In Vitro Activity of Cefiderocol against Clinical Gram-Negative Isolates Originating from Germany in 2016/17

Antimicrobial resistance poses a global threat to public health. Of great concern are Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacterales with resistance to carbapenems or third-generation cephalosporins. The aim of the present study was to investigate the in vitro activity of the novel siderophore cephaloporin cefiderocol (CID) and four comparator β-lactam-β-lactamase-inhibitor combinations and to give insights into the genetic background of CID-resistant isolates. In total, 301 clinical Enterobacterales and non-fermenting bacterial isolates were selected for this study, including randomly chosen isolates (set I, n = 195) and challenge isolates (set II, n = 106; enriched with ESBL and carbapenemase producers, as well as colistin-resistant isolates). Isolates displayed CID MIC50/90 values of 0.12/0.5 mg/L (set I) and 0.5/1 mg/L (set II). Overall, the CID activity was superior to the comparators against A. baumannii, Stenotrophomonas maltophilia and set II isolates of P. aeruginosa. There were eight CID-resistant isolates detected (MIC > 2 mg/L): A. baumannii (n = 1), E. cloacae complex (n = 5) and P. aeruginosa (n = 2). Sequencing analyses of these isolates detected the acquired β-lactamase (bla) genes blaNDM-1, blaSHV-12 and naturally occurring blaOXA-396, blaACT-type and blaCMH-3. In conclusion, CID revealed potent activity against clinically relevant organisms of multidrug-resistant Enterobacterales and non-fermenters.


Introduction
The emergence of antibiotic-resistant bacteria has been described as one of the biggest threats to global health and food safety [1,2]. It is a consequence of selective pressure caused by a range of factors such as the overuse of antibiotics in human and veterinary medicine, as well as insufficient hygiene precautions and the release of antibiotics into the environment [1][2][3][4]. The three most critical pathogens defined by the World Health Organization (WHO) for finding new treatment options are the Gram-negative pathogens Acinetobacter baumannii (carbapenemresistant), Pseudomonas aeruginosa (carbapenem-resistant) and the order of Enterobacterales (carbapenem-resistant and ESBL-producing) [5]. Various established antibiotics have been suggested as an appropriate therapy for severe infections caused by carbapenem-resistant Gram-negative bacteria, such as high dose meropenem and colistin [6,7]. New antimicrobial agents, especially β-lactam-β-lactamase-inhibitor combinations such as ceftazidime-avibactam, ceftolozane-tazobactam, imipenem-relebactam or meropenem-vaborbactam, have been considered for the effective treatment of severe infections caused by carbapenem-resistant Gramnegative bacteria [8,9]. However, none of these agents address all resistant Gram-negative bacteria or show sufficient activity against Stenotrophomonas maltophilia [7][8][9].
Cefiderocol (CID) is a novel parenteral cephalosporin carrying a catechol moiety at the 3-position side chain [10]. It has been shown recently that the compound forms a chelating complex with extracellular trivalent iron, leading to active transport of the drug into the periplasmatic space of P. aeruginosa [11]. It is thus considered a siderophore cephalosporin. CID shows promising antimicrobial activity against critical Gram-negative pathogens, such as carbapenem-resistant non-fermenters such as A. baumannii (CRAB), P. aeruginosa and S. maltophilia, as well as carbapenem-resistant Enterobacterales [12], with high stability against β-lactamases of all Ambler classes. Clinically relevant carbapenemhydrolyzing β-lactamases, such as class A KPC, class B metallo-enzymes or class D OXA enzymes (e.g., OXA-48 in Klebsiella pneumoniae or OXA-23 in A. baumannii), show the weak hydrolysis of CID [13]. Furthermore, CID shows a low tendency to induce chromosomal AmpC β-lactamases of P. aeruginosa and Enterobacter cloacae complex, which could otherwise cause resistance development under therapy [14][15][16]. CID has been approved by the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for the treatment of infections (i.e., complicated urinary-tract infections and hospital-acquiredand ventilator-associated bacterial pneumonia) caused by Gram-negative bacteria in adult patients with limited treatment options [17,18].
In vitro data of CID from Germany are scarce. In our previous study published in 2020, CID was found to inhibit 97.2% of a randomly chosen collection of 213 Gramnegative clinical isolates of different species at the investigational susceptibility breakpoint of ≤2 mg/L [19]. The isolates were obtained from patients in intensive care units during a multicentre surveillance study conducted by the Paul-Ehrlich-Society for Infection Therapy (PEG) in 2013. Furthermore, CID was shown to inhibit 88.1% of a collection of 80 carbapenemase-producing clinical isolates from different sources at the investigational susceptibility breakpoint of ≤2 mg/L during that same study [19].
The present study aimed (I) to investigate the in vitro activity of CID against Gramnegative pathogens recovered from patients during a more recent multicentre surveillance study conducted by the PEG in 2016/17 and (II) to compare it with the susceptibility against novel β-lactam-β-lactamase-inhibitor (BL-BLI) combinations.
Compared to Enterobacterales, MEV was less effective against the 58 P. aeruginosa isolates, with 89.7% of the isolates being inhibited at the respective breakpoint (R > 8 mg/L). The susceptibility rates of P. aeruginosa against the other agents were comparable to that seen with the Enterobacterales: 98.3% (CID), 96.6% (IMR), 94.8% (CTV) and 91.4% (CTT) ( Table 2)

Challenge Organisms (Set II)
The results of set II isolates are displayed together with set I in Table 1 (CID MIC distributions) and Table 2 (MIC 50/90 and susceptibility/resistance rates, where applicable).
Among the 53 Enterobacterales isolates, 100% were susceptible to MEV, 98.1% were susceptible to both CTV and IMR and 77.4% were susceptible to CTT. With a susceptibility rate of 94.3% (50/53), CID exhibited slightly lower activity compared to CTV and IMR in Enterobacterales. Overall, the comparator agents were similar or less effective in the 39 P. aeruginosa isolates, with susceptibility rates of 61.5% (CTT), 51.3% (CTV), 46.2% (IMR) and 35.9% (MEV), while CID inhibited 97.4% (38/39) of P. aeruginosa isolates. Among the CRAB isolates, CID revealed the most potent activity compared to the other compounds with MIC 50/90 values of 0.12/2 mg/L as opposed to ≥16/≥16 mg/L.

Resistant Isolates (Set I and II)
The CID MIC distributions of ESBL-producing isolates, carbapenemase-producing isolates and colistin-resistant isolates from set I and II are summarized in Table 3, sorted by their respective resistance patterns and mechanisms. ESBL-and carbapenem-resistance determinants are correlated with bacterial species and CID susceptibility in Table 4.  The CID susceptibility in 47 ESBL-encoding isolates ranged from ≤0.03 mg/L to ≥64 mg/L, with MIC 50/90 values of 0.5/1 mg/L. There was no difference in CID MIC distribution with regard to different resistance genes, with the exception of two bla NDM-1-encoding isolates (A. baumannii and P. aeruginosa) and two SHV-12-encoding E. cloacae complex isolates with CID MICs > 2 mg/L. With the exception of CTT, the comparator compounds revealed similar activity against ESBL-producing isolates compared to CID. The MIC 50/90 values were 0.25/0.5 mg/L for CTV, 0.12/0.25 mg/L for IMR and ≤0.06/0.12 mg/L for MEV.
The overall CID susceptibility of the 47 ESBL-encoding Enterobacterales isolates was 95.7% (45/47). CTV, IMR and MEV were able to inhibit 100% of the isolates at their respective breakpoints, with only the CTT susceptibility being lower at 85.1%.

Discussion
The WHO has designated antimicrobial resistance as one of the top ten global public health threats. Of great concern are carbapenem-resistant non-fermenting Gram-negative bacteria such as A. baumannii and P. aeruginosa, as well as Enterobacterales species with acquired resistance against carbapenems or third-generation cephalosporins, all possessing a high risk of severely limited treatment options. In contrast to resistance against third-generation cephalosporins, carbapenem resistance is still rarely encountered in Enterobacterales species such as E. coli and K. pneumoniae in Germany. For example, the surveillance study originated by the Paul-Ehrlich-Society for Infection Therapy in 2016/17 revealed resistance rates of 0% against imipenem and meropenem in 571 E. coli isolates and 1.6% (5/318) against both substances in K. pneumoniae isolates. According to the annual surveillance data for Germany reported to the European Centre for Disease Prevention and Control (ECDC), the rates of carbapenem-resistant E. coli and K. pneumoniae isolates were 0.0% and 0.8% in 2021 (out of a total of 29,105 and 6538 isolates tested, respectively) compared to resistance rates against third-generation cephalosporins of 9.1% (2641/29,021) and 10.4% (678/6538) (Surveillance Atlas of Infectious Diseases (europa.eu); data source: invasive isolates). In Acinetobacter spp. and P. aeruginosa, carbapenem-resistance was more frequently detected with 4.3% (26/605) and 14.8% (425/2864), respectively.
The antimicrobial agents compared in this study are considered promising compounds in the treatment of infections with the above-mentioned organisms when no other options are available. In contrast to the comparators, CID possesses activity against a variety of Gram-negative species and β-lactamases of all Ambler classes, including OXA-encoding A. baumannii, MBL-producing organisms and S. maltophilia, which is intrinsically resistant against multiple antimicrobial agents, including carbapenems [20]. Unlike the other compounds, MEV is not available in Germany yet.
In the current study, CID showed broad activity with overall inhibition rates of 98.5% (set I, MIC 50/90 s 0.12/0.5 mg/L) and 95.3% (set II, MIC 50/90 s 0.5/1 mg/L) at ≤2 mg/L (Table 1), which was in accordance with our previous study [19]. The other compounds showed comparable potent activity in Enterobacterales (susceptibility rates >92%), with reduced activity of CTT in challenge isolates (set II) ( Table 2). This result might have been expected due to the compound-specific spectrum. In P. aeruginosa, MEV displayed reduced activity in set I isolates compared to CID (Table 2). In set II isolates of P. aeruginosa, all comparators showed decreased activity, with inhibition rates ranging from 36% to 62%. In accordance with our study, the SENTRY surveillance study reported similar CID MIC 50/90 values based on larger strain collections of 8047 Enterobacterales and 2282 P. aeruginosa isolates from Europe and the United States with 0.06/0.5 mg/L in Enterobacterales and 0.12/0.5 mg/L in P. aeruginosa [21].
Our study observed more potent CID activity against A. baumannii and S. maltophilia isolates than the comparators (Table 2). Similar results were reported previously in different studies investigating bacterial isolates from the United States and from Europe [21][22][23]. In the current study, 100% of S. maltophilia and A. baumannii random isolates (set I) were inhibited by CID at a concentration of ≤2 mg/L, while there was only one isolate detected among fourteen CRAB isolates (set II) with an MIC of >2 mg/L. However, due to the small sample size of S. maltophilia and A. baumannii, these data should be considered with caution.  [25]. In this study, CID at 2 mg/L was only able to inhibit 80.8% of the investigated isolates. In contrast, Delgado-Valverde et al. only reported reduced CID efficacy in OXA-24/40 expressing A. baumannii (n = 25), while isolates harbouring OXA-58 or OXA-23 were all susceptible (n = 75) [23].
Overall, our study revealed the broad activity of CID against ESBL-and carbapenemaseproducing isolates, as well as colistin-resistant isolates, with the majority of isolates inhibited at ≤2 mg/L (Table 3). Isolates with CID MIC values > 2 mg/L harboured acquired β-lactamases such as NDM-1-like (A. baumannii and P. aeruginosa) and SHV-12 (E. cloacae complex), or the naturally occurring β-lactamases such as class C ACT-type, CMH-3 (both E. cloacae), as well as PDC-8 together with class D OXA-396 (P. aeruginosa) ( Table 4). The correlation of NDM production with CID non-susceptibility has been observed previously [10,25]. In accordance, some studies showed that the cloning of bla NDM-1 in E. coli resulted in an increase in the CID MIC from 0.5 mg/L to 4 mg/L, while the cloning of other β-lactamase genes such as bla ACT-type or bla OXA-23 revealed lower MICs of 0.125 to 0.5 mg/L [26,27]. In our study, bla NDM-1 -carrying isolates also revealed CID MICs > 2 mg/L, but as only two isolates were included, our data are of limited value in further support of the association between the presence of NDM-1 and reduced CID susceptibility. Of note, the CID-resistant A. baumannii isolate PEG-16-19-65 revealed the disruption of the piuA gene, which encodes a siderophore receptor that might be needed for efficient CID uptake, as has been shown previously for its homologue in P. aeruginosa [28,29]. Furthermore, the disruption of oprD porin genes was detected in PEG-16-19-65 and the CID-resistant P. aeruginosa PEG-16-14-45. However, an association between CID resistance and disrupted oprD genes has not been described yet.
In conclusion, CID revealed potent activity against Enterobacterales and non-fermenting Gram-negative bacterial isolates from Germany. The association of CID non-susceptibility with a particular resistance determinant seemed to be unlikely, while the presence of bla NDM-1 might be an exception to this and requires further investigation. CID activity was superior to the comparators against A. baumannii, S. maltophilia and challenge isolates of P. aeruginosa. In addition to ESBL-producing isolates, the majority of CP-producing and colistin-resistant isolates were inhibited at a CID concentration ≤2 mg/L, indicating good activity of CID in clinically relevant organisms.

Bacterial Isolates
In total, 301 Gram-negative bacterial isolates were investigated in this study. All isolates were obtained from patient samples collected at 22 German microbiological laboratories during a multicentre surveillance study conducted by the PEG in 2016/17. The majority of laboratories were affiliated with tertiary-care medical centres. Two sets of isolates were selected: random samples (set I) and challenge organisms (set II).

Species Identification
The verification of species identification was performed via matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MALDI Biotyper, Microflex, Bruker Daltonics GmbH, Bremen, Germany).

Molecular Analysis of CID-Resistant Isolates
Isolates with CID MICs > 2 mg/L were sent to the International Health Management Associates (IHMA) for whole-genome sequencing.

Statistical Evaluation
The statistical significance of differences in susceptibility rates was judged by comparing 95% confidence intervals (CIs). Intervals were constructed using the Newcombe-Wilson method without continuity correction. If no rate was contained in the CI of the other one, significance of p < 0.05 was assumed.