Comparing the activity of novel antibiotic agents against carbapenem-resistant Enterobacterales clinical isolates

Abstract Objective: We compared the activity of 8 novel β-lactam and tetracycline-derivative antibiotics against a cohort of clinical carbapenem-resistant Enterobacterales (CRE) isolates and investigated the incremental susceptibility benefit of the addition of an aminoglycoside, fluoroquinolone, or polymyxin to the β-lactam agents to assist with empiric antibiotic decision making. Methods: A collection of consecutive CRE clinical isolates from unique patients at 3 US hospitals (2016–2021) was assembled. Broth microdilution was performed to obtain antimicrobial susceptibility testing results. Mechanisms of carbapenem resistance were investigated through short-read and long-read whole-genome sequencing. Results: Of the 603 CRE isolates, 276 (46%) were carbapenemase producing and 327 (54%) were non–carbapenemase producing, respectively. The organisms most frequently identified were Klebsiella pneumoniae (38%), Enterobacter cloacae complex (26%), and Escherichia coli (16%). We obtained the following percent susceptibility to novel β-lactam agents: ceftazidime-avibactam (95%), meropenem-vaborbactam (92%), imipenem-relebactam (84%), and cefiderocol (92%). Aminoglycosides and the polymyxins provided greater incremental coverage as second agents, compared to fluoroquinolones. Amikacin and plazomicin exhibited the greatest additive value. Ceftazidime-avibactam, meropenem-vaborbactam, and cefiderocol were active against 94% of the 220 KPC-producing isolates. Cefiderocol was active against 83% of the 29 NDM-producing isolates. Ceftazidime-avibactam had 100% activity against the 9 OXA-48-like–producing isolates. Tigecycline had the highest activity compared to other tetracyclines against KPC, NDM, or OXA-48-like–producing isolates. Conclusion: Selection among novel agents requires a nuanced understanding of the molecular epidemiology of CRE. This work provides insights into the comparative activity of novel agents and the additive value of a second antibiotic for empiric antibiotic decision making.

Conclusion: Selection among novel agents requires a nuanced understanding of the molecular epidemiology of CRE. This work provides insights into the comparative activity of novel agents and the additive value of a second antibiotic for empiric antibiotic decision making. Within the past decade, 3 novel β-lactam-β-lactamase inhibitor combinations with activity against carbapenem-resistant Enterobacterales (CRE) have received US Food and Drug Administration (FDA) approval: ceftazidime-avibactam, meropenem-vaborbactam, and imipenem-cilastatin-relebactam. [1][2][3] Furthermore, a novel siderophore-cephalosporin conjugate (ie, cefiderocol), 4 aminoglycoside (ie, plazomicin), 5 and tetracycline derivatives (ie, eravacycline and omadacycline) 6,7 have also been introduced into the clinical arena in recent years. Understanding the comparative activity of these novel antibiotics is critical to avoiding unnecessary delays in effective therapy, particularly because CRE tend to infect vulnerable medical populations at high risk of mortality. Moreover, the introduction of rapid molecular diagnostics capable of identifying carbapenemase genes prior to antimicrobial susceptibility testing (AST) results further underscores the importance of understanding the likelihood of each novel agent's activity against specific carbapenemase families.
Comprehensive investigations into the relative activity percentages of various novel agents against CRE are limited. Studies funded by pharmaceutical companies often limit evaluation to novel agents they have developed and marketed, and generally select traditional agents as comparators, rather than other novel ones. We evaluated the activity of 8 novel antibiotics against a cohort of consecutive CRE clinical isolates and investigated the incremental benefit in susceptibility percentage with the addition of a second agent (ie, aminoglycosides, fluoroquinolones, or polymyxins) to novel β-lactam agents to assist with empiric antibiotic decision making.

Description of isolates
From June 1, 2016, to June 30, 2021, a cohort of consecutive CRE clinical isolates was assembled. CRE were defined as isolates (1) exhibiting resistance to at least 1 carbapenem agent or (2) carrying at least 1 carbapenemase gene. 8 CRE isolates were obtained from clinical specimens collected from patients receiving medical care at The Johns Hopkins Hospital, Bayview Medical Center, and Howard County General Hospital, all located in Maryland. Only the first isolate was included for patients who had multiple cultures growing the same species (eg, carbapenem-resistant Escherichia coli recovered in multiple specimens from the same patient). However, if different carbapenem-resistant species were recovered from the same patient (eg, E. coli and Klebsiella pneumoniae), the first isolate of each species was included.
Bacterial genus and species were identified using matrixassisted laser-desorption ionization time-of-flight mass spectrometry (Bruker Daltonics, Billerica, MA). Antimicrobial susceptibility testing (AST) results were determined using the BD Phoenix Automated System (BD Diagnostics, Sparks, MD) and interpreted following Clinical Laboratory and Standards Institute (CLSI) guidelines. 9 All CRE isolates were stored at −80°C in glycerol until further testing was performed.

Statistical analysis
The χ 2 test was used to evaluate differences between susceptibility proportions across drug-organism combinations for each of the 4 novel β-lactams and 4 tetracycline derivatives, followed by post-hoc tests of pairwise comparisons between agents. Bonferroni corrections of P values were applied for the 4 β-lactams (group 1) and for the 4 tetracycline-derivatives (group 2), separately. The χ 2 test was also used to compare susceptibilities between CP-CRE and non-carbapenemase-producing CRE (non-CP-CRE). Statistical analyses were performed using R version 4.1.1 software (R Foundation for Statistical Learning, Vienna, Austria).

CP-CRE and non-CP-CRE
Of the 603 CRE isolates, 276 (46%) and 327 (54%) were CP-CRE and non-CP-CRE, respectively. Of the 327 non-CP-CRE isolates, the most common identified resistance mechanisms included the presence of extended-spectrum β-lactamase (ESBL) genes and/or ampC genes in conjunction with porin mutations or loss (eg, ompK35 and ompK36), which were identified in 249 (76%) of non-CP-CRE isolates. Table 1 describes the species recovered categorized by the presence of carbapenemase production and the percent susceptibility to 8 last-resort antibiotic agents. No significant differences were detected between susceptibilities in CP-CRE and non-CP-CRE across any of the 8 β-lactam or tetracycline-derivative antibiotics.

Additive value of combination therapy
The incremental benefit of agents frequently combined as components of combination therapy (ie, aminoglycosides, fluoroquinolones, or polymyxins) when added to a novel β-lactam agent was investigated (Fig. 1). The calculations displayed in Figure 1 reflect isolates with in vitro susceptibility to either the β-lactam or the additive agent. Organisms known to be intrinsically resistant to the polymyxins were removed from the analysis, including Morganella spp, Proteus spp, Providencia spp, and Serratia spp. Generally, aminoglycosides and polymyxins provided greater incremental benefit as second agents compared to the fluoroquinolones. The percentages of susceptibility to ciprofloxacin and levofloxacin were identical, and neither agent provided substantial additive value to any of the β-lactam agents. Of aminoglycosides, plazomicin, and amikacin provided the greatest additive value, providing nearly identical incremental benefits ranging from an additional 4%-11% compared to β-lactam therapy alone (all P values <.001). The β-lactam that benefitted the most from the addition of a second agent was imipenemrelebactam.

Discussion
Evaluating a cohort of 603 consecutive clinical CRE isolates, ceftazidime-avibactam and tigecycline were the β-lactam and tetracycline-derivatives, respectively, with the highest likelihood of activity, regardless of whether organisms were carbapenemase producing or not. When specific carbapenemase genes were identified, the following β-lactams had the highest activity: KPC-producing (ceftazidime-avibactam, meropenem-vaborbactam, and cefiderocol at 94%), NDM-producing (cefiderocol, 83%), and OXA-48-like-producing (ceftazidime-avibactam, 100%). These findings underscore the important role of carbapenemase gene identification in guiding antibiotic decision making. 12 Moreover, we investigated the incremental benefit of adding an aminoglycoside, fluoroquinolone, or polymyxin to each of the novel β-lactams to determine whether they substantively increased the likelihood of activity against CRE isolates for empiric antibiotic decision making. Clinical trial data comparing the outcomes of patients with CRE infections treated with combination therapy (eg, ceftazidime-avibactam and amikacin) versus β-lactam monotherapy (eg, ceftazidime-avibactam) are not available. An observational study comparing the outcomes of 577 patients receiving ceftazidime-avibactam or ceftazidime-avibactam plus a second agent for the treatment of KPC-producing infections did not identify a mortality benefit with this approach. 13 However, for ill-appearing patients known to be colonized with CRE or in regions of high CRE endemicity, the addition of a second agent to a novel β-lactam may still have a role to increase the likelihood that at least 1 active antibiotic agent is being administered while awaiting AST results. In our cohort, while the addition of a fluoroquinolone to a novel β-lactam was generally of limited additive value, the addition of an aminoglycoside to a β-lactam, particularly amikacin or plazomicin, increased the likelihood of activity across all β-lactams.
Our study had several limitations. Our cohort consisted of isolates from patients in the mid-Atlantic United States and may not be reflective of other regions of the United States or other regions of the world. Region-specific antibiograms are necessary to understand local susceptibility data. Moreover, for regions with a high prevalence of CRE clinical isolates, the development of regional combination antibiograms specific for CRE organisms can provide data on the combinations of antibiotics associated with the highest likelihood of adequate coverage when novel agents need to be administered on an empiric basis. [14][15][16] We included the first CRE species isolated from unique patients. Therefore, our results do not reflect the potential emergence of resistance in subsequent isolates after exposure to novel agents. As an example, >90% of all KPC-producing isolates were susceptible to ceftazidime-avibactam and meropenem-vaborbactam. Estimates of the emergence of resistance after clinical exposure of CRE isolates to ceftazidime-avibactam and meropenem-vaborbactam have been described to be ∼20% 13,17-21 and 5%, [21][22][23] respectively. With the inclusion of subsequent isolates, susceptibility percentages would likely be lowered, particularly to ceftazidime-avibactam, in which acquired resistance due to amino acid substitutions in the KPC carbapenemase are not rare events. 24 Notably, Sensititer MDRGNX2F panels were used to generate cefiderocol MICs for the current study. In early 2022, an investigation from the manufacturer found that these panels may produce lower cefiderocol MICs compared to reference BMD for E. coli and Klebsiella isolates. Importantly, in vitro susceptibility does not necessarily translate into improved clinical outcomes. Factors such as adequate and sustained antibiotic penetration to the site of infection and drug-specific toxicities need to be considered when selecting amongst antibiotics. For example, colistin enhanced CRE coverage by 4%-9% across novel β-lactam agents in our cohort. However, colistin is administered as a prodrug, leading to unreliable plasma concentrations. 25 Additionally, its associated nephrotoxicity often precludes its use for patients with existing renal disease. 26 As a second example, although tigecycline exhibited 94% activity against CRE isolates, tetracycline-derivatives achieve rapid tissue distribution following administration, resulting in limited concentrations in urine and poor serum concentrations, 27 limiting their effectiveness for certain sites of infection.
In conclusion, selecting among novel agents can be challenging because it requires a nuanced understanding of the molecular epidemiology of gram-negative resistance mechanisms. This research provides insights into the comparative activity of novel β-lactam and tetracycline-derivate agents against CRE isolates and the additive value of a second agent as empiric therapy. However, in vitro activity is just one component of the complex decision-making process of selecting the most effective antibiotic or combination of antibiotic agents.