Evaluation of the in vitro activity of ceftazidime-avibactam and ceftolozane-tazobactam

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1 AAC Accepted Manuscript Posted Online 14 March 2016 Antimicrob. Agents Chemother. doi: /aac Copyright 2016, American Society for Microbiology. All Rights Reserved. 1 2 Evaluation of the in vitro activity of ceftazidime-avibactam and ceftolozane-tazobactam against meropenem-resistant Pseudomonas aeruginosa isolates Deanna J. Buehrle 1, Ryan K. Shields 2,3, Liang Chen 4, Binghua Hao 3, Ellen G Press 2, Ammar Alkrouk 2, Brian A. Potoski 1, Barry N. Kreiswirth 4, Cornelius J. Clancy 2,3,5*, M. Hong Nguyen 2,3 1 Department of Pharmacy & Therapeutics, University of Pittsburgh, Pittsburgh, Pennsylvania 2 Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 3 XDR Pathogen Laboratory, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania 4 Rutgers University-New Jersey Medical School, Newark, New Jersey 5 VA Pittsburgh Healthcare System, Pittsburgh, Pennsylvania Keywords: Pseudomonas aeruginosa; ceftazidime-avibactam; ceftolozane-tazobactam, carbapenem-resistant Running Title: New β-lactam/β-lactamase inhibitors against P. aeruginosa *Corresponding Author: Cornelius J. Clancy, MD Associate Professor of Medicine University of Pittsburgh 3550 Terrace Street S867 Scaife Hall Pittsburgh, PA Phone: (412) Fax: (412) cjc76@pitt.edu 1

2 30 ABSTRACT We compared ceftazidime-avibactam, ceftolozane-tazobactam, ceftazidime, cefepime and piperacillin-tazobactam minimum inhibitory concentrations (MICs) against 38 meropenemresistant P. aeruginosa. No isolates harbored carbapenemases; 74% were OprD mutants. Ceftazidime-avibactam and ceftolozane-tazobactam were active against 92%, including 80% that were resistant to all three β-lactams. Forty-three percent of ceftazidime-avibactam and 6% of ceftolozane-tazobactam-susceptible isolates exhibited MICs at respective breakpoints. Ceftolozane-tazobactam and ceftazidime-avibactam are therapeutic options against meropenemresistant P. aeruginosa infections, which should be used judiciously to preserve activity. 53 2

3 Pseudomonas aeruginosa has a remarkable propensity to develop antibiotic resistance (1). β-lactam resistance in P. aeruginosa is mediated through several mechanisms, including β- lactamase production, altered membrane permeability, MexA-MexB-OprM efflux pump overexpression, and penicillin-binding protein alterations. Inducible extended-spectrum AmpC cephalosporinases may confer reduced susceptibility to all cephalosporins (2, 3). Carbapenems are stable to AmpC cephalosporinases alone, but activity may be attenuated by combinations of Ambler class A or B β-lactamases, AmpC production, efflux pump up-regulation, and OprD porin mutations (4, 5). Carbapenem-resistant P. aeruginosa are often resistant to antipseudomonal agents like ceftazidime, cefepime and piperacillin-tazobactam (6). Avibactam, a new non-β-lactam β-lactamase inhibitor, inactivates extended-spectrum β- lactamases (ESBLs), AmpC cephalosporinases, and class A (including K. pneumoniae carbapenemases (KPC)), class C, and some class D β-lactamases (7). Ceftazidime-avibactam, an agent recently approved by the United States Food and Drug Administration (FDA), shows promising activity against carbapenem-resistant Enterobacteriaceae (CRE) like KPC-producing K. pneumoniae and E. coli. Carbapenemase production is the primary determinant of carbapenem resistance among CRE. Ceftazidime-avibactam is less certain to be active against carbapenem-resistant P. aeruginosa since resistance mechanisms are multifactorial. Ceftolozane, a novel cephalosporin, has less affinity for hydrolysis by Amp C cephalosporinases, is a weak substrate for drug efflux systems, and is not affected by OprD loss (4, 8-10). The addition of the β-lactamase inhibitor tazobactam broadens the activity of ceftolozane to include most ESBLproducing Gram negative bacilli (11). In this study, we measured ceftazidime-avibactam and ceftolozane-tazobactam activity in vitro against meropenem-resistant P. aeruginosa isolates that 3

4 76 77 exhibited a range of susceptibility and resistance to ceftazidime, cefepime and piperacillin- tazobactam Bloodstream (n=20) and respiratory tract (n=18) isolates were collected from unique patients at the University of Pittsburgh Medical Center. Minimum inhibitory concentrations (MICs) of all agents except ceftolozane-tazobactam were determined in triplicate by reference broth microdilution methods (12). Avibactam was tested at a fixed concentration (4 μg/ml). Ceftolozane-tazobactam MICs were measured by Etest according to manufacturer s recommendations (Biomerieux). MICs were interpreted using CLSI reference breakpoints for ceftazidime, cefepime, piperacillin-tazobactam and meropenem; isolates classified as intermediate or resistant by CLSI criteria were defined as resistant. FDA-approved susceptibility breakpoints of 4 µg/ml and 8 µg/ml were applied for ceftolozane-tazobactam and ceftazidime-avibactam, respectively. E. coli ATCC and P. aeruginosa ATCC were used for quality controls. Polymerase chain reaction (PCR) was used to detect Amber class A (TEM, SHV, CTX-M, GES, PER, VEB and KPC), class B (metallo β-lactamases VIM, IMP, NDM), class C (CMY, MOX, FOX, ACT, DHA) and class D (OXA) β-lactamases. OprD mutations were detected by PCR and DNA sequencing. The contribution of efflux to ceftazidime-avibactam and ceftolozane-tazobactam resistance was assessed with the efflux pump inhibitor carbonyl cyanide m-chlorophenyl hydrazine (CCCP) at a fixed concentration of 12.5 µg/ml. Comparisons between categorical or continuous variables were made by Fisher's exact and Mann-Whitney tests, respectively We tested 38 meropenem resistant-p. aeruginosa isolates [Table 1]. None of the isolates harbored class A, B, C or D β-lactamases. Seventy-four percent (28/38) of isolates carried oprd mutations compared to reference strain PAO1, including non-synonymous mutations (34%, 4

5 /38), premature stop codons (13%, 5/38), absence of oprd (11%, 4/38), and various point mutations (9%, 6/38) Sixty-six percent (25/38), 55% (21/38) and 47% (18/38) of isolates were resistant to piperacillin-tazobactam, ceftazidime and cefepime, respectively [Table 1]. Eighteen percent (7/38), 16% (6/38) and 39% (15/38) of isolates were resistant to 1, 2 or 3 of these agents, respectively. Twenty-six percent (10/38) of isolates were susceptible to all three agents. Median ceftazidime-avibactam and ceftolozane-tazobactam MICs were 4 µg/ml (range: 1 to >32) and 1 µg/ml (range: 0.25 to 64), respectively (p<0.0001). MIC 90 s of ceftazidimeavibactam and ceftolozane-tazobactam were 8 µg/ml and 4 µg/ml, respectively. Ceftazidimeavibactam and ceftolozane-tazobactam MICs were strongly correlated (Spearman R=0.91, p < ). Ceftazidime-avibactam and ceftolozane-tazobactam MICs were moderately to strongly correlated with MICs of cefepime, ceftazidime, piperacillin-tazobactam (R range: 0.86 to 0.93 and 0.84 to 0.98, respectively) [Table 2]. Median ceftazidime-avibactam and ceftolozanetazobactam MICs were higher against isolates that were resistant to cefepime, ceftazidime and piperacillin-tazobactam than against susceptible isolates [Table 2]. Median ceftazidimeavibactam and ceftolozane-tazobactam MICs increased as the number of inactive antipseudomonal drugs increased [Figure 1]. Ceftazidime-avibactam and ceftolozane-tazobactam MICs did not correlate with meropenem MICs (R=0.06 and 0.02, respectively; p=0.70 and 0.91, respectively). Eight percent (3/38) of isolates were resistant to ceftazidime-avibactam, and 8% (3/38) were resistant to ceftolozane-tazobactam; two isolates were resistant to both agents, and one isolate was resistant to each agent but not the other. Among ceftazidime-avibactam susceptible 5

6 isolates, 43% (15/35) exhibited MICs that were at the susceptibility breakpoint (8 µg/ml). In contrast, 6% (2/35) of isolates susceptible to ceftolozane-tazobactam exhibited MICs at the susceptibility breakpoint (4 µg/ml) (p=0.0005, 15/35 vs 2/35). Overall, 47% (18/38) and 13% (5/38) of isolates exhibited ceftazidime-avibactam and ceftolozane-tazobactam MICs that were their respective breakpoints (p=0.003). All isolates that were resistant to either ceftazidime-avibactam or ceftolozane-tazobactam were also resistant to all 3 β-lactam agents. Ceftazidime-avibactam MICs were susceptibility breakpoint against 0% (0/10), 57% (4/7), 67% (4/6) and 67% (10/15) of isolates that were resistant to none, one, two and three β-lactam agents, respectively [Figure 1]. The corresponding rates for ceftolozane-tazobactam MICs susceptibility breakpoint were 0% (0/10), 0% (0/7), 0% (0/6) and 33% (5/15) of isolates, respectively [Figure 1]. Isolates resistant to 2 β-lactams were significantly more likely to exhibit MICs breakpoint for ceftazidime-avibactam 67% (14/21) than ceftolozane-tazobactam (24% (5/21) (p=0.03)). Isolates resistant to three β-lactams were also more likely to exhibit MICs breakpoint for ceftazidime-avibactam (67% (10/15)) than ceftolozane-tazobactam (33% (5/15); p=0.14). Sixty-four percent (18/28) and 30% (3/10) of oprd mutants and wild-type isolates were resistant to cefepime (p=0.08), respectively; 57% (16/28) and 20% (2/10), respectively, were resistant to ceftazidime (p=0.07). There was no difference in piperacillin-tazobactam resistance among oprd mutant (71%, 20/28) and wild-type isolates (50%, 5/10; p=0.26). There were also no differences in ceftazidime-avibactam and ceftolozane-tazobactam MICs or resistance rates among oprd mutant and wild-type isolates. 6

7 To assess efflux, we tested all isolates that were resistant to ceftazidime-avibactam and ceftolozane-tazobactam, as well as randomly selected susceptible isolates. The median fold change in MIC against ceftazidime-avibactam was 0 (range: 0 2) with the addition of CCCP, which did not differ between resistant (n=3) or susceptible (n=4) isolates. Likewise, among ceftolozane-tazobactam resistant (n=3) or susceptible (n=4) isolates, MICs were not significantly reduced in combination with CCCP (median 0-fold change, range: 0 2). To our knowledge, this is the first study to compare in vitro activities of ceftazidimeavibactam and ceftolozane-tazobactam against P. aeruginosa clinical isolates. Our most encouraging finding was that each agent was active against 92% (35/38) of meropenem-resistant P. aeruginosa, including 80% (12/15) of isolates that were resistant to all three anti-pseudomonal β-lactams. Overall, at least one of the new agents retained activity against 92% (23/25), 90% (19/21) and 89% (16/18) of isolates that were resistant to piperacillin-tazobactam, ceftazidime and cefepime, respectively. At the same time, our data provide important cautionary notes. Resistance rates to ceftazidime-avibactam and ceftolozane-tazobactam were 8% (3/38), prior to the introduction of these agents to our hospital. Moreover, ceftazidime-avibactam and ceftolozane-tazobactam MICs correlated with each other and with ceftazidime, cefepime and piperacillin-tazobactam MICs, consistent with some degree of cross-resistance. MICs of both ceftazidime-avibactam and ceftolozane-tazobactam were significantly higher as isolates became resistant to more β-lactams. These results corroborate recent reports of decreased susceptibility to ceftazidime-avibactam and ceftolozane-tazobactam among P. aeruginosa isolates that are resistant to other β-lactams, compared with isolates that are susceptible (3) (13) (14) (15). Taken together, the data demonstrate that ceftazidime-avibactam and ceftolozane-tazobactam are 7

8 important additions to the antimicrobial armamentarium, but suggest that they will need to be used judiciously to preserve their activity Our data suggest that ceftolozane-tazobactam may be more active than ceftazidimeavibactam against meropenem resistant-p. aeruginosa. Isolates were significantly more likely to exhibit MICs susceptibility breakpoint for ceftazidime-avibactam than ceftolozane-tazobactam. In particular, significantly higher percentages of isolates that were resistant to 2 antipseudomonal β-lactams exhibited MICs ceftazidime-avibactam breakpoint than the ceftolozane-avibactam breakpoint. These results should be interpreted with the understanding that definitive breakpoint MICs have not been determined for either agent. The ceftazidimeavibactam breakpoint, for example, is based on a ceftazidime dosing regimen of 1 g every 8 hours as a 30 minute infusion (12), whereas the drug has been administered as 2 g of ceftazidime and 500 mg of avibactam over 2 hours in clinical trials (16) ( It is also important to appreciate that the superior performance of ceftolozane-tazobactam may reflect the particular resistance mechanisms of our P. aeruginosa isolates. The isolates in this study did not carry ESBLs or carbapenemases, which is consistent with previous reports in the United States (17, 18), and efflux was not a significant contributor to either ceftazidimeavibactam or ceftolozane-tazobactam resistance. On the other hand, a sizeable majority exhibited oprd mutations. Avibactam restores the activity of ceftazidime against Gram-negative bacilli with resistance mediated through ESBLs, class A and some class D β-lactamases, and chromosomal and acquired AmpC class C enzymes (19-22). It s reasonable to hypothesize that avibactam restores susceptibility to ceftazidime through inhibition of AmpC enzymes. Indeed, a recent study showed 91% (31/34) of P. aeruginosa strains with unique AmpC sequences demonstrated restored susceptibility to ceftazidime following the addition of avibactam (23). 8

9 Tazobactam extends the activity of ceftolozane against most class A and some class C β- lactamases, but the combination is less active than ceftazidime-avibactam against ESBL- or KPC-producing Gram negative bacteria. Therefore, the isolates in this study were likely better suited to inhibition by ceftolozane-tazobactam than ceftazidime-avibactam; results may differ at centers where ESBLs or carbapenemases are more prominent. Clinicians must understand susceptibility patterns at their institutions. Since resistance mechanisms among P. aeruginosa are complex and multifactorial, detailed molecular characterization of isolates should be incorporated into future studies of antimicrobial regimens (24). We anticipate that ceftolozane-tazobactam will be most useful at our center against infections that are caused by P. aeruginosa resistant to carbapenems and all β-lactams, as the agent is likely to be more active than ceftazidime-avibactam and less toxic than colistin or gentamicin. We anticipate that ceftazidime-avibactam will be most useful against infections caused by CRE, for which β-lactamases and carbapenemases are predominant resistance determinants. Indeed, ceftazidime-avibactam was more active against ESBL- and KPCproducing, carbapenem-resistant K. pneumoniae from our center (range: to 4 µg/ml), than reported here against meropenem-resistant P. aeruginosa (range: 1 to > 32 µg/ml) (25). Further studies are needed to understand how ceftolozane-tazobactam and ceftazidime-avibactam can be best incorporated into clinical practice, in a manner that optimizes effectiveness while minimizing the emergence of resistance

10 209 Acknowledgements This project was supported by funding provided to the XDR Pathogen Laboratory by the University of Pittsburgh Medical Center, and by the National Institutes of Health (NIH) under award numbers K08AI (R. K. S.) and R21AI (C. J. C.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. Downloaded from on October 29, 2018 by guest 10

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15 Table 1. In vitro susceptibility data on 38 meropenem-resistant P. aeruginosa isolates Antimicrobial Median MIC MIC 90 Range % Resistance agents (µg/ml) (µg/ml) (µg/ml) Meropenem % Cefepime >512 47% Ceftazidime > % Piperacillintazobactam > % Ceftazidimeavibactam >32 8% Ceftolozanetazobactam %

16 Table 2. Correlations between MICs of ceftazidime-avibactam and ceftolozane-tazobactam MICs and those of other β-lactam agents. Cefepime Median ceftazidimeavibactam MIC (µg/ml) Median ceftolozanetazobactam MIC (µg/ml) Susceptible isolates (n=20) Resistant isolates (n=18) 8 2 p-values < Correlation between cefepime and indicated agent 1 Ceftazidime Susceptible isolates (n=17) Resistant isolates (n=21) p-values < Correlation between ceftazidime and indicated agent 1 Piperacillin-tazobactam Susceptible isolates (n=13) Resistant isolates (n=25) 8 1 p-values < Correlation between piperacillintazobactam and indicated agent 1 Correlation between 2 agents were determined using Spearman correlation coefficient R.

17 Figure 1. Correlation between ceftazidime-avibactam (A) and ceftolozanetazobactam (B) MICs against meropenem-resistant P. aeruginosa and the number of inactive β-lactam agents A. B. P=0.05 P=0.56 P=0.003 P=0.70 P=0.29 P= Footnote. Horizontal red lines represent the FDA-proposed susceptibility breakpoint for each agent. Median MICs were compared between isolates resistant to zero, one, two, or three β-lactam agents. P=0.04 P=0.76 P=0.04 P=0.02 P=0.008 P=0.003