Comparison of Broth Microdilution, Disk Diffusion and Strip Test Methods for Cefiderocol Antimicrobial Susceptibility Testing on KPC-Producing Klebsiella pneumoniae

The aim of this study was to compare the reference broth microdilution (BMD) method with the Disk Diffusion (DD) test and strip test against a collection of 75 well-characterized Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae (KPC-Kp) clinical strains to assess cefiderocol (CFD) antimicrobial activity. Whole-genome sequencing was performed on KPC-Kp strains by Illumina iSeq100 platform. The Categorical Agreement (CA) between the BMD method and DD test was 92% (69/75) with a Major Error (ME) of 16.7% (6/36). Additionally, the CA between the BMD method and test strip was 90.7% (68/75) with a Very Major Error (VME) of 17.9% (7/39) and 82.7% (62/75) between the strip test and DD with a ME of 30.2%. KPC-Kp strains showing resistance to CFD were 27 out of 75 (36%) by three methods. Specifically, 51.9% (14/27) of KPC-Kp resistant to CFD harbored blaKPC-3, while 48.1% (13/27) harbored mutated blaKPC-3. Moreover, KPC-Kp strains carrying a mutated blaKPC-3 gene exhibited high MIC values (p value < 0.001) compared to wild-type blaKPC-3. In conclusion, the DD test resulted as a valid alternative to the BMD method to determine the in vitro susceptibility to CFD, while the strip test exhibited major limitations.

In order to overcome these limitations, cefiderocol (CFD), formerly S-649266, an intravenously infused siderophoric cephalosporin [5,8], was approved by the US Food and Drug Administration (FDA) in 2019 for the treatment of UTIs and in 2020 for the Antibiotics 2023, 12, 614 2 of 8 treatment of bacterial hospital-acquired pneumonia (HAP) and VAP [2]. Subsequently, it was approved in Europe for the treatment of infections caused by gram-negative bacteria in adults with limited treatment options [5,9].
CFD exhibited structural stability against most β-lactamase resistance mechanisms of gram-negative bacteria [2,10], since it is able to enter the periplasmic space through the active iron transport system, as well as through passive diffusion through the porin channels of the outer membrane [5,[8][9][10]. Once inside, it dissociates from iron, and the cephalosporin moiety binds primarily to penicillin-binding protein 3 (PBP3), inhibiting bacterial cell wall synthesis [2]. CFD revealed high in vitro bactericidal activity against K. pneumoniae, Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumannii [9] and showed intrinsic structural stability against most gram-negative bacteria producing β-lactamases, such as KPC [11], oxacillin carbapenemase (OXA), New Delhi metallo-β-lactamase (NDM) and Verona integron metallo-β-lactamase (VIM) [3,10]. However, in vitro sensitivity procedures used to evaluate this molecule against MDR microorganisms have wide limitations, due to its demanding preparation procedure, which includes iron-depleted cation-adjusted Mueller-Hinton broth (ID-CAMHB).
Hence, the aim of this work is to compare the broth microdilution reference method (BMD) currently used to assess CFD antimicrobial activity against KPC-producing Enterobacteriales and disk diffusion (DD) test and strip test sensitivity methods, describing the degree of concordance (Categorical Agreement) and errors (Very Major Error, Major Error and Minor Error) observed between the three different techniques analyzed.

Results
In this study, we selected 75 strains of KPC-producing K. pneumoniae (KPC-Kp) genotypically characterized by WGS.

Discussion
Currently, CFD represents an effective antimicrobial agent for the treatment of infections caused by gram-negative bacteria in adults with limited treatment options available [5]. In particular, CFD is effective against gram-negative bacteria exhibiting resistance to carbapenem and could be considered for the treatment of difficult-to-treat (DTR) infections [12,13]. Although the BMD method is recommended by EUCAST as the reference method for the in vitro susceptibility assessment of CFD, other suitable and less laborious methods are available, such as the DD test and the strip test. Therefore, in this study we evaluated the susceptibility of CFD against 75 clinical KPC-Kp strains by comparing the accuracy of the DD test and MIC test strip to BMD method.
According to EUCAST criteria, the DD test compared to the reference method showed a degree of concordance of 92% and a ME of 16.7%. VME was not detected, thus suggesting that the DD method is more reliable in avoiding false negatives results. In addition, excluding values within the ATU range set by EUCAST (18-22 mm), the categorical agreement rises to 98.6%, and the ME drops to 2.8%, positioning the DD test as a notable alternative to the BMD method. However, particular attention must be placed on strains that fall inside the ATU range, which should be retested by the reference method, reported as resistant or ignored [10]. Moreover, potentially susceptible strains, showing a MIC of 2 mg/L but a DD test value between 18 and 22 mm, can be erroneously classified as resistant [10]. Considering the limited treatments options for patients affected by MDR pathogens or unable to tolerate aggressive drug regimens due to renal insufficiency, this could lead to negative implications for treatment of patients [10]. The comparison between the BMD and MIC test strip results showed a good degree of agreement. However, the MIC test strip incorrectly classified the BMD method-resistant isolates as CFD sensitive (i.e., VME). In particular, following the EUCAST interpretative criteria, the CA was 90.7%, and the VME 17.9%. Previous studies showed that KPC-Kp strains harboring mutations within the Ω-loop of the KPC-3 enzyme can exhibit increased MIC values for CFD and resistance to CAZ-AVI [11,14,15]. Indeed, mutations in the bla KPC-3 gene are likely responsible for extending the resistance spectrum by increasing CFD MIC values. In agreement with previous studies, our results revealed that strains harboring mutated bla KPC-3 showed higher MICs for CFD and acquisition of resistance to CAZ-AVI, compared to wild type bla KPC-3 . In this context, further analyses are needed in order to better understand the emergence of resistance mechanisms, which can lead to the development of cross-resistance to the latest generation of antibiotics in KPC-Kp strains.
In conclusion, our results demonstrated that the DD test is a valid alternative for determining in vitro susceptibility to CFD, since the BMD method has proved to be challenging for routine laboratory analysis, especially for those laboratories managing large numbers of patients. The ATU values can be used as an indication of uncertainty in the test procedure and therefore as an index of difficulty in interpreting results. In these cases, it may be useful