Disc Diffusion and ComASP® Cefiderocol Microdilution Panel to Overcome the Challenge of Cefiderocol Susceptibility Testing in Clinical Laboratory Routine

Cefiderocol susceptibility testing represents a major challenge for clinical microbiology. Although disc diffusion showed robustness to test cefiderocol susceptibility, large areas of technical uncertainty (ATU) are reported by current EUCAST breakpoints. Herein, we evaluated the in vitro activity of cefiderocol on a collection of 286 difficult-to-treat Gram-negative isolates using disc diffusion and ComASP® cefiderocol microdilution panel. Broth microdilution (BMD) in iron-depleted Mueller–Hinton broth was used as reference method. Following the EUCAST guidelines, disc diffusion allowed to determine cefiderocol susceptibility (susceptible or resistant) in 78.6%, 88.1%, 85.4% and 100% of Enterobacterales, P. aeruginosa, A. baumannii and S. maltophilia isolates tested, respectively. ComASP® cefiderocol panel showed 94% and 84% of overall categorical agreement and essential agreement. Only one very major error and two major errors were observed, for MIC values nearly close to the resistance breakpoint (2 mg/L). Overall, 20.5% of the carbapenemase-producing Enterobacterales that achieved ATU results by the disc diffusion method tested resistant by both ComASP® panel and reference BMD. Conversely, all VIM-producing P. aeruginosa showed MIC values in the susceptible range (≤2 mg/L). Lastly, only six out of seven (85.7%) A. baumannii isolates showing inhibition zones <17 mm tested resistant by both ComASP® panel and the reference BMD suggesting that inhibition zone <17 mm are not unequivocally suggestive of resistance. Our results, although obtained on a limited number of isolates, suggest that the combination of disc diffusion with a ComASP® cefiderocol microdilution panel could be a viable solution to overcome the challenge of cefiderocol susceptibility testing in routine microbiology laboratories.


Introduction
The spread of multidrug-resistant (MDR) bacteria is a major public health threat. Carbapenemase-resistant Gram-negative bacteria, including Enterobacterales, Pseudomonas aeruginosa and Acinetobacter baumannii are considered superbugs in health care settings since they exhibit a multiresistance phenotype toward almost all commonly used classes of antibiotics [1,2]. They have been then recognized as high-priority pathogens for which development of new drugs are urgently needed by the World Health Organization [3]. In recent years, several antimicrobials have been approved for clinical use, including new β-lactam/β-lactamase inhibitor combinations, e.g., ceftolozane-tazobactam, ceftazidimeavibactam, meropenem-vaborbactam and imipenem-relebactam [4,5].
Knowledge of the antimicrobial susceptibility and local prevalence of specific carbapenemase enzymes is of paramount importance when selecting new β-lactam/β-lactamase inhibitor combinations, as not all β-lactamase inhibitors have activity against all classes of enzymes [6,7]. Avibactam, vaborbactam and relebactam have activity against class A βlactamases but not against metallo-β-lactamase producers and OXA-23-like carbapenemase. Furthermore, only avibactam has activity on OXA-48-like carbapenemase [8].
Cefiderocol is a novel siderophore cephalosporin approved for the treatment of a broad spectrum of MDR Gram-negative pathogens, including metallo-β-lactamase producers and OXA-23-like-producing A. baumannii. It is structurally similar to cefepime (pyrrolidium group on the C-3 side chain) and ceftazidime (carboxypropyl-oxymino group on the C-7 side chain), characteristics that enhance hydrolytic stability against β-lactamases and transport across the bacterial outer membrane [9]. Moreover, binding to extracellular free ferric ions allows cefiderocol to be transported across the outer membrane via the iron transport system of Gram-negative organisms, overcoming resistance mechanisms such as efflux pump upregulation and porin channel mutations [9]. Cefiderocol has been evaluated in large international surveillance studies, revealing promising activity against carbapenemresistant Gram-negative isolates [10]. However, reports on cefiderocol resistance are steadily increasing [11][12][13].
Based on the mechanism of cefiderocol, mutations affecting the iron transporter systems are associated with clinical resistance. Mutations in piuD and pirR, pirA and piuA, and cirA have been identified in P. aeruginosa, A. baumannii and K. pneumoniae clinical isolates, respectively [14]. However, the role of several β-lactamases in reducing susceptibility or inducing resistance to cefiderocol has been supported by several recent reports [14][15][16]. In this context, in vitro antimicrobial susceptibility testing is essential to enable the appropriate use of this last-resort drug. MIC determination by broth microdilution (BMD) represents the gold standard for cefiderocol susceptibility evaluation. However, it requires the use of cation-adjusted iron-depleted Mueller-Hinton medium (ID-CAMHB), whose preparation is time-consuming and difficult to implement in routine clinical microbiology laboratories. Recently, a CE-IVD BMD plate using a normal Mueller-Hinton broth was proposed, but it was subsequently withdrawn from the market due to lack of accuracy (https://www.eucast.org/ast-of-bacteria/warnings, accessed on 5 January 2023). Disc diffusion was shown to be robust and applicable to test cefiderocol susceptibility in Gram-negative bacteria [17] and was proposed by EUCAST guidelines as a first-line method (https://www.eucast.org/ast-of-bacteria/warnings, accessed on 5 January 2023) in routine testing. However, large areas of technical uncertainty (ATU) are reported by current EUCAST breakpoint tables (e.g., 18-22 mm for Enterobacterales and 14-22 mm for Pseudomonas spp.). Moreover, EUCAST MIC and inhibition zone diameter breakpoints for Acinetobacter spp. and Stenotrophomonas malthophilia are currently not available. A note indicates that inhibition zone diameters ≥17 mm and ≥20 mm for the cefiderocol 30 µg disc correspond to MIC values below the PK-PD breakpoint of susceptibility (≤2 mg/L) for Acinetobacter spp. and S. malthophilia, respectively. Inside the ATU, and as long as there is no alternative method to resolve interpretative uncertainties (e.g., routine laboratory MIC testing or support by a reference laboratory), EUCAST recommends ignoring the ATU and interpreting the inhibition zone diameter according to the table breakpoints. Therefore, alternative methods feasible in routine laboratories are urgently needed to resolve disc diffusion interpretative uncertainties.
The purpose of this study was to assess cefiderocol susceptibility on MDR Gramnegative isolates collected in Italian hospitals during a four-year period. For this aim, a diagnostic testing algorithm based on disc diffusion and the novel ComASP ® cefiderocol microdilution panel (Liofilchem ® , Roseto degli Abruzzi, Italy) was implemented and evaluated.

Discussion
Cefiderocol susceptibility testing represents a major challenge for clinical microbiology since reference microdilution requires ID-CAMHB, whose preparation is difficult to implement in the vast majority of laboratories. This technical requirement is also an obstacle to the inclusion of the cefiderocol test in commercial antimicrobial susceptibility panels. Lyophilized panels (Sensititre TM , ThermoFisher Scientific, Waltham, MA, USA) containing scalar concentrations of cefiderocol and a proprietary chelator in the wells bypassed the requirement for ID-CAMHB. First evaluations showed that Sensititre TM panels were substantially equivalent to reference broth microdilution and received FDA clearance [18][19][20]. In August 2022, EUCAST evaluated the commercially available tests for cefiderocol susceptibility reporting that all presented problems related to accuracy, reproducibility, bias and/or for some, skipped wells. Consequently, Sensititre TM panels have been withdrawn from the market. In such conditions, it is very complicated to find a solution to interpret the ATU results in laboratory routines. Considering the current limitations, EUCAST recommends starting testing cefiderocol with disc diffusion, which is predictive of susceptibility and resistance outside the ATU. Inside the ATU, alternative methods to resolve interpretative uncertainties are desirable (https://www.eucast.org/ast-of-bacteria/warnings, accessed on 5 January 2023). Recently, Liofilchem has launched the ComASP ® Cefiderocol microdilution panel, a two-test panel containing the dried antibiotic in 15 two-fold dilutions for the quantitative determination of the cefiderocol MIC against Gram-negative non-fastidious organisms, such as Enterobacterales, P. aeruginosa, Acinetobacter spp. and S. maltophilia (https://www.liofilchem.com, accessed on 5 January 2023). Unlike previous Sensititre TM panels, this system uses ID-CAMHB, thus representing the first commercial method that mirrors the reference BMD.
Herein, we evaluated the in vitro activity of cefiderocol on a collection of difficult-totreat bacterial isolates collected in a four-year period in different Italian hospitals following an algorithm based on disc diffusion and the ComASP ® cefiderocol panel. Following the EUCAST guidelines, disc diffusion allowed us to determine cefiderocol susceptibility (susceptible or resistant) in 78.6%, 88.1%, 85.4% and 100% of Enterobacterales, P. aeruginosa, A. baumannii and S. maltophilia isolates tested, respectively. Thus, excluding S. malthophilia, the rate of uninterpretable results ranged from 12 to 22% depending on the bacterial species. Moreover, a higher percentage of ATU results was found in the subset of ceftazidime/avibactam-resistant KPC producer isolates than in the ceftazidime/avibactamsusceptible isolates (43% vs. 11.4%). Similarly, a higher rate of cefiderocol resistance emerged in the same ceftazidime/avibactam-resistant group. These data were consistent with recent evidence highlighting the involvement of mutations in the omega loop of the KPC enzyme in determining co-resistance toward ceftazidime/avibactam and cefiderocol [11,21,22]. The ComASP ® cefiderocol microdilution panel was shown to be a valid method to determine cefiderocol MIC on isolates for which the disc diffusion results were uninterpretable. In fact, among the 50 isolates tested, three categorical errors were obtained, of which, only one was a VME, exhibiting 94% and 84% of overall categorical and essential agreement, respectively. Both MEs and VME were observed for MIC values nearly close to the resistance breakpoint (2 mg/L).
Overall, 20.5% of the carbapenemase-producing Enterobacterales that achieved ATU results by the disc diffusion method tested resistant by both the ComASP ® cefiderocol microdilution panel and the reference BMD. Conversely, all VIM-producing P. aeruginosa showed MIC values in the susceptible range (≤2 mg/L). Lastly, six out of seven (85.7%) A. baumannii tested by the ComASP ® cefiderocol microdilution panel and the reference BMD were resistant to cefiderocol according to EUCAST PK/PD breakpoints (susceptible ≤2 mg/L, resistant >2 mg/L) suggesting that zone diameters <17 mm were not unequivocally suggestive of resistance. Overall, among resistant isolates, cefiderocol MIC values ranged from 4 to 16 mg/L in both Enterobacterales and A. baumannii isolates.
Although surveillance studies have reported potent activity with low MIC90 and MIC50 values, even on MDR isolates, reports of cefiderocol resistance are steadily increas-ing [11][12][13]. The combination of several mechanisms, including β-lactamases and mutations affecting siderophore receptor expression or function, appears to be the main cause of reduced susceptibility or resistance to cefiderocol [14]. Considering both the disc diffusion and MIC determination methods, our study showed resistance rates to cefiderocol of 4.3%, 61.4%, 47.4% and 13% in ceftazidime/avibactam-susceptible KPC-producing Enterobacterales, ceftazidime/avibactam-resistant KPC-producing K. pneumoniae, NDM-producing Enterobacterales and carbapenemase-producing A. baumannii isolates, respectively. The high rates of resistance in ceftazidime/avibactam-resistant KPC-producing K. pneumoniae are already known, as well as among NDM producers [11,12,14,23]. A systematic review by Wang et al. showed that MIC values for NDM-producing Enterobacterales isolates were significantly higher than those harboring other β-lactamase genes, with a cefiderocol susceptibility rate of 83.4% [10]. In accordance with published data [10,24], our results showed that cefiderocol is highly active against VIM-producing Enterobacterales, carbapenemase-resistant P. aeruginosa and S. maltophilia, reaching 100% susceptibility. We should acknowledge that the results are limited to the Italian Piedmont epidemiology, which may be close to the epidemiology of other Italian regions and Western European countries.

Study Design
We investigated the in vitro activity of cefiderocol on a collection of 286 MDR Gramnegative clinical isolates collected from various clinical specimens (blood, rectal swabs, urine, respiratory samples) of patients admitted at six Italian Piedmont hospitals in the period 2019-2022. Disc diffusion was used as first-line antimicrobial susceptibility method. Subsequently, Enterobacterales and P. aeruginosa isolates that presented zone diameters within ATU (18-22 mm and 14-22 mm, respectively) were tested in parallel by the ComASP ® cefiderocol microdilution panel and the reference BMD. Likewise, A. baumannii and S. maltophilia that presented zone diameters <17 mm and <20 mm, respectively, were tested in parallel by the ComASP ® cefiderocol microdilution panel and the reference BMD.

Antimicrobial Susceptibility Testing
Antimicrobial susceptibility to main antimicrobials was tested using panel NMDR on a Microscan WalkAway automated microdilution system (Beckman Coulter, Nyon, Switzerland). Cefiderocol testing was performed using disc diffusion according to EUCAST guidelines. Cation-adjusted Mueller-Hinton agar (Becton-Dickinson, Franklin Lakes, NJ, USA) and a cefiderocol 30 µg disc (Oxoid Ltd., Basingstoke, UK) were used. Sequentially, the ComASP ® cefiderocol microdilution panel and the reference BMD were used in parallel to test cefiderocol on isolates that presented ATU or non-interpretable results by disc diffusion. Reference broth microdilution was performed by using an ID-CAMHB broth as recommended by EUCAST guidelines and using 0.015-32 mg/L of cefiderocol as concentration range [27]. The ComASP ® cefiderocol microdilution panel was used according to the manufacturer's instructions. Briefly, 0.4 mL of a 1:20 diluted 0.5 McF bacterial solution was added to the ID-CAMHB tube provided in the kit. Subsequently, 100 µL of the obtained solution was dispensed into each well of the panel, and it was incubated at 37 • C for 16-20 h in ambient air. At the end of the incubation period, the MIC values were visually read as the lowest concentration of antibiotic that completely inhibits organism growth. If trailing was observed the cefiderocol MIC was read as the first well in which growth was significantly reduced corresponding to a button of ≤1 mm or the presence of light haze/faint turbidity. For each test, the validity of the positive control was checked.
Antimicrobial susceptibility testing results were inspected and interpreted according to EUCAST recommendations by two operators. E. coli ATCC 25922 and P. aeruginosa ATCC 27853 were used as quality control strains on each experimental sitting, checking that the quality control results were within the specified ranges. Susceptibility data were interpreted according to EUCAST clinical breakpoints (v. 13.0 2023) [28].

Statistical Analysis
Cefiderocol MIC values obtained by the ComASP ® cefiderocol microdilution panel and the reference BMD were compared to delineate categorical agreement. Two different types of error were defined: VME and ME, indicating a variation of the interpretation from resistant to susceptible and from susceptible to resistant, respectively. Variations in scalar dilutions were analyzed in order to delineate EA, corresponding to the number of MIC values within one doubling the dilution of the reference results. Comparisons involving distributions of inhibition zone diameters were tested using the Mann-Whitney test (two-tailed). The X 2 test was used to compare resistance rates between isolates subsets. For all tests, a p ≤ 0.05 was considered significant.

Conclusions
To the best of our knowledge, this is the first study evaluating a commercial version of the reference BMD for cefiderocol susceptibility testing. Our results, although obtained on a limited number of isolates, suggest that the combination of disc diffusion with the ComASP ® cefiderocol microdilution panel could be a viable solution to overcome the challenge of cefiderocol susceptibility testing in routine microbiology laboratories. Further studies on high numbers of multicenter bacterial isolates are needed to strengthen these findings and to evaluate the test in cases where the inhibition zones fall outside the areas of uncertainty.

Data Availability Statement:
The dataset analyzed during the current study is available from the corresponding author upon reasonable request.

Conflicts of Interest:
The authors declare no conflict of interest.