Activity of cefepime/taniborbactam and comparators against whole genome sequenced ertapenem-non-susceptible Enterobacterales clinical isolates: CANWARD 2007–19

Abstract Objectives This study assessed in vitro activities of cefepime/taniborbactam and comparator antimicrobial agents against ertapenem-non-susceptible Enterobacterales (ENSE) clinical isolates collected from the CANWARD study 2007–19, and associations between MIC and various mechanisms of β-lactam resistance identified using WGS. Methods A total of 179 ENSE (MIC ≥ 1 mg/L) isolates underwent susceptibility testing using reference CLSI broth microdilution. WGS was performed using the Illumina NextSeq platform. Carbapenemases, ESBLs and other β-lactamases were identified using ResFinder 4.0. Alterations in ompC/F and ftsI (PBP3) were identified by comparing extracted sequences to the appropriate NCBI reference gene. Porin alterations were analysed with Provean v1.1.3. Specific alterations of interest in PBP3 included a YRIN or YRIK insertion after P333. Results Cefepime/taniborbactam was highly active (MIC50/MIC90, 0.5/2 mg/L; 177/179 isolates inhibited at ≤ 8 mg/L) against ENSE with various antimicrobial resistance phenotypes. Thirteen (7.3%) of the 179 ENSE isolates demonstrated cefepime/taniborbactam MIC values ≥ 4 mg/L and possessed combinations of β-lactam resistance mechanisms, including a carbapenemase and/or ESBL and/or other β-lactamase genes, as well as alterations in OmpC and/or OmpF and/or PBP3. Of the two Escherichia coli isolates that demonstrated a cefepime/taniborbactam MIC of 32 mg/L, one possessed NDM-5, OXA-181 and TEM-1B, an OmpC alteration and P333_Y334insYRIN in PBP3, while the second contained CTX-M-71, a truncated OmpF and a large alteration in OmpC (F182_R195delinsMTTNGRDDVFE). Conclusions Cefepime/taniborbactam was highly active against ENSE with various antimicrobial resistance phenotypes/genotypes. ENSE isolates with cefepime/taniborbactam MIC values ≥ 4 mg/L possessed combinations of β-lactam resistance mechanisms, including β-lactamase genes, as well as alterations in OmpC and/or OmpF and/or PBP3.


Antimicrobial susceptibility testing
Following two subcultures from frozen stock, the in vitro activities of cefepime/taniborbactam (cefepime doubling dilution range 0.03-128 mg/L with taniborbactam fixed at 4 mg/L) and comparator agents were determined by reference CLSI broth microdilution (M07, 11th edition, 2018) using 96-well custom designed microtitre plates. 3,11 Antimicrobial agents were obtained as laboratory grade powders from their respective manufacturers or commercial sources. Stock solutions were prepared and dilutions made as described by CLSI. 3,11 MICs were interpreted using CLSI M100 (30th edition, 2020) or FDA breakpoints. 12,13 Isolates with cefepime/taniborbactam MIC ≤ 8 mg/L were deemed susceptible.

WGS
ENSE isolates, plus 51 ertapenem-susceptible Enterobacterales controls, were sequenced using the Illumina NextSeq platform. Quality control was assessed using the FastQC tool (https://www.bioinformatics. babraham.ac.uk/projects/fastqc/) and contigs were assembled using SPAdes software. 14 Sequencing yielded an average of 2 637 395 reads per genome and an average genome coverage of 82×. De novo assembly yielded an average contig length and N50 length of 128583 and 318 077 bp, respectively.
MLST alleles and STs were identified by scanning assembled contigs against available PubMLST databases (https://github.com/tseemann/ mlst). Carbapenemases, ESBLs and other β-lactamases were identified using ResFinder 4.0 at an identity threshold of 90%. 15 Alterations in genes ompC/F (encoding major porins), ompK37 (in K. pneumoniae only) and ftsI (encoding PBP3) were identified by comparing extracted sequences to the appropriate NCBI reference gene. PBP3 alterations of interest included four amino acid insertions after P333, as previously described. 16 Porin alterations were analysed with Provean v1.1.3 (default settings) to predict those that may have a negative impact on biological protein function. 17 Only alterations predicted by Provean as having a negative impact on Omp function are discussed further in this study.

Nucleotide sequence accession numbers
The WGS data reported in this study have been deposited in the NCBI Short Read Archive under BioProject PRJNA736690.

Specific gene content of ENSE with cefepime/ taniborbactam MICs ≥ 4 mg/L
Although the CLSI susceptible, dose-dependent (SDD) breakpoint for cefepime/taniborbactam for Enterobacterales is ≤ 8 mg/L, this analysis also examined the characteristics of isolates within the cefepime SDD range (cefepime/taniborbactam MICs of 4-8 mg/L) as well as above 8 mg/L. Only 7.2% (n = 13) of ENSE isolates demonstrated a cefepime/taniborbactam MIC ≥ 4 mg/L, including seven K. pneumoniae, three E. coli, two E. cloacae and one S. marcescens ( Table 3). Each of the 13 isolates possessed at least one β-lactamase gene and at least one altered or truncated Omp (including OmpK37 for one K. pneumoniae isolate) ( Table 3). Only two isolates with cefepime/taniborbactam MIC ≥ 4 mg/L possessed a carbapenemase (OXA-48; NDM-5 + OXA-181); more commonly, isolates possessed an ESBL with multiple additional β-lactamase genes. Seven isolates possessed a truncated Omp, while eight had Omps with insertions of two or more amino acids (with or without accompanying deletions) ( Table 3). Only one isolate possessed a four amino acid insertion after P333 in PBP3 (see below). Frequently these 13 isolates also demonstrated elevated MICs or resistance to ceftazidime/ Shown are numbers for the lowest common concentration; actual MICs of some isolates may be lower than indicated. b Shown are numbers for the highest common concentration; actual MICs of some isolates may be higher than indicated.

Discussion
Of the 18 027 Enterobacterales isolates collected from CANWARD from 2007 to 2019, we obtained and tested the Shown are numbers for the lowest common concentration; actual MICs of some isolates may be lower than indicated. d Shown are numbers for the highest common concentration; actual MICs of some isolates may be higher than indicated. Golden et al. 0.99% (179/18 027) of isolates that were ENSE (ertapenem MIC ≥1 mg/L). 3 In this study we assessed the activity of cefepime/taniborbactam against this highly selected cohort of ENSE clinical isolates which using WGS were found to contain carbapenemase genes (8.9%), ESBL genes (21.8%) and other β-lactamases (e.g. AmpC) (97.2%) as well as porin alterations (88.3%) and insertions in PBP3 (1.1%) ( Table 3, Table S1). The low number of ENSE isolates with a carbapenemase (8.9%) may reflect that we studied ertapenem-non-susceptible isolates rather than meropenem-resistant isolates. Not surprisingly, the 179 ENSE isolates demonstrated low susceptibilities to other β-lactam and β-lactam-like agents (Table 1). Against this MDR cohort, cefepime/taniborbactam demonstrated a . 64-fold reduction in MIC 90 compared with cefepime (MIC 90 , 2 mg/L versus . 64 mg/ L, respectively) and was active against subsets of isolates with various β-lactam and non-β-lactam antimicrobial resistance phenotypes (Table 1). These data are consistent with Hamrick et al. 10 who reported that the addition of taniborbactam (fixed concentration at 4 mg/L) potentiated cefepime activity 8-to . 1024-fold.
carbapenemases. 17 However, cefepime/taniborbactam was not active against strains harbouring IMP, demonstrating MICs of 128 mg/L. 20 Abdelraouf et al. 21 demonstrated that the in vitro activity observed with cefepime/taniborbactam against resistant Enterobacterales was translated to the in vivo setting. Using a cefepime/taniborbactam human-simulated regimen equivalent to 2 g/0.5 g q8 h administered as a 2 h infusion (which is the dose used in clinical trials) in mice, against 26 clinical Enterobacterales expressing ESBLs, plasmid-mediated AmpC and/or class A (KPC) or D carbapenemases (OXA-48), the combination exerted potent in vivo activity (. 1 log 10 killing among all the isolates examined with cefepime/taniborbactam MICs up to 16 mg/L) against cefepime-resistant isolates, including serinecarbapenemase producers.
Although the number of isolates tested in the present study was small, we report that cefepime/taniborbactam demonstrated MICs of 0.5-4 mg/L for 75% (6 of 8), 50% (2 of 4) and 50% (2 of 4) of isolates resistant to imipenem/relebactam, ceftazidime/avibactam and meropenem/vaborbactam, respectively. In our study, one isolate containing NDM-1 was resistant to imipenem/relebactam, ceftazidime/avibactam and meropenem/vaborbactam, but was provisionally susceptible to cefepime/taniborbactam (MIC 1 mg/L). This is not surprising as, unlike ceftazidime/avibactam and meropenem/vaborbactam, cefepime/taniborbactam inhibits Enterobacterales with MBL NDM and VIM as well as serine-β-lactamases KPC and OXA-48. 7,9,10,20,22 Only 13 (7.2%) ENSE isolates demonstrated a cefepime/ taniborbactam MIC ≥ 4 mg/L. The 13 isolates represented both microbiological species diversity (7 K. pneumoniae, 3 E. coli, 2 E. cloacae and 1 S. marcescens) and clonal diversity (STs). All isolates possessed combinations of β-lactam resistance mechanisms, including a carbapenemase and/or ESBL and/or other β-lactamase genes, as well as alterations in OmpC and/or OmpF (reduced uptake into the periplasmic space) and/or PBP3 (reduced binding to target site) ( Table 3). Wang et al. 18 analysed 29 NDM-5-producing E. coli isolates from China with cefepime/ taniborbactam MICs . 8 mg/L (taniborbactam fixed at 4 mg/L) and documented the presence of PBP3 mutations in 28/29 isolates. A variety of different mutations in PBP3 were documented. 15 Unfortunately, other (non-PBP3 and non-β-lactamasemediated) resistance mechanisms, such as porin changes or efflux pump expression, were not characterized in that study. Mushtaq et al. 9 analysed Enterobacterales with cefepime/ taniborbactam MICs . 8 mg/L (taniborbactam fixed at 4 mg/L) (E. coli n = 15, Klebsiella spp. n = 19 and Enterobacter spp. n = 1). These researchers noted both genetic diversity (a variety of STs) as well as no universal resistance mechanism in all isolates but rather combinations of carbapenemases (e.g. NDM-5, NDM-7) along with PBP3 insertions (e.g. after amino acid 333), and/or porin changes (e.g. OmpF). 9 Kloezen et al. 19 analysed three isolates of Enterobacterales with cefepime/taniborbactam MICs . 4 mg/L (taniborbactam fixed at 4 mg/L). One isolate harboured a VIM gene while the other two carried VIM-1, CMY-13 and qnrA1 genes. The authors concluded that the presence of Includes eight K. pneumoniae, six E. coli and three E. cloacae. b Shown are numbers for the lowest common concentration; actual MICs of some isolates may be lower than indicated here. Cefepime/taniborbactam CANWARD 2007-19 VIM and AmpC may reduce cefepime/taniborbactam activity against Enterobacterales. The presence of other underlying resistance mechanisms such as porin alterations, which may reduce periplasmic uptake, or target site binding to PBP3 or efflux were not assessed.
There are limitations to the data presented here that deserve attention. Only a limited number of isolates resistant to ceftazidime/avibactam, imipenem/relebactam and meropenem/vaborbactam were available for testing, thus the promising results showing cefepime/taniborbactam activity against ceftazidime/ avibactam-, imipenem/relebactam-and meropenem/vaborbactam-resistant Enterobacterales need to be confirmed by others. In addition, it should be mentioned that the results of our WGS provide genetic associations with phenotypic resistance but have not been proven to result in MIC increases or resistance using complementation studies. Finally, though we assessed β-lactam resistance, porin alterations (which may or may not affect periplasmic uptake) and putative binding to the target site (PBP3), we did not assess efflux pump expression, which is known to confer increased cefepime MICs in Enterobacterales and may affect cefepime/taniborbactam activity. 8 In summary, the current study demonstrated that cefepime/ taniborbactam was highly active against whole genome sequenced ENSE isolates with various antimicrobial resistance phenotypes/genotypes. ENSE isolates with cefepime/taniborbactam MIC values ≥ 4 mg/L possessed combinations of β-lactam resistance mechanisms, including a carbapenemase and/or ESBL and/or other β-lactamase genes, as well as alterations in OmpC and/or OmpF and/or PBP3.