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Occurrence of High Levels of Cefiderocol Resistance in Carbapenem-Resistant Escherichia coli before Its Approval in China: a Report from China CRE-Network

ABSTRACT Cefiderocol has been approved in the United States and Europe but not in China. We aim to evaluate carbapenem-resistant Enterobacterales (CRE) susceptibility to cefiderocol to provide baseline data and investigate the resistance mechanism. From 2018 to 2019, 1,158 CRE isolates were collected from 23 provinces and municipalities across China. The MICs of antimicrobials were determined via the agar dilution and broth microdilution methods. Whole-genome sequencing was performed for 26 cefiderocol-resistant Escherichia coli isolates to investigate the resistance mechanism. Clone transformations were used to explore the function of cirA, pbp3, and blaNDM-5 in resistance. Among the 21 antimicrobials tested, aztreonam-avibactam had the highest antibacterial activity (98.3%), followed by cefiderocol (97.3%) and colistin (95.3%). A total of 26 E. coli isolates harboring New Delhi metallo-beta-lactamase 5 (NDM-5) showed high levels of cefiderocol resistance, of which sequence type 167 (ST167) accounted for 76.9% (20/26). We found 4 amino-acid insertions (YRIN/YRIK) at position 333 of penicillin-binding protein 3 (PBP3) in the 26 E. coli isolates, and 22 isolates had a siderophore receptor cirA premature stop codon. After obtaining the wild-type cirA supplementation, the MIC of the transformants decreased by 8 to 16 times in two cefiderocol-resistant isolates. A cefiderocol-susceptible isolate harboring NDM-5 has an MIC increased from 1 μg/mL to 64 μg/mL after cirA deletion, and the MIC decreased from 64 μg/mL to 0.5 μg/mL after blaNDM-5 deletion. The MIC of the E. coli DH5α, from which the pbp3 mutant was obtained, increased from 0.064 μg/mL to 0.25 μg/mL. Cefiderocol showed activity against most CRE in China. The resistance of ST167 E. coli to cefiderocol is a combination of the premature stop codon of cirA, pbp3 mutation, and blaNDM-5 existence. IMPORTANCE Cefiderocol, a new siderophore cephalosporin, has been approved in the United States and Europe but not in China. At present, there are almost no antimicrobial susceptibility evaluation data on cefiderocol in China. We evaluated the in vitro susceptibility of 1,158 strains of carbapenem-resistant Enterobacterales to cefiderocol and other antibiotics. We found that a high proportion of Escherichia coli showed high-level resistance to cefiderocol. Whole-genome sequencing (WGS) and molecular cloning experiments confirmed that the synergistic effect of the cirA gene premature stop codon, blaNDM-5 existence, and the pbp3 mutation is associated with high levels of cefiderocol resistance.

Characteristics of cefiderocol-resistant E. coli. Among the 1,158 CRE isolates, 30 isolates were resistant to cefiderocol, comprising 26 E. coli, 2 K. pneumoniae, and 2 E. cloacae complex isolates. We evaluated the phenotypic characteristics and resistance mechanisms of the E. coli isolates which represented the majority. Table 3 shows the general characteristics of the 26 cefiderocol-resistant E. coli isolates. Chronologically, 13 of the 26 cefiderocol-resistant isolates were isolated in 2018 and 13 in 2019. Geographically, 26 isolates were collected from 9 provinces in China; 9 isolates were from Sichuan, 5 were from Shandong, 3 were from Guangxi, 3 were from Hebei, and 2 were from Beijing, one each from Jiangsu, Shanxi, Hunan, and Guangdong, respectively. A total of 6 different multilocus sequence types (MLSTs) were identified, among  Notably, all 26 cefiderocol-resistant E. coli isolates harbored NDM-5, and 1 ST167 strain harbored both KPC-2 and NDM-5. AST showed that high-level cefiderocol-resistant isolates (MIC $32 mg/mL) accounted for 92.3% (24/26), and only 3 isolates had an MIC of 16 mg/mL. The MIC values of tigecycline against these 26 isolates were all lower than 1 mg/mL. The MIC of colistin for all isolates was ,0.5 mg/mL, except for one isolate with an mcr-1 gene, which exhibited an MIC of 2 mg/mL.  Tables S2 and S3 in the supplemental material show the comparison results of all PBPs and TBDTs using protein-BLAST using E. coli K-12 as a reference strain. Mutants with four amino acids (YRIN or YRIK) inserted at the 333rd amino acid of PBP3 in all cefiderocol-resistant isolates were observed ( Table 3). The TBDTs of cirA of the 22 isolates contained a stop codon for the cirA gene of the catecholamine siderophore receptor. The TBDT gene encoding CirA has a stop codon in 22 isolates.
A phylogenetic tree based on the core genome showed that the distance of the 26 strains is very similar to that of the MLST. Differences in the resistance genes between ST167 isolates and non-ST167 isolates were observed, especially in extended-spectrum beta-lactamase-related genes (bla CTX-M ), tetracycline resistance genes (tetA, tetB), and fosfomycin resistance genes related to resistance (fosA) (Fig. 2). As shown in Fig. S1, the phylogenetic tree based on the core genome of 20 strains of ST167 E. coli can be seen that the strains from different regions and have large differences between strains. There is no statistically significant difference in the copy number of bla NDM between the resistant and susceptible groups. See Fig. S2 for details.
We selected two cefiderocol-resistant E. coli isolates, C5492 and C6346, for which the MIC values of cefiderocol were 16 mg/mL and 64 mg/mL, respectively. The cirA genes in these two isolates were premature stop codons. We introduced a wild-type cirA gene into these drug-resistant isolates using a vector and induced the expression using IPTG (isopropyl-b-D-thiogalactopyranoside). AST showed that the MIC of cefiderocol for C5492 and C6346 decreased from 16 to 2 mg/mL and from 64 to 4 mg/mL, decreasing 8-and 16-fold, respectively. Therefore, the isolates recovered their susceptibility after wild-type cirA was introduced. We also knocked out the cirA and bla NDM genes with the CRISPR-Cas9 genome editing system in a cefiderocol (FDC)-susceptible (MIC = 1 mg/mL) strain (C7310). It was found that, compared with C7310, the MIC of cefiderocol to C7310DcirA increased by 64 times, from 1 mg/mL to 64 mg/mL. When both cirA and bla NDM genes were knocked out, the MIC of C7310DcirA Dbla NDM decreased from 64 mg/mL to 0.5 mg/mL. ATCC 25922 did not increase the MIC of cefiderocol after deletion of the cirA gene.

DISCUSSION
Recent research and development of new antimicrobial agents have provided additional treatment options for CRE infection, especially in European countries and the United States (14). In the United States, the clinical application of new beta-lactam combination agents has resulted in clinical benefits, including reducing mortality related to infections with carbapenem-resistant Gram-negative bacilli (15). In contrast, relatively fewer new anti-CRE drugs have been approved in China. Therefore, there is a great demand for new anti-CRE drugs.
Iron is essential for the survival and metabolism of pathogenic bacteria (16). Siderophores are natural small-molecule compounds produced by bacteria and secreted extracellularly. A transport system transfers the siderophore-Fe 31 chelate into the cell to support bacterial survival and metabolism (17). Natural siderophores are classified into four types according to functional groups chelated with iron ions: hydroxamic acid, catechol, hydroxycarboxylic acid, and mixed type (18). The siderophore component of cefiderocol is catecholamine.
Our results showed that among the 1,158 CRE isolates tested, the resistance rates of carbapenem-resistant K. pneumoniae and Enterobacter cloaceae against cefiderocol were 0.3% and 1.9%, respectively. Our results are consistent with the data of many previous studies (10,11,15). Surprisingly, 14.4% of carbapenem-resistant E. coli were resistant to cefiderocol, which was not reported previously. In 2015, Kohira et al. reported the in vitro susceptibility of clinically typical Enterobacterales to cefiderocol. Only seven NDM-producing E. coli isolates show reduced susceptibility to cefiderocol; however, the resistance mechanism remains unclear (19). The data of SIDERO-WT from 2014 to 2016 show that among the 8,307 isolates of Enterobacterales tested, only 44 isolates are resistant to cefiderocol, and most of them are moderately resistant with an MIC value between 8 and 16 mg/mL; isolates with an MIC of 64 mg/mL are rare. Our data showed that there are more high-level cefiderocol-resistant isolates among carbapenem-resistant E. coli in China. All these isolates harbored NDM-5, which has not been reported before. Previous CRE-Network data reports indicate that NDM-producing E. coli is the second-most prevalent CRE strain in China (20,21). Therefore, determining the resistance mechanism of E. coli to cefiderocol may support the rational use of antimicrobial agents, delaying the development of resistance to new drugs. It is quite striking that all cefiderocol-resistant E. coli isolates harbored NDM. Nurjadi et al. have reported that NDM facilitated the emergence of cefiderocol resistance in E. cloacae (22). Therefore, we should be alert to the high risk of resistance to cefiderocol in the treatment of NDM-producing CRE.
PBPs are essential enzymes in cell wall peptidoglycan synthesis and are also important targets for beta-lactam drugs (23). In 2017, Ito et al. selected typical ATCC isolates and tested the affinity of cefiderocol for different PBPs of E. coli. They found that among E. coli PBPs, cefiderocol had the highest affinity for PBP3, with an 50% inhibitory concentration (IC 50 ) of 0.04 mg/L, followed by PBP2 with an IC 50 of 2.12 mg/L (24). In 2015, Alm et al. reported that the 333rd position of PBP3 in an NDM-producing E. coli strain contained YRIN or YRIK, a four-amino acid insertion associated with aztreonamavibactam resistance (25). Similarly, a point mutation in Acinetobacter baumanniiderived PBP3 may cause cefiderocol resistance (26). The results of our cloning transformation showed that the MIC of cefiderocol against DH5a containing a PBP3 mutation (YRIN or YRIK) increased by 4-fold; however, it did not reach the resistance level. Mutations in PBP3 do not considerably affect cefiderocol resistance significantly. This result demonstrates that the PBP3 mutation may not serve as an efficient determinant of cefiderocol resistance and is only an auxiliary effect. An essential issue in the Trojan Horse strategy is if the trojan horse is unable to enter the city. Most pathogenic bacteria have multiple approaches for acquiring iron, and the more genes involved in iron transport, the higher the possibility of the bacteria developing drug resistance. Currently, seven TBDTs in E. coli rely on the TonB system, namely, FepA, FecA, FhuA, CirA, Fiu, BtuB, and FhuE. FepA, CirA, and Fiu are mainly responsible for transporting catechol-type siderophores (17,27). In our study, 23 cefiderocol-resistant E. coli isolates had cirA gene termination codes. After obtaining a wild-type cirA gene and inducing expression, the MIC of cefiderocol against the transformant strain decreased to the susceptible range (#4 mg/mL); the strain also carried the PBP3 mutation. This result confirmed that the truncation of cirA may be the main reason for cefiderocol resistance. Ito et al. showed that the MIC of cefiderocol against the E. coli K-12 strain increases by 16-fold when the iron transporter cirA and fiu genes are knocked out simultaneously (24). Klein et al. reported that rapid development of cefiderocol resistance in E. cloacae during treatment is associated with heterogeneous mutations in the catechin siderophore receptor cirA (28). Unlike this study, our resistant strains were not subjected to antibiotic pressure from cefiderocol. Kohira et al. reported that beta-lactamase PER is associated with cefiderocol resistance, and our study did not find such extended-spectrum beta-lactamase (ESBL) genes (29). Simner et al. also recently reported an increase in bla NDM copy number under antibiotic pressure, resulting in high expression of NDM, leading to cefiderocol resistance (30). Our data show that the presence of the bla NDM gene in cirA knockout strains plays an important role in cefiderocol resistance.
This study has a few limitations. (i) Although our study is a multicenter study, it is not comprehensive enough at the regional and hospital level, and there is still some bias in the data. Follow-up studies with larger and more comprehensive sample sizes are needed. (ii) We could not determine the entire resistance mechanism of the four wild-type cirA-encoding cefiderocol-resistant E. coli isolates. (iii) Also, non-E. coli resistant isolates were not investigated. The resistance mechanism of K. pneumoniae and the Enterobacter cloacae complex still needs follow-up research and exploration.
To the best of our knowledge, this is the first large-scale in vitro AST study of CRE isolates against cefiderocol and other drugs in China. Carbapenem-resistant E. coli shows resistance to cefiderocol by the termination of cirA coding combined with PBP3 insertion mutation. This study provides a reference for the application of cefiderocol in China.

MATERIALS AND METHODS
Bacterial isolates. From January 2018 to December 2019, 1,158 CRE isolates were collected from 48 hospitals in 23 provinces and municipalities across China, including 38 tertiary hospitals and 10 secondary hospitals, as part of the China CRE-Network research (20). The definition of CRE refers to the definition published by the U.S. CDC (https://www.cdc.gov/hai/organisms/cre/technical-info.html#Definition). The infection types of the isolates include respiratory tract infection, accounting for 41.6% (482/1158), intra-abdominal infection, accounting for 13.4% (155/1158), urinary tract infection, accounting for 13.3% (154/1158), bloodstream infection, accounting for 13.1% (152/1158), and other infection type, accounting for 18.6% (215/1158). CRE were defined as members of the Enterobacterales resistant to imipenem, meropenem, ertapenem, or doripenem, or any one of the carbapenems, or producers of carbapenemase based on laboratory experiments, such as modified carbapenem inactivation method (mCIM) according to CLSI (31). The species of all Enterobacterales were reconfirmed according to matrix-assisted laser desorption ionization-time of flight mass spectrometry (Bruker Daltonik, Bremen, Germany) at a central laboratory (Peking University People's Hospital) and were stored at 280°C for further use.
Antimicrobial susceptibility testing. Antimicrobial susceptibility testing was performed via the agar dilution and broth microdilution methods at Peking University People's Hospital according to the Clinical and Laboratory Standards Institute (CLSI) guideline (32), and the results were interpreted according to CLSI M100, 30th edition, categories and MIC breakpoints (33). The MIC of cefiderocol was determined using irondepleted cation-adjusted Mueller Hinton broth (ID-CAMHB) as described in CLSI M100, 30th edition, and previous studies (34). Susceptibility to aztreonam, aztreonam/avibactam, cefoxitin, cefotaxime, ceftazidime, ceftazidime/avibactam, cefepime, piperacillin/tazobactam, cefoperazone/sulbactam, fosfomycin, ertapenem, imipenem, meropenem, amikacin, ciprofloxacin, minocycline, chloramphenicol, and levofloxacin was tested via the agar dilution method. Tigecycline and colistin susceptibility was tested via the broth microdilution method (32). The breakpoint of tigecycline for Enterobacterales was obtained from the U.S. FDA standard (https://www.fda.gov/drugs/development-resources/tigecycline-injection-products). The breakpoints of colistin were used in the breakpoint tables to interpret the MIC version 11.0 published by the European Committee on AST. Pseudomonas aeruginosa ATCC 27853 and Escherichia coli ATCC 25922 were used as quality control standards for AST. Susceptibility data were analyzed using WHONET v5.6 (http://www.whonet .org/contact.html).
Investigation of carbapenem resistance mechanisms. All 1,158 isolates were subjected to mCIM to determine whether carbapenemase is phenotypically produced. PCR analysis was used to detect the genes encoding carbapenemases (bla KPC , bla NDM , bla IMP , bla VIM , bla SIM , and bla OXA-48 ) as previously described (20,35,36). PCR products were purified using a QIAquick PCR purification kit (Qiagen, Valencia, CA, USA) and sequenced via Sanger sequencing on an ABI PRISM 3730XL system (Applied Biosystems, Foster City, CA, USA). The full-length sequence obtained was submitted to the Beta-Lactamase Database (BLDB) for comparison and analysis to obtain the carbapenemase genotype (37).
Whole-genome sequencing, assembly, and annotation. A total of 26 cefiderocol-resistant CRE were subjected to whole-genome sequencing. Total DNA was extracted using the TIANamp bacterial DNA kit DP302 (Tiangen Biotech, Beijing, China), and genomic DNA was sequenced using the Illumina NextSeq 550 platform, which produced 150-bp paired-end reads and at least 100-fold coverage of raw reads. The short-read sequence was assembled de novo using SPAdes v3.10.0 (38). The resulting assemblies were annotated using Prokka v1.12 (39), and the core and accessory genomes were defined and extracted using Roary v3.11.2 (40). We constructed a phylogenetic tree in RAxML by using a general time reversible (GTR) model and 1,000 bootstrap replicates (41). All resistance genes, including beta-lactamase and colistin resistance (mcr-1) and other resistance genes, were detected using ResFinder (https://cge.cbs.dtu.dk/services/ResFinder/) and the Basic Local Alignment Search Tool (BLAST). We selected 18 ST167 cefiderocol-susceptible NDM-5 E. coli strains that had previously undergone WGS as the control group and 20 ST167 NDM-5 producing strains in the cefiderocol-resistant group for bla NDM copy number comparison. bla NDM copy numbers were estimated by mapping the raw short-read data to our short-read de novo contig assemblies using the Burrows-Wheeler Aligner (BWA) v3.1 (42). The absolute number of reads that mapped to the bla NDM gene, normalized by sequence depth, was used to generate a sequence depth of coverage.
Subcloning experiments. Two different recombinant plasmids were constructed to determine the impact of a pbp3 gene sequence containing a 12-bp insertion and truncated cirA gene on cefiderocol resistance. The gene sequences for the two amino acid insertions of PBP3 were amplified from C5881 (PBP3: YRIK) and C5492 (PBP3: YRIN). A cirA wild-type gene sequence was amplified from E. coli DH5a (derived from E. coli K-12). The plasmid pEASY-T1 was used as a template to amplify all sequences except the coding sequence of lacZ to retain the promoter region of lacZ. The primers and PCR cycling conditions used are listed in Table S1. PCR amplification was performed using a PrimeSTAR high-sensitivity (HS) kit (TaKaRa Biomedical Technology, Beijing, China). The vector PCR product and insertion PCR product were ligated using NEBuilder high-fidelity (HiFi) DNA assembly master mix (New England Biolabs, Ipswich, MA, USA). The assembly reagent (2 mL) containing recombinant plasmids pEASY-T1-PBP3 (YRIK), pEASY-T1-PBP3 (YRIN), pEASY-T1-PBP3 (W.T.), and pEASY-T1-CirA (W.T.) were added to competent E. coli DH5a (100 mL) via incubation on ice for 30 min followed by incubation for 45 s at 42°C. Subsequently, 1 mL of prewarmed SOC medium (37°C; TaKaRa Biomedical Technology) was added to the competent cells. The solution was incubated for 1 h at 37°C with shaking at 200 rpm, and then 100 mL of the bacterial solution was inoculated onto Luria-Bertani (LB) agar containing 100 mg/mL of ampicillin following overnight incubation. Positive clones were selected for subsequent PCR to verify the success of the transformation. E. coli DH5a carrying plasmid W.T. was enriched in LB broth containing 100 mg/mL of ampicillin at 37°C overnight. For plasmid extraction, the bacterial solution (1 mL) was processed using a plasmid minikit (Omega Bio-tek, Norcross, GA, USA). Subsequently, 50 ng of DNA was transformed into cefiderocol-resistant isolates C5492 and C6346. The transformation was performed via electroporation using the MicroPulser electroporator (Bio-Rad, Hercules, CA, USA) using the program EC3 (3.0 kV, 5.5 ms). The cells were then immediately treated with 1 mL of SOC culture (preheated at 37°C) for 1 h. Then, 100 mL of the bacterial solution was inoculated onto LB agar containing 100 mg/mL of kanamycin following overnight incubation. Positive clones were selected for subsequent PCR analysis to verify the success of the transformation. All transformants and parental isolates were subjected to AST for cefiderocol under induction using 1 mM isopropyl beta-D-1-thiogalactopyranoside (IPTG).
cirA and bla NDM gene deletion. The E. coli cirA and bla NDM gene deletion strain was obtained from E. coli ATCC 25922 and a clinically derived carbapenem-resistant E. coli C7310 by the CRISPR-Cas9 genome editing system according to methods described previously (43,44). C7310 was a cefiderocol-susceptible (MIC = 1 mg/mL) E. coli harbored NDM-5 with a wild-type cirA gene. Briefly, the pCas plasmid (#62225) carrying kanamycin resistance was transformed into ATCC 25922 and C7310 strains using electroporation. The colonies were selected on an LB agar plate containing 50 mg/mL kanamycin at 30°C. Annealed cirA or bla NDM spacer oligonucleotides and the repair arms of the cirA gene (;1 kb) were inserted into the pSGKP-spe plasmid (#117234) by Golden Gate assembly (New England Biolabs, Ipswich, MA, USA). Then, the cirA spacer with the repair arms of the cirA gene (;1 kb) and introduced pSGKP-spe plasmid was transformed into the ATCC 25922 and C7310 strains with pCas plasmid using electroporation. The colonies were selected on an LB agar plate containing 100 mg/mL spectinomycin and 50 mg/mL kanamycin at 30°C with 0.2% L-arabinose. The successful cirA deletion strain was confirmed by PCR and sequencing. After confirmation, the pCas and pSGKP-spe plasmids were cured by culturing the cells with at 37°C and in the presence of sucrose. The bla NDM gene deletion was performed based on C7310DcirA, following the same steps as described above. For information on primers, please refer to Table S1.
Data availability. The complete sequences of the 26 cefiderocol-resistant Escherichia coli isolates and assembled data have been deposited on NCBI with BioProject no. PRJNA756960 (Table S4).

SUPPLEMENTAL MATERIAL
Supplemental material is available online only. SUPPLEMENTAL FILE 1, PDF file, 0.5 MB.