In Vitro Discordance with In Vivo Activity: Humanized Exposures of Ceftazidime-Avibactam, Aztreonam, and Tigecycline Alone and in Combination against New Delhi Metallo-β-Lactamase-Producing Klebsiella pneumoniae in a Murine Lung Infection Model

TEXT

New Delhi metallo-β-lactamase (NDM) is a carbapenemase that efficiently hydrolyzes carbapenems and other β-lactams (1). As the prevalence of NDM carbapenemases continues to rise globally, effective antibiotic therapy is limited, increasing the demand for novel antimicrobials and synergistic combinations (2). Ceftazidime-avibactam (CZA) is novel non-β-lactam-β-lactamase inhibitor-cephalosporin combination, which is shown to be stable in vitro against Ambler class A and C (and some class D) β-lactamases; yet, against NDM-producing organisms, the antibiotic often displays resistant MICs (3). However, the expression of the NDM in vivo was shown to be unremarkable against CZA, previously demonstrating antibacterial activity independent of high MICs in a murine thigh infection model (4). Other antimicrobials with potential in vivo activity are tigecycline, given its in vitro susceptibility to the organism of interest, and aztreonam (ATM), which is not hydrolyzed by NDM (5).

Humanized doses of tigecycline (lot AJP312; Wyeth Pharmaceuticals, Inc., Dallas, TX) of 50 mg every 12 h (q12h) and 100 mg q12h, ATM (lot 6010677; Fresenius Kabi USA, LLC., Lake Zurich, IL) of 2 g q6h, and CZA (lot V005; GlaxoSmithKline, Verona, Italy; and lot 150901; Tecoland Corp., Irvine, CA) of 2.5 g q8h were prepared to produce exposures consistent with previously published data for each agent (6–8).

One Klebsiella pneumoniae strain provided by Jackson Health System in Miami, FL, was selected for this study. Previous molecular characterizations of the K. pneumoniae isolate revealed bla_NDM, bla_OXA-48, and bla_CTX-M expression (9). MICs for CZA, tigecycline, and ATM were initially determined using Etest strips on inoculated Mueller-Hinton (MH) agar according to the manufacturer's guidelines. Comparator antibiotic MICs, as well as those for CZA, tigecycline, and ATM, were subsequently confirmed in triplicates via broth microdilution according to the 2016 Clinical and Laboratory Standards Institute (CLSI) guidelines (10). For synergy testing, the combinations of Etest strips were placed on the same culture medium in a cross formation, with a 90° angle at the intersection between the scales at the respective MICs for K. pneumoniae, and the plates were incubated at 37°C for 24 h. The MICs were interpreted at the point of intersection between the inhibition zone and the Etest strip (9, 10). The fractional inhibitory concentration (FIC) index (ΣFIC) was calculated on the basis of the resultant zone of inhibition as follows: ΣFIC = FIC A + FIC B, where FIC A is the MIC of the combination/MIC of drug A alone, and FIC B is the MIC of the combination/MIC of drug B alone (11).

In the neutropenic lung model, specific-pathogen-free female ICR (CD-1) mice weighing 20 to 22 g were obtained from Envigo RMS, Inc. (Indianapolis, IN). The protocol was reviewed and approved by the Institutional Animal Care and Use Committee at Hartford Hospital, Hartford, CT. A bacterial suspension of approximately 10⁷ CFU/ml was made for the inoculations. Mice were anesthetized with isoflurane and inoculated with 0.05 ml of the infecting K. pneumoniae isolate in 3% porcine stomach mucin (Sigma-Aldrich, St. Louis, MO). The inoculum was administered into the nares of the mice using a pipette with an appropriate tip attached.

Two hours postinfection, groups of six mice received human simulated regimens of CZA, ATM, and tigecycline alone and in combination administered as 0.2-ml subcutaneous injections. Control animals (24 h) received 0.2 ml of 0.9% normal saline solution subcutaneously in a frequency identical to the most frequently dosed drug regimen. After sacrifice, lungs were aseptically harvested, rinsed with normal saline, and then homogenized in 1.0 ml of normal saline. Serial dilutions of lung homogenate were plated onto Trypticase soy agar with 5% sheep blood (BD Biosciences, Sparks, MD) and incubated at 37°C for 24 h. The change in bacterial density was calculated as the difference in log₁₀ CFU from the antibiotic-treated mice after 24 h from the level in the 0-h control animals. Synergy was defined as a ≥2-log₁₀ drop in CFU/ml from the count obtained with the more active agent at 24 h (12).

The molecular characteristics of K. pneumoniae strain 518 were NDM, OXA-48, and CTX-M positive. This strain demonstrated resistance to all antimicrobials tested, with the exception of tigecycline (MIC, 1 μg/ml), with MICs of >64 μg/ml for ceftazidime, ceftriaxone, cefepime, imipenem, meropenem, and tobramycin, MICs of >16 μg/ml for ciprofloxacin, colistin, and ertapenem, and MICs of >512 μg/ml for amikacin, 64 μg/ml for aztreonam, >128/4 μg/ml for ceftazidime-avibactam, >64/4 μg/ml for ceftolozane-tazobactam, and >256/4 μg/ml for piperacillin-tazobactam. Figure 1 demonstrates K. pneumoniae 518 Etest MICs for CZA, tigecycline, and ATM before synergy testing. Etest MICs were ≥256, 1, and ≥256 μg/ml for CZA, tigecycline, and ATM, respectively. Figure 2 shows in vitro synergy between CZA and ATM (ΣFIC < 0.5), but not between CZA and tigecycline.

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FIG 1

K. pneumoniae 518 MIC determinations for ceftazidime-avibactam (A), tigecycline (B), and aztreonam (C).

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FIG 2

K. pneumoniae 518 synergy interpretations for ceftazidime-avibactam and tigecycline (A) and ceftazidime-avibactam and aztreonam (B).

Figure 3 illustrates the antibacterial efficacy of human simulated doses of CZA, aztreonam, tigecycline 50 mg (50 mg q12h), and tigecycline 100 mg (100 mg q12h) alone and in combination against K. pneumoniae 518. For K. pneumoniae 518, the bacterial density increased by 2.66 log₁₀ CFU in control animals at 24 h. CZA alone produced a 2.43-log₁₀ CFU reduction at 24 h. When administered with aztreonam, the log₁₀ CFU reduction increased to 2.54. CZA plus tigecycline 100 mg or CZA plus tigecycline 100 mg plus ATM did not improve bacterial reduction compared with that from CZA alone producing log₁₀ CFU values of 2.15 and 2.18, respectively. ATM, tigecycline 50 mg, and tigecycline 100 mg demonstrated increases in bacterial growth at 24 h compared with that at 0 h of 2.33, 2.36, and 1.44 log₁₀ CFU, respectively.

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FIG 3

Efficacy (mean change in log₁₀ CFU ± standard deviation) of human simulated CZA at 2.5 g q8h, ATM at 2 g q6h, tigecycline at 50 mg q12h (TGC 50), and tigecycline at 100 mg q12h (TGC 100) alone and in combination against K. pneumoniae 518 (n = 6 for each group).

Despite the in vitro resistance observed for individual compounds, in vitro synergy was demonstrated with CZA and ATM, similar to previous in vitro work, providing us with a “zone of hope” regarding viable therapeutic approaches for this extensively drug resistant (XDR) organism (9, 13). Consistent with high rates of clinical failure reported in cancer patients with pneumonia treated with tigecycline, the K. pneumoniae 518 in vitro susceptibility to tigecycline was not translated to the neutropenic murine lung model at humanized doses reflective of 50 and 100 mg q12h (14). These tigecycline doses were based on a previous murine thigh model regimen that demonstrated in vivo efficacy against extended-spectrum-β-lactamase-producing Escherichia coli and K. pneumoniae. Furthermore, tigecycline demonstrated efficacy in a murine pneumonia model against Staphylococcus aureus, thereby eliminating an inadequate pharmacokinetic model or animal model selection as the cause of tigecycline failure (6, 15). The humanized dose of CZA monotherapy resulted in extensive in vivo killing, and as a result, synergy was not observed with combinations. This in vitro/in vivo discordance may be attributed to the broad serine β-lactamase inhibition by CZA and the poor in vivo expression of NDM, as previously described (4). Based on these and previous in vivo observations, additional investigation should be undertaken to determine the clinical utility of CZA as a monotherapy or in combination with other agents in the face of evolving metallo-β-lactamases. Furthermore, the in vivo activity of this in vitro synergistic combination in other sterile body fluids is unknown and should be further investigated.