carbapenems by virtue of nonenzymatic mechanisms, such as reduced outer membrane permeability. An MBL-positive isolate was defined as an isolate for which PCR detected the presence of one of the MBL genes (IMP, VIM, NDM, GIM, and SPM) that was included in the screening algorithm. These isolates could also contain one or more additional serine β-lactamases.

Molecular identification of β-lactamase genes in Enterobacteriaceae and P. aeruginosa.

Enterobacteriaceae isolates were chosen for PCR testing to detect the presence of β-lactamase genes when they met at least one of the following criteria: (i) the isolates demonstrated a positive result for ESBL production by a confirmatory test, (ii) the isolates demonstrated a negative result for ESBL production by a confirmatory test but were resistant to ceftazidime (MIC, ≥16 μg/ml), and (iii) the isolates had meropenem, doripenem, or imipenem MICs of ≥2 μg/ml (nonsusceptible) (isolates of Proteeae with imipenem MICs of 2 or 4 μg/ml but meropenem and doripenem MICs of <2 µg/ml were excluded) (16). A multiplex PCR assay followed by DNA sequencing was performed as previously described (35) to molecularly characterize β-lactamase genes encoding MBLs (i.e., IMP, VIM, NDM, GIM, and SPM) and other β-lactamases (i.e., SHV, TEM, CTX-M, VEB, PER, GES, ACC, CMY, DHA, FOX, ACT, MIR, MOX, KPC, and OXA-48-like). P. aeruginosa isolates with meropenem, doripenem, or imipenem MICs of ≥4 μg/ml (nonsusceptible) were screened for the presence of β-lactamase genes encoding MBLs (IMP, VIM, NDM, GIM, and SPM) and other β-lactamases (SHV, TEM, VEB, PER, GES, KPC, and OXA-24-like) as previously described (36).

Supplementary Material

Supplemental material:

ACKNOWLEDGMENTS

This study was sponsored by AstraZeneca Pharmaceuticals LP, which also included compensation fees for manuscript preparation. AstraZeneca's rights to aztreonam-avibactam were acquired by Pfizer in December 2016.

Medical writing and editorial support were provided by J.A.K., a consultant for IHMA and an employee of the University of Manitoba and Diagnostic Services Manitoba, and K.M.K, an employee of IHMA. All authors provided analysis input and have read and approved the final manuscript.

M.A.H. and D.F.S. are employees of IHMA. None of the IHMA authors or J.A.K. has a personal financial interest in the sponsor of this paper (AstraZeneca Pharmaceuticals). B.L.M.D.J. was an employee of and shareholder in AstraZeneca at the time of the study and is currently an employee of Pfizer. P.A.B. is a former employee of AstraZeneca.

We gratefully acknowledge the contributions of the study investigators, laboratory personnel, and all members of the AstraZeneca Surveillance program.

Footnotes

Supplemental material for this article may be found at https://doi.org/10.1128/AAC.00472-17.

REFERENCES

1. Walsh TR, Toleman MA, Poirel L, Nordmann P. 2005. Metallo-β-lactamases, the quiet before the storm? Clin Microbiol Rev 18:306–325. 10.1128/CMR.18.2.306-325.2005. [Europe PMC free article] [Abstract] [CrossRef] [Google Scholar]
2. Li H, Estabrook M, Jacoby GA, Nichols WW, Testa RT, Bush K. 2015. In vitro susceptibility of characterized β-lactamase-producing strain tested with avibactam combinations. Antimicrob Agents Chemother 59:1789–1793. 10.1128/AAC.04191-14. [Europe PMC free article] [Abstract] [CrossRef] [Google Scholar]
3. Livermore DM, Mushtaq S, Warner M, Zhang J, Maharjan S, Doumith M, Woodford N. 2011. Activities of NXL104 combinations with ceftazidime and aztreonam against carbapenemase-producing Enterobacteriaceae. Antimicrob Agents Chemother 55:390–394. 10.1128/AAC.00756-10. [Europe PMC free article] [Abstract] [CrossRef] [Google Scholar]
4. Pérez A, Canle D, Latasa C, Poza M, Beceiro A, del Mar Tomás M, Fernández A, Mallo S, Pérez S, Molina F, Villanueva R, Lasa I, Bou G. 2007. Cloning, nucleotide sequencing, and analysis of the AcrAB-TolC efflux pump of Enterobacter cloacae and determination of its involvement in antibiotic resistance in a clinical isolate. Antimicrob Agents Chemother 51:3247–3253. 10.1128/AAC.00072-07. [Europe PMC free article] [Abstract] [CrossRef] [Google Scholar]
5. Adler M, Anjum M, Andersson DI, Sandegren L. 2016. Combinations of mutations in envZ, ftsI, mrdA, acrB, and acrR can cause high-level carbapenem resistance in Escherichia coli. J Antimicrob Chemother 71:1188–1198. 10.1093/jac/dkv475. [Abstract] [CrossRef] [Google Scholar]
6. Queenan AM, Bush K. 2007. Carbapenemases: the versatile β-lactamases. Clin Microbiol Rev 20:440–458. 10.1128/CMR.00001-07. [Europe PMC free article] [Abstract] [CrossRef] [Google Scholar]
7. Bradford PA. 2001. Extended-spectrum β-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat Clin Microbiol Rev 14:933–951. 10.1128/CMR.14.4.933-951.2001. [Europe PMC free article] [Abstract] [CrossRef] [Google Scholar]
8. Rafailidis PI, Falagas ME. 2014. Options for treating carbapenem resistant Enterobacteriaceae. Curr Opin Infect Dis 27:479–483. 10.1097/QCO.0000000000000109. [Abstract] [CrossRef] [Google Scholar]
9. Biedenbach DJ, Kazmierczak KM, Bouchillon SK, Sahm DF, Bradford PA. 2015. In vitro activity of aztreonam-avibactam against a global collection of Gram-negative pathogens from 2012 and 2013. Antimicrob Agents Chemother 59:4239–4248. 10.1128/AAC.00206-15. [Europe PMC free article] [Abstract] [CrossRef] [Google Scholar]
10. Ehmann DE, Jahic H, Ross PL, Gu RF, Hu J, Kern G, Wakup GK, Fisher SL. 2012. Avibactam is a covalent, reversible, non-β-lactam β-lactamase inhibitor. Proc Natl Acad Sci U S A 109:11663–11668. 10.1073/pnas.1205073109. [Europe PMC free article] [Abstract] [CrossRef] [Google Scholar]
11. Ehmann DE, Jahic H, Ross PL, Gu RF, Hu J, Curand-Reville TF, Lahiri S, Thresher J, Livchak S, Gao N, Palmer T, Walkup GK, Fisher SL. 2013. Kinetics of avibactam inhibition against class A, C, and D β-lactamases. J Biol Chem 288:27960–27971. 10.1074/jbc.M113.485979. [Europe PMC free article] [Abstract] [CrossRef] [Google Scholar]
12. Giani T, Cannatelli A, Di Pilato V, Testa R, Nichols WW, Rossolini GM. 2016. Inhibitory activity of avibactam against selected β-lactamases expressed in an isogenic Escherichia coli strain. Diagn Microbiol Infect Dis 86:83–85. 10.1016/j.diagmicrobio.2016.03.002. [Abstract] [CrossRef] [Google Scholar]
13. Lahiri SD, Johnstone M, Ross PL, McLaughlin R, Olivier NB, Alm RA. 2014. Avibactam and class C β-lactamases: mechanisms of inhibition, conservation of binding pocket and implications for resistance. Antimicrob Agents Chemother 58:5704–5713. 10.1128/AAC.03057-14. [Europe PMC free article] [Abstract] [CrossRef] [Google Scholar]
14. Kazmierczak KM, Rabine S, Hackel M, McLaughlin RE, Biedenbach DJ, Bouchillon SK, Sahm DF, Bradford PA. 2016. Multiyear, multinational survey of the incidence and global distribution of metallo-β-lactamase-producing Enterobacteriaceae and Pseudomonas aeruginosa. Antimicrob Agents Chemother 60:1067–1078. 10.1128/AAC.02379-15. [Europe PMC free article] [Abstract] [CrossRef] [Google Scholar]
15. Clinical and Laboratory Standards Institute. 2015. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standards, 10th ed CLSI document M07-A10. Clinical and Laboratory Standards Institute, Wayne, PA. [Google Scholar]
16. Clinical and Laboratory Standards Institute. 2017. Performance standards for antimicrobial susceptibility testing; 27th informational supplement. CLSI document M100-S27. Clinical and Laboratory Standards Institute, Wayne, PA. [Google Scholar]
17. Drawz SM, Papp-Wallace KM, Bonomo RA. 2014. New β-lactamase inhibitors: a therapeutic renaissance in an MDR world. Antimicrob Agents Chemother 58:1835–1846. 10.1128/AAC.00826-13. [Europe PMC free article] [Abstract] [CrossRef] [Google Scholar]
18. Papp-Wallace KM, Bonomo RA. 2016. New β-lactamase inhibitors in the clinic. Infect Dis Clin North Am 30:441–464. 10.1016/j.idc.2016.02.007. [Europe PMC free article] [Abstract] [CrossRef] [Google Scholar]
19. ClinicalTrials.gov. 2016. A study to investigate the safety and tolerability of aztreonam-avibactam (ATM-AVI). Registration no. NCT02655419. https://clinicaltrials.gov/ct2/show/NCT02655419?term=NCT02655419&rank=1. Accessed 19 October 2016.
20. Boucher HW, Talbot GH, Bradley JS, Edwards JE, Gilbert D, Rice LB, Scheld M, Spellberg B, Bartlett J. 2009. Bad bugs, no drugs: no ESKAPE! An update from the Infectious Diseases Society of America. Clin Infect Dis 48:1–12. 10.1086/595011. [Abstract] [CrossRef] [Google Scholar]
21. World Health Organization. 2014. Antimicrobial resistance: global report on surveillance. World Health Organization, Geneva, Switzerland: http://www.who.int/drugresistance/documents/surveillancereport/en/. [Google Scholar]
22. Wang X, Zhang F, Zhao C, Wang Z, Nichols WW, Testa R, Li H, Chen H, He W, Wang Q, Wang H. 2014. In vitro activities of ceftazidime-avibactam and aztreonam-avibactam against 372 Gram-negative bacilli collected in 2011 and 2012 from 11 teaching hospitals in China. Antimicrob Agents Chemother 58:1774–1778. 10.1128/AAC.02123-13. [Europe PMC free article] [Abstract] [CrossRef] [Google Scholar]
23. Testa R, Cantón R, Giani T, Morosini MI, Nichols WW, Seifert H, Stefanik D, Rossolini GM, Nordmann P. 2015. In vitro activity of ceftazidime, ceftaroline, and aztreonam alone and in combination with avibactam against European Gram-negative and Gram-positive clinical isolates. Int J Antimicrob Agents 45:641–646. 10.1016/j.ijantimicag.2014.12.033. [Abstract] [CrossRef] [Google Scholar]
24. Dupont H, Gaillot O, Goetgheluck A-S, Plassart C, Emond J-P, Lecuru M, Gaillard N, Derdouri S, Lemaire B, Girard de Courtilles M, Cattoir V, Mammeri H. 2015. Molecular characterization of carbapenem-nonsusceptible enterobacterial isolates collected during a prospective interregional survey in France and susceptibility to the novel ceftazidime-avibactam and aztreonam-avibactam combinations. Antimicrob Agents Chemother 60:215–221. 10.1128/AAC.01559-15. [Europe PMC free article] [Abstract] [CrossRef] [Google Scholar]
25. Vasoo S, Cunningham SA, Cole NC, Kohner PC, Menon SR, Krause KM, Harris KA, De PP, Koh TH, Patel R. 2015. In vitro activities of ceftazidime-avibactam, aztreonam-avibactam, and a panel of older and contemporary antimicrobial agents against carbapenemase-producing Gram-negative bacilli. Antimicrob Agents Chemother 59:7842–7846. 10.1128/AAC.02019-15. [Europe PMC free article] [Abstract] [CrossRef] [Google Scholar]
26. Kazmierczak KM, Biedenbach DJ, Hackel M, Rabine S, de Jonge BL, Bouchillon SK, Sahm DF, Bradford PA. 2016. Global dissemination of blaKPC into bacterial species beyond Klebsiella pneumoniae and in vitro susceptibility to ceftazidime-avibactam and aztreonam-avibactam. Antimicrob Agents Chemother 60:4490–4500. 10.1128/AAC.00107-16. [Europe PMC free article] [Abstract] [CrossRef] [Google Scholar]
27. Bradford PA, Kazmierczak KM, Biedenbach DJ, Wise MG, Hackel M, Sahm DF. 2015. Correlation of β-lactamase production and colistin resistance among Enterobacteriaceae isolates from a global surveillance program. Antimicrob Agents Chemother 60:1385–1392. 10.1128/AAC.01870-15. [Europe PMC free article] [Abstract] [CrossRef] [Google Scholar]
28. Alm RA, Johnstone MR, Lahiri SD. 2015. Characterization of Escherichia coli NDM isolates with decreased susceptibility to aztreonam/avibactam: role of a novel insertion in PBP3. J Antimicrob Chemother 70:1420–1428. 10.1093/jac/dku568. [Abstract] [CrossRef] [Google Scholar]
29. Winkler ML, Papp-Wallace KM, Taracila MA, Bonomo RA. 2015. Avibactam and inhibitor-resistant SHV β-lactamases. Antimicrob Agents Chemother 59:3700–3709. 10.1128/AAC.04405-14. [Europe PMC free article] [Abstract] [CrossRef] [Google Scholar]
30. Lahiri SD, Bradford PA, Nichols WW, Alm RA. 2016. Structural and sequence analysis of class A β-lactamases with respect to avibactam inhibition: impact of Ω-loop variations. J Antimicrob Chemother 71:2848–2855. 10.1093/jac/dkw248. [Abstract] [CrossRef] [Google Scholar]
31. Papp-Wallace KM, Winkler ML, Taracila MA, Bonomo RA. 2015. Variants of β-lactamase KPC-2 that are resistant to inhibition by avibactam. Antimicrob Agents Chemother 59:3710–3717. 10.1128/AAC.04406-14. [Europe PMC free article] [Abstract] [CrossRef] [Google Scholar]
32. Masuda N, Sakagawa E, Ohya S, Gotoh N, Tsujimoto H, Nishino T. 2000. Substrate specificities of MexAB-OprM, MexCD-OprJ, and MexXY-OprM efflux pumps in Pseudomonas aeruginosa. Antimicrob Agents Chemother 44:3322–3327. 10.1128/AAC.44.12.3322-3327.2000. [Europe PMC free article] [Abstract] [CrossRef] [Google Scholar]
33. Pfizer Inc. 2016. Tygacil. U.S. FDA product information. Pfizer, Inc., Collegeville, PA. [Google Scholar]
34. European Committee on Antimicrobial Susceptibility Testing (EUCAST). 2017. Clinical breakpoints. http://www.eucast.org/clinical_breakpoints.
35. Lob S, Kazmierczak K, Badal R, Hackel M, Johnson A, Bouchillon S, Biedenbach D, Sahm D. 2015. Trends in susceptibility of Escherichia coli from intra-abdominal infections to ertapenem and comparators in the United States according to data from the SMART program, 2009 to 2013. Antimicrob Agents Chemother 59:3606–3610. 10.1128/AAC.05186-14. [Europe PMC free article] [Abstract] [CrossRef] [Google Scholar]
36. Nichols WW, de Jonge BLM, Kazmierczak KM, Karlowsky JA, Sahm DF. 2016. In vitro susceptibility of global surveillance isolates of Pseudomonas aeruginosa to ceftazidime-avibactam (INFORM 2012 to 2014). Antimicrob Agents Chemother 60:4743–4749. 10.1128/AAC.00220-16. [Europe PMC free article] [Abstract] [CrossRef] [Google Scholar]
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