In Vitro and In Vivo Evaluations of β-Lactam/β-Lactamase Mono- and Combined Therapies against Carbapenem-Nonsusceptible Enterobacteriaceae in Taiwan

Increasing carbapenem resistance rates worldwide underscored the urgent need of novel antimicrobials. Ceftazidime–avibactam and aztreonam–avibactam combinations are developed to combat carbapenem resistance, but biological and geographic variations must be considered for antibiotic susceptibility patterns varied. Thus, we sought to assess the susceptibilities of ceftazidime–avibactam and aztreonam–avibactam against 660 carbapenem-nonsusceptible Enterobacteriaceae isolates (472 Klebsiella pneumoniae and 188 Escherichia coli) collected during an earlier Taiwan surveillance study. Agar dilution method was used to determine ceftazidime–avibactam and aztreonam–avibactam susceptibility. Metallo-carbapenemase’s contribution to resistance were investigated with EDTA addition. The in vivo efficacies were evaluated using a Caenorhabditis elegans model. High susceptibility rates were observed for ceftazidime–avibactam and aztreonam–avibactam against the 472 carbapenem-nonsusceptible K. pneumoniae (CnsKP) (85.2% and 95.3%, respectively) and 188 carbapenem-nonsusceptible E. coli (CnsEC) isolates (91.5% and 94.1%, respectively). For non-metallo-carbapenemase producers, the susceptibility rates for ceftazidime–avibactam were 93.6% for CnsKP and 97.7% for CnsEC, whereas only 7.1% CnsKP and 11.1% CnsEC in metallo-carbapenemase producers were susceptible to ceftazidime–avibactam. Of all isolates, 95.3% CnsKP and 94.1% CnsEC were susceptible to aztreonam–avibactam. In C. elegans model, ceftazidime–avibactam and aztreonam–avibactam revealed effective against a blaKPC-producing K. pneumoniae isolate in vivo. Our results propose a positive therapeutic approach for both combinations against carbapenem-nonsusceptible Enterobacteriaceae in Taiwan.


Introduction
Due to the rapid dissemination of resistant genes and the over-prescription and overconsumption of carbapenems, health care professionals all over the world are facing challenges associated with carbapenem-resistant Enterobacteriaceae (CRE) infections, with treatments costing billions of dollars [1]. Carbapenem resistance mechanisms are associated with the production of transmittable carbapenemases, the loss of porins in combination with bla AmpC β-lactamase overexpression, and active efflux pumps [2]. Global epidemiological studies in the Asia-Pacific region, the Indian subcontinent, Europe, North America, and Latin America indicate carbapenem resistance rates of up to 58.6% in Enterobacteriaceae, with significantly higher rates in Europe and India [3]. In Taiwan, 10.5% (71/673) K. pneumoniae bloodstream isolates collected in 2017 were not susceptible to at least one carbapenem [4].
Ceftazidime-avibactam, a β-lactam-plus-β-lactamase inhibitor combination that received US FDA approval in 2015, has been described as having anti-CRE efficacy, except for metallo-beta-lactamase producers [5]. Another CRE infection treatment option that is currently in phase III clinical trials is the combination of aztreonam and avibactam (NCT03580044 and NCT03329092) [6]. However, local antibiotic susceptibility patterns are important when prescribing these new agents empirically and before the metallo-beta-lactamase producers were identified [7]. In a SIDERO-WT-2014 study, different resistance rates in North America (3.3%) and Europe (28.1%) were observed for ceftazidime-avibactam in meropenem-nonsusceptible Enterobacteriaceae [7]. In another SIDERO-WT-2014 study, the authors reported that the KPC-type enzymes were the dominant carbapenemase carriage in both North America and Europe, but metallo-carbapenemases (NDM, VIM, or IMP) were mainly found in European isolates [8]. From their results of antimicrobial susceptibility testing, ceftazidime-avibactam was noted with poor activities against metallo-carbapenemase producers. For the present research we assessed the in vitro and in vivo efficacies of ceftazidime-avibactam and aztreonam-avibactam against 660 carbapenem-nonsusceptible Enterobacteriaceae isolates collected as part of a nationwide surveillance project in Taiwan. Bioinformatic analyses were performed to clarify our results and to identify factors affecting susceptibility.
Standard agar dilution tests were used to measure the MICs of β-lactam/β-lactamase inhibitor combinations. Avibactam (AVI) was assessed at a concentration of 4 mg/L in combination with 2-fold dilutions of ceftazidime (CAZ) or aztreonam (AZT) [11]. CAZ and AZT monotherapy MIC values were also determined using the agar dilution method. In all, 23 isolates carrying various metallo-carbapenemases were used to estimate MIC values with or without EDTA at 320 mg/L [12].

In Vivo Caenorhabditis elegans Study
C. elegans strain N2 was used to evaluate the treatment effects of CAZ-AVI and AZT-AVI combinations against the KPC-producing K. pneumoniae clinical isolate CRE-1462, a member of sequence type 11, the most prevalent in Taiwan [10]. Nematodes were maintained at 20 • C on growth medium agar plates with the OP50 non-toxic E. coli laboratory strain. Protocols are described in detail in an earlier report [15]. Briefly, 700-1000 growth-synchronized L4 worms were infected with CRE-1462 for 3 days, and 40 infected worms were transferred onto nematode growth medium (NGM) agar with either a placebo, β-lactam alone (CAZ or AZT), or a β-lactam/β-lactamase inhibitor combination (CAZ-AVI or AZT-AVI). Antibiotic concentrations were 8 mg/L for CAZ and 4 mg/L for AZT, alone or in combination. Avibactam was examined at a fixed concentration of 4 mg/L in combination with individual antibiotics. Nematode survival was monitored daily; surviving worms were transferred onto new plates and treated at the same concentrations. Assays were performed in triplicate.

Statistical Analyses
Antimicrobial susceptibility test and gene detection results were visualized as ggplot2 package heatmaps using RStudio (version 1.1.453). The log 2 -transformed MIC values were used for statistical analyses using GraphPad Prism Version 7.0 software (San Diego, CA, USA) with paired t-tests. Log-rank (Mantel-Cox) tests in the same software package were used to create Kaplan-Meier survival test curves.

Combination Therapy Efficacy Against Metallo-Carbapenemase Producers
Low antibacterial activity for the combination of ceftazidime with avibactam was observed in 23 class B metallo-carbapenemase producers ( Table 1). The heatmap shown as Figure 4 presents MIC values for all 23, along with their species and carbapenemase classifications. Among them, blaIMP and blaVIM were found in 9 and 5 CnsKP isolates, respectively, and blaIMP, blaVIM and blaNDM were found in 1, 3, and 5 CnsEC isolates, also respectively ( Figure 4). Similar MIC patterns were observed for ceftazidime alone and ceftazidime with avibactam, indicating weak effectiveness against metallocarbapenemase producers. In contrast, strong in vitro activity against the same isolates was observed for the combination of aztreonam with avibactam.

Combination Therapy Efficacy Against Metallo-Carbapenemase Producers
Low antibacterial activity for the combination of ceftazidime with avibactam was observed in 23 class B metallo-carbapenemase producers ( Table 1). The heatmap shown as Figure 4 presents MIC values for all 23, along with their species and carbapenemase classifications. Among them, bla IMP and bla VIM were found in 9 and 5 CnsKP isolates, respectively, and bla IMP , bla VIM and bla NDM were found in 1, 3, and 5 CnsEC isolates, also respectively ( Figure 4). Similar MIC patterns were observed for ceftazidime alone and ceftazidime with avibactam, indicating weak effectiveness against metallo-carbapenemase producers. In contrast, strong in vitro activity against the same isolates was observed for the combination of aztreonam with avibactam. EDTA was added to agar at various concentrations for each regimen to assess metallocarbapenemase contributions to the efficacies of the three combination therapies. A statistically significant difference was observed between ceftazidime and ceftazidime with avibactam in the presence of EDTA (p < 0.0001), but not in its absence (Figure 5a). We found that EDTA inhibited metallo-carbapenemase and recovered the strength of ceftazidime with avibactam in vitro. The presence of blaESBL genes in the 23 metallo-carbapenemase producers might explain the reduction in activity observed in ceftazidime alone. Avibactam was capable of restoring the antibacterial efficacy of aztreonam in either the presence or absence of EDTA, with significant decreases in MIC values (both p < 0.0001) (Figure 5b). It did not have the same effect when added to ceftazidime (Figure 5a).  EDTA was added to agar at various concentrations for each regimen to assess metallo-carbapenemase contributions to the efficacies of the three combination therapies. A statistically significant difference was observed between ceftazidime and ceftazidime with avibactam in the presence of EDTA (p < 0.0001), but not in its absence (Figure 5a). We found that EDTA inhibited metallo-carbapenemase and recovered the strength of ceftazidime with avibactam in vitro. The presence of bla ESBL genes in the 23 metallo-carbapenemase producers might explain the reduction in activity observed in ceftazidime alone. Avibactam was capable of restoring the antibacterial efficacy of aztreonam in either the presence or absence of EDTA, with significant decreases in MIC values (both p < 0.0001) (Figure 5b). It did not have the same effect when added to ceftazidime (Figure 5a).  EDTA was added to agar at various concentrations for each regimen to assess metallocarbapenemase contributions to the efficacies of the three combination therapies. A statistically significant difference was observed between ceftazidime and ceftazidime with avibactam in the presence of EDTA (p < 0.0001), but not in its absence (Figure 5a). We found that EDTA inhibited metallo-carbapenemase and recovered the strength of ceftazidime with avibactam in vitro. The presence of blaESBL genes in the 23 metallo-carbapenemase producers might explain the reduction in activity observed in ceftazidime alone. Avibactam was capable of restoring the antibacterial efficacy of aztreonam in either the presence or absence of EDTA, with significant decreases in MIC values (both p < 0.0001) (Figure 5b). It did not have the same effect when added to ceftazidime (Figure 5a).

In Vivo C. Elegans Study
A C. elegans model was used to evaluate the in vivo efficacies of the two combination therapies against a randomly selected carbapenem-resistant K. pneumoniae isolate (CRE-1462) carrying the bla KPC gene, the most common carbapenemase gene in Taiwan. Compared to CRE-1462-infected nematodes subjected to ceftazidime monotherapy, the median survival time of nematodes treated with the ceftazidime-avibactam combination increased significantly (p < 0.0001) (Figure 6a). A strong treatment effect was also noted for the aztreonam with avibactam group (p < 0.0001), with a significant right-shift curve compared to the single-agent therapy group (Figure 6b).

In Vivo C. Elegans Study
A C. elegans model was used to evaluate the in vivo efficacies of the two combination therapies against a randomly selected carbapenem-resistant K. pneumoniae isolate (CRE-1462) carrying the blaKPC gene, the most common carbapenemase gene in Taiwan. Compared to CRE-1462-infected nematodes subjected to ceftazidime monotherapy, the median survival time of nematodes treated with the ceftazidime-avibactam combination increased significantly (p < 0.0001) (Figure 6a). A strong treatment effect was also noted for the aztreonam with avibactam group (p < 0.0001), with a significant right-shift curve compared to the single-agent therapy group (Figure 6b). Median survival time for infected nematodes either treated with ceftazidime monotherapy or untreated was two days ( Table 2). Treatment with the ceftazidime-avibactam combination extended median survival to 4 days, with a significant 0.472 hazard ratio (HR) reduction (95% confidence interval (CI) 0.295 to 0.756) (p < 0.0001). Compared to the aztreonam monotherapy group, median time for the combined aztreonam-avibactam group increased from 2 to 4 days (HR 0.420; 95% CI 0.260 to 0.679) (p < 0.0001). In sum, our data indicate that both combination therapies were capable of rescuing the C. elegans model infected with a carbapenem-resistant K. pneumoniae isolate.

Discussion
There are currently many reports of carbapenem-resistant Enterobacteriaceae (CRE) worldwide, with limited clinical therapeutic options due to multidrug resistance [2,[16][17][18]. In one international study of 267 metallo-carbapenemase Enterobacteriaceae isolates, resistance rates to ceftazidime, Median survival time for infected nematodes either treated with ceftazidime monotherapy or untreated was two days ( Table 2). Treatment with the ceftazidime-avibactam combination extended median survival to 4 days, with a significant 0.472 hazard ratio (HR) reduction (95% confidence interval (CI) 0.295 to 0.756) (p < 0.0001). Compared to the aztreonam monotherapy group, median time for the combined aztreonam-avibactam group increased from 2 to 4 days (HR 0.420; 95% CI 0.260 to 0.679) (p < 0.0001). In sum, our data indicate that both combination therapies were capable of rescuing the C. elegans model infected with a carbapenem-resistant K. pneumoniae isolate.
The aztreonam-avibactam combination is currently undergoing phase III clinical trials as an option for treating carbapenem-resistant Enterobacteriaceae infections [23]. In a study involving Gram-negative pathogens collected in 2012 and 2013 from 190 medical centers in 39 countries, 577 of 23,516 Enterobacteriaceae isolates were identified as meropenem-nonsusceptible (aztreonam MIC 50 and MIC 90 values both >128 mg/L) [24]. In that study, avibactam effectively restored aztreonam efficacy and reduced MIC 50 and MIC 90 values to 0.25 mg/L and 1 mg/L, respectively. A separate global study of aztreonam-avibactam antimicrobial activity involved 1498 meropenem-nonsusceptible Enterobacteriaceae clinical isolates collected in 40 countries in 2017 [19]. MIC 50 and MIC 90 values for the aztreonam monotherapy were both >128 mg/L. Aztreonam-avibactam results included an MIC 50 of 0.25 mg/L and MIC 90 of 1 mg/L (99.2% susceptibility, 1486/1498). In a study involving 177 carbapenemase-producing Enterobacteriaceae isolates collected in Singapore and the US [25], MIC 50 and MIC 90 values for aztreonam alone against different carbapenemase classes were 128-512 mg/L and >512 mg/L, respectively. For the combination of aztreonam and avibactam they ranged from 0.12 to 0.25 mg/L and from 0.5 to 1 mg/L, also respectively. We found that avibactam significantly restored aztreonam activity (p < 0.0001) ( Table 1 and Figure 2), with high levels of CnsEC (94.1%) and CnsKP (95.3%) susceptibility (Figure 3b,d).
Based on evidence showing β-lactamases (both bla AmpC and carbapenemases) as contributing to carbapenem resistance [2], β-lactamase inhibitors such as avibactam and relebactam have been examined as candidates for treating carbapenem-resistant Gram-negative bacilli [5]. Avibactam and relebactam belong to a class of bi-cyclic diazabicyclooctane β-lactamase inhibitors that only act against serine β-lactamases [22,26]. Specifically, avibactam is active against class A, C, and D β-lactamases [27], and relebactam mostly inhibits class A and C and a small number of class D β-lactamases [28]. Aztreonam, which is active against metallo-β-lactamase-(MBL-) producing bacteria, is subject to hydrolyzation by class A or D β-lactamases [23]. The combination of aztreonam with avibactam (a class A or D β-lactamase inhibitor) expresses antimicrobial activity against bacteria that carry MBL with class A or D β-lactamases [29].
In a previous study, 177 carbapenemase-producing Gram-negative bacilli isolates (116 class A, 47 class B, and 14 class D) were examined to determine ceftazidime-avibactam and aztreonam-avibactam susceptibilities [25]. Both combinations were found to be effective against all class A (108 bla KPC , 5 bla IMI and 3 bla SME ) and class D carbapenemase isolates, with susceptibilities ranging from 93% to 100%. In comparison, low susceptibility values were noted for all 47 class B carbapenemase isolates (32 bla NDM , 11 bla IMP , and 4 bla VIM ) treated with ceftazidime-avibactam (0-9%). High susceptibility values were observed following aztreonam-avibactam treatment (94-100%). In summary, we found that the ceftazidime-avibactam combination was generally ineffective against class B carbapenemase-producing isolates, while the aztreonam-avibactam combination exhibited robust efficacy in all carbapenemase-producing isolate classes (Table 1; Figure 4). Ceftazidime-avibactam activity was restored by the addition of EDTA, further evidence of the MBL effect ( Figure 5). Several research teams have described the efficacy of the ceftazidime-avibactam combination in vivo [30][31][32], but little is known about the combination of aztreonam with avibactam. In one study involving mice infected with carbapenemase-producing K. pneumoniae, 100% of those treated with ceftazidime-avibactam survived, and 70% treated with a placebo died within 4 days [30]. In a retrospective clinical study, the 30-day mortality rate for 104 patients infected with bla KPC -carrying K. pneumoniae decreased significantly following treatment with ceftazidime-avibactam (p = 0.005, 36.5% vs. 55.8% for other therapies) [31]. For our study we infected a C. elegans model in vivo with a randomly selected CRE-1462 bla KPC -containing K. pneumoniae clinical isolate and measured the effects of treatment with either ceftazidime-avibactam or aztreonam-avibactam. Significant right-shifts in survival curves were observed in both treatment groups (both p < 0.0001) ( Figure 5), with extended median survival times of 2-4 days ( Table 2). In addition to suggesting the in vivo efficacy of ceftazidime-avibactam, our data also indicate in vivo aztreonam-avibactam efficacy against a bla KPC -producing K. pneumoniae clinical isolate.