Exploring mutational possibilities of KPC variants to reach high level resistance to cefiderocol

KPC beta-lactamase, clinical KPC variants, mutagenesis, cefiderocol resistance, 11 cirA gene, fiu gene, ybiX gene 12 13 Corresponding Author: 14 * André Birgy, [email protected] 15 16 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 15, 2025. ; https://doi.org/10.1101/2025.02.14.638246doi: bioRxiv preprint

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Klebsiella pneumoniae carbapenemase (KPC) is one of the most 18 widespread carbapenemases, with over 242 variants identified. Some, like KPC -33 (D179Y), 19 resist both ceftazidime -avibactam (CZA) and cefiderocol but show only a moderate 20 cefiderocol MIC increase. KPC's success stems from its genetic adaptability, enabling 21 mutation ac cumulation and necessitating proactive resistance monitoring. This study 22 investigates the mutational possibilities for three KPC variants to develop high -level 23 cefiderocol resistance. 24

Using random mutagenesis and enrichment selection over a 10-day 25 cefiderocol exposure at increasing concentration , we investigated the mutational potential 26 of KPC -2, KPC -3, and KPC -33 to develop high -level cefiderocol resistance. Resistance 27 mechanisms were then analyzed through phenotypic testing and sequencing. 28 Results:Random mutagenesis generated 10⁵, 10⁴, and 105 mutants for blaKPC-2, blaKPC-29 3, and blaKPC-33, respectively. After ten days of increasing cefiderocol exposure, MICs rose 30 significantly, with KPC -2 mutants reaching >32 mg/L. Phenotypic analysis revealed a 31 common resistance profile across all tested mutants, with resistance to ceftazidime, 32 ceftazidime-avibactam, cefixime, and piperacillin, but restored susceptibility to 33 carbapenems and most other β-lactams. Sequencing identified key mutations (D179Y-34 D209V in K PC-2, L169P in KPC -3), while chromosomal changes, particularly cirA and ybiX 35 disruptions, played a crucial role in resistance evolution. 36

Our results show that the enriched mutations in KPC genes are not 37 sufficient to confer high cefiderocol MICs, suggesting that, in the studied variants, no simple 38 mutational pathway allows the KPC enzyme to efficiently hydrolyze cefiderocol. These 39 findings underscore the interplay between enzymatic and iron transport mutations in 40 cefiderocol resistance, high lighting the importance of surveillance to anticipate emerging 41 resistance in KPC-producing pathogens. 42 43 44 45 46 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 15, 2025. ; https://doi.org/10.1101/2025.02.14.638246doi: bioRxiv preprint Klebsiella pneumoniae carbapenemase (KPC) stands as the most prevalent 47 carbapenemase enzyme globally, with an endemic presence across North and South 48 America, China, Israel, Greece, and Italy (1, 2) . KPC exhibits a wide -ranging resistance 49 profile, encompassing most beta -lactams including carbapenems and clas sical beta -50 lactamase inhibitors . New beta -lactamase inhibitors are effective against KPC beta -51 lactamase (avibactam, vaborbactam , relebactam …) and showed effectiveness against KPC -52 producing Enterobacterales(3–5). However, KPC diversified rapidly with 242 clinical variants 53 described to date ( January 2025). The clinical success of KPC variants is attributed to 54 intricate molecular mechanisms and a remarkable genetic adaptability, though our 55 understanding of these processes remains incomplete (6, 7) . While the combination of 56 ceftazidime-avibactam (CZA) is recommended as the first -line treatment for systemic 57 infections caused by KPC-producing Enterobacteriaceae (KPE), many KPC variants have been 58 reported to be resistant to CZA (6, 8, 9) . Recent studies highlight the emergence of clinical 59 variants that confer cross -resistance to newly introduced therapeutic agents such as 60 meropenem-vaborbactam (MEV) or imipenem -relebactam (IMR) (10–12). In t his context, 61 Cefiderocol has become a therapeutic option since its introduction in 2020 (9). 62 Cefiderocol is a siderophore cephalosporin sharing side chains similar to those of both 63 cefepime and ceftazidime conferring stability against most beta -lactamases (13–15). The 64 chlorocatechol group binds covalently to extracellular Fe3+, allowing its fast and active 65 transport into the bacterial periplasm by iron TonB -dependent transporters (TBDT) such as 66 FepA, Fiu or CirA (13, 14, 16) . However, recently cross-resistance to both CZA and 67 Cefiderocol has been described in some KPC variants and notably in KPC -33 (D179Y) and 68 KPC-31 (D179Y-H274) (17–21). The D179Y mutation is observed in 26 out of 242 (11%) of 69 KPC variants with KPC-33 and KPC -31, being prominent in globa l epidemiology (6). The 70 structural analogy between Cefiderocol and Ceftazidime could be one of the origins of this 71 cross-resistance, corroborated by the ide ntification of common molecular and enzymatic 72 mechanisms (18, 22) . While described mutants currently confer a modest increase in 73 cefiderocol MICs – close to EUCAST breackpoints (2 mg/L), it is possible that the natural 74 evolution of KPC -2 or one of it’s variant , along with the accumulation of mutational events 75 with time, could increase the level of resistance in the future. 76 This evolutionary adaptability, similar to the one observed with TEM beta-lactamases and 77 3rd generation cephalosporins would pose an escalating threat to healthcare systems, 78 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 15, 2025. ; https://doi.org/10.1101/2025.02.14.638246doi: bioRxiv preprint underscoring the need to anticipate future resistance (23) . In this context, we aimed to test 79 the evolutionary possibilit ies for 3 KPC variants (KPC-2 and KPC-3 being the most frequent 80 and KPC-33: a variant with increased cefiderocol MIC) to confer a high level of resistance to 81 cefiderocol. To achieve this , we generated KPC mutant libraries through random 82 mutagenesis to assess the potential for selecting a combination of mutations that confer 83 high levels of resistance to Cefiderocol. 84 85

86 87 Mutant libraries from random mutagenesis experiment 88 Libraries of mutants w ere generated by random mutagenesis experiment, resulting 89 in a total of 1.3 x 10⁵ colonies from blaKPC-2, 3 x 10⁴ colonies from blaKPC-3, and 10 5 colonies 90 from blaKPC-33. Colonies were scraped off the agar into LB -glycerol (40%), and this cell 91 suspension was aliquoted and stored at −80°C after thorough mixing. The average 92 mutagenesis efficiency resulted in 3.6 substitution -type mutations per allele. It was 93 determined on 100 randomly chosen colonies before selection on which the KPC gene was 94 sanger sequenced. 95 96 In vitro enrichment experiment and selection of mutants 97 Cefiderocol MICs of the ancestral variants blaKPC-2, blaKPC-3 and blaKPC-33 were 0.25, 98 0.12, and 2 mg /L respectively. The increase of MICs over the ten -day exposure period is 99 shown in Figure 1. By day 10, an increase in MICs was observed for all three exposed mutant 100 libraries: >32, 2 and 8 mg /L corresponding to an increase by >128-fold, 17-fold, and 4 -fold 101 respectively for the librar ies derived from blaKPC-2, blaKPC-3 and blaKPC-33. Starting from the 102 well that grew at the highest cefiderocol concentration, we re -isolated 10 µL of culture by 103 plating on LB -tetracycline. Ten colonies from each of these enrichment experiment s were 104 randomly selected for phenotypic characterization. 105 106 Phenotypic characterization: Antibiotic susceptibility testing allowed the 107 identification of a common resistance phenotypic profile for all tested mutants after the 108 ten-day exposure to increasing concentrations of cefiderocol. Mutants were all resistant to 109 ceftazidime and CZA, cefixime, and piperacillin but were susceptible to carbapenems (Table 110 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 15, 2025. ; https://doi.org/10.1101/2025.02.14.638246doi: bioRxiv preprint 1, Supplementary table 2 and Figure 2 ). So, mutations enriched in our experiment 111 (especially from KPC-2 and KPC-3) modified the resistance capacity of the KPC protein. Also, 112 the Cefiderocol MICs increased differently depending on the initial variant (table 1). 113 Genotypic characterization: Sanger sequencing was performed on the 10 isolated 114 colonies from each enrichment experiment. From KPC-2 mutants’ library, we identified only 115 the combination of D179Y and D209V mutations on all tested mutants (n=10) which had 116 cefiderocol MIC > 32 mg/L (corresponding to a > 128-fold increase compare d to wild -type 117 KPC-2). From KPC-3 (H274Y), we identified only the L169P mutation (n=10) with cefiderocol 118 MIC o f 2 mg/L (17-fold increase compare to wild -type KPC -3) and from KPC -33 (D179Y) 119 mutants’ library, we identified either the F20L (n=6), the F20L-G291S (n=2) or the R6H-F20L 120 (n=2) mutations, a ll conferring a modest increase in cefiderocol MIC of 8 mg/L 121 corresponding to a 4-fold increase compare to KPC-33. 122 123 To assess the direct impact of these mutations in the KPC gene on cefiderocol and other 124 beta-lactams resistance, mutated pBR322 plasmids were extracted from selected strains. 125 Antibiotic susceptibility testing and cefiderocol MIC were then determined after re -126 electroporating the plasmids into fresh E. coli TOP10 (Table 1). A decrease in cefiderocol 127 MICs was observed following re-electroporation: from > 32 mg/L to 2 mg/L for KPC-2 with 128 D179Y and D209V mutations, from 2 to 0.5 mg/L for KPC-3 with L169P and from 8 to 2 mg/L 129 for KPC-33 (either with F20L, F20L -G291S or R6H -F20M mutations) while the susceptibility 130 to other beta-lactams was not modified. This show that the KPC mutations alone do not fully 131 explain the increase in cefiderocol MIC suggesting the contribution of other molecular 132 mechanisms within the genomes to confer high levels of resistance to cefiderocol. 133 Exploration of associated molecular mechanisms impacting Cefiderocol resistance 134 To explore the presence of underlying molecular mechanisms, WGS was performed 135 on one representative isolate of each mutant libraries (KPC-2n2 from KPC -2, KPC-3n4 from 136 KPC-3 and KPC-33n6 from KPC-33). 137 First, a snip comparison to the reference genome was performed. No newly acquired 138 mutations were detected and notably in genes encoding porins , iron TonB -dependent 139 transporters (TBDT) such as FepA, Fiu or CirA or penicillin-binding proteins (PBPs). 140 In comparison to the reference genome, all analyzed genomes exhibited at least one 141 insertion in the c irA gene, resulting in a truncated protein. In details, insertion sequences 142 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 15, 2025. ; https://doi.org/10.1101/2025.02.14.638246doi: bioRxiv preprint (IS) identified with ISFinder as ISKpn72, ISKpn8 and IS1X2 were present in cirA gene of KPC -143 2n2, KPC-3n4 and KPC -33n6 isolates respectively. The integration occurred in the same 144 region of the gene but at different sites. 145 In the KPC -2n2 we observed the insertion of another IS, ISKpn8 in the intergenic region 146 between the fiu and ybiX genes (upstream of ybiX), both being part of an operon. 147 148 Deciphering the impact of each gene in cefiderocol MICs 149 150 To assess the contribution of cirA, ybiX and the combination of cirA/ybiX in 151 cefiderocol resistance, MICs were assessed on Keio (E. coli K12) WT, and the same strain 152 with ΔcirA, ΔybiX, or ∆cirA/∆ybiX knockout mutants (the latter has been constructed for this 153 study) with and without the p BR322-KPC-2n2 plasmid containing blaKPC-2 (D179Y-D209V), 154 and pBR322-KPC-33 (D179Y) (Table 2). 155 Cefiderocol MICs for both Keio WT and ΔybiX cells were initially very low (< 0.03 mg.L -1). In 156 contrast the ΔcirA resulted in a higher baseline MIC (0.12 mg/L) compared to Keio WT. Upon 157 introducing the blaKPC-33 (D179Y) or the blaKPC-2 (with D179Y-D209V) plasmids, the MIC 158 increased from < 0.03 to 0.5 mg /L (for both plasmids) in Keio WT cells, from < 0.03 to 1 or 159 0.5 mg/L in Keio ΔybiX cells and from 0.12 to 2 or 4 mg/L in Keio ΔcirA cells respectively for 160 blaKPC-33 (D179Y) and the blaKPC-2 (with D179Y-D209V). The double deletion of cirA and ybiX 161 led to a major increase in cefiderocol MICs from 0.5 mg/L without plasmid to > 32mg/L with 162 both blaKPC-33 (D179Y) or the blaKPC-2 (with D179Y-D209V) plasmids (Table 2). 163 164

165 166 Using random mutagenesis, we introduced diversity into three KPC alleles: KPC -2, KPC -3, 167 and KPC -33 (the latter being already described with increased cefiderocol MICs (18)) to 168 evaluate mutational possibilities to confer high -level resistance to cefiderocol . Our 169 mutagenesis allowed the generation of approximately 10⁴ to 10⁵ mutants per allele (with 170 mean 3.6-mutations/alleles), corresponding in total to about 3 -4 × 10⁵ different mutations. 171 The hypothesis driving this work is that the accumulation of mutations in blaKPC could 172 enhance the cefiderocol resistance conferred by the KPC enzyme. A scenario similar to what 173 was observed with TEM β-lactamases in response to ceftazidime a few decades earlier (23). 174 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 15, 2025. ; https://doi.org/10.1101/2025.02.14.638246doi: bioRxiv preprint 175 Enrichment experiment under increasing concentrations of cefiderocol resulted in MIC 176 increases, reaching >32, 2, and 8 mg/L for libraries derived from KPC -2, KPC-3, and KPC -33, 177 respectively. When analysing the phenotypes of mutants selected at the highest cefiderocol 178 concentrations, we observed a phenotypic convergence . The restoration of susceptibility to 179 carbapenems among all mutants suggests an evolutionary trade -off, where the acquisition 180 of specific mutations conferring resistance to cefiderocol may simultaneously enhance 181 sensitivity to carbapenems and influence susceptibility to most other beta-lactams. Overall, 182 the mutants exhibited resistance to ceftazidime and ceftazidime -avibactam, along with 183 increased cefiderocol MICs. The cross-resistance to CZA and cefiderocol has recently been 184 documented in the literature among clinical KPC variants, sometimes associated with other 185 resistance mechanisms or beta -lactamases (17, 24, 25) . Although these variants can be 186 selected during CZA treatment, our results raise the concerning possibility of rapid selection 187 of CZA resistant-mutants also under cefiderocol treatment (21, 24). This likely stems from 188 shared structural features between ceftazidime and cefiderocol and a similar hydrolytic 189 mechanism by KPC variants for the two drugs (22). 190 191 Among mutants derived from KPC -2 or KPC -3, only a single clone was enriched and 192 subsequently dominated the population, as evidenced by identical sequences in all analysed 193 mutants (10/10 clones). Interestingly, the D179Y mutation, frequently observed in clinical 194 KPC variants and known to increase cefiderocol resistance, was select ed in KPC-2 alongside 195 the D209V mutation. The catalytic efficiency of KPC -2 has been described to be 196 approximately 20 times lower for Cefiderocol compared to Ceftazidime (22). Nevertheless, 197 the acquisition of the D179Y substitution significantly enhances the hydrolytic capacity of 198 KPC-2 towards Cefiderocol by increasing the enzyme's affinity for this an tibiotic substrate. 199 The D209V mutation is not located directly on the essential loops involved in the 200 conformation of the active site of the enzyme, and has not been previously reported . To 201 investigate the role of the D209V substitution in conferring high levels of resistance to 202 Cefiderocol, Keio WT cells were transformed with the pBR322 -KPC-33 plasmid (harboring 203 only the D179Y mutation) and the pBR322 -CKPC-2n2 plasmid (containing both D179Y and 204 D209V mutations) and their MICs were assessed and compared. The Cefiderocol MICs were 205 similar for both strains, suggesting that the D209V mutation did not contribute much to the 206 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 15, 2025. ; https://doi.org/10.1101/2025.02.14.638246doi: bioRxiv preprint increased Cefiderocol MIC. This highlights the adaptive importance of D179Y in cefiderocol 207 resistance, as already suggested with clinical variants (6, 18) . Of n ote, mutant libraries 208 derived from KPC -33 (D179Y) did not yield other mutations that further enhanced 209 significantly the resistance capabilities of the KPC enzyme. 210 Finally, the L169P substitution selected from KPC-3 is associated with a moderate increase in 211 Cefiderocol MICs. This mutation has been previously reported in five clinical KPC variants 212 and is often associated with other mutations (KPC-35, KPC-46, KPC-48, KPC-138, and KPC -213 155) exhibiting varying resistance phenotypes to beta -lactams (26). Strains producing KPC -214 35, KPC-46, and KPC-48 have been reported as susceptible to carbapenems, and KPC -46 and 215 KPC-48 exhibit an ESBL -like resistance profile including resistance to ceftazidime -avibactam 216 (6, 27, 28). 217 218 To disentangle the respective contributions of KPC mutations and chromosomal 219 mechanisms to cefiderocol resistance, we assessed their individual impacts. For that, t he 220 impact of newly acquired KPC mutations on Cefiderocol resistance was subsequently 221 assessed by isolating the plasmid containing the mutated blaKPC gene from enriched mutant 222 and reintroducing it into a naïve E. coli TOP10 strain, devoid of prior evolutionary changes. 223 We found that the resulting Cefiderocol MICs attributed to the mutated KPC protein s, were 224 elevated compared to the ancestral variant s, though the increase s were considerably more 225 moderate (maximum an 8-fold increase with the plasmid from the KPC-2n2 mutant) (Table 226 1). These findings strongly suggest that additional molecular mechanisms are likely at play, 227 and that chromosomal mutations are essential for achieving high -level cefiderocol 228 resistance in these KPC variants . However, we started from most prevalent KPC variants 229 (KPC-2, KPC -3, and KPC -33) but we cannot rule out that our results could have been 230 different starting from other variants. 231 232 A comprehensive analysis of the mutants via WGS did not reveal any newly 233 mutational events involving PBPs or outer membrane proteins, such as porin channels. 234 Recent literature aligns with this perspective, indicating that mutations affecting genes 235 encoding porins or PBP3 transpeptidases a lone are insufficient to confer resistance 236 thresholds to cefiderocol in Enterobacter ales (29, 30) . But WGS analysis and results from 237 transformation of knockout mutants with plasmids harboring KPC variants strongly suggest 238 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 15, 2025. ; https://doi.org/10.1101/2025.02.14.638246doi: bioRxiv preprint the involvement of the c irA gene in the acquisition of in vitro resistance in our mutants. 239 Indeed, t he introduction of the pBR322 -KPC-2n2 plasmid into Keio WT cells raised the 240 Cefiderocol MIC from 0.03 to 0.5 mg/L corresponding to a 16-fold increase, showing a direct 241 impact of the D179Y and D209V mutations on Cefiderocol resistance, though the MIC 242 remained below the EUCAST resistance threshold (2 mg /L) (31). The deletion of cirA gene in 243 Keio strain resulted in a 4 -fold increase in cefiderocol MIC (0.12 vs. < 0.03 mg /Lin WT), 244 suggesting that the absence of CirA transport restricts Cefiderocol uptake into the bacterial 245 periplasm, thus elevating MIC values as already shown (32). Furthermore, the integration of 246 the pBR322-KPC-2n2 plasmid into Keio cirA-deficient cells led to an even greater increase in 247 resistance, with MICs rising 16 -fold relative to ΔcirA cells (from 0,12 to 2 mg /L) and more 248 than 66 -fold compared to WT (from < 0,03 to 2 mg /L). These results are consistent with 249 existing data, as TBDT are the primary targets for in vitro and in vivo resistance against 250 cefiderocol in Enterobacter ales (29, 33) . Recent studies have demonstrated the impact of 251 loss of function of CirA in acquiring high levels of resistance, particularly in Enterobactera les 252 producing New Delhi Metallo -beta-lactamase (NDM) (16, 30, 34, 35) . Moreover, a Chinese 253 multicentric study suggested that restoring normal CirA function can reverse resistance in 254 NDM-producing E. coli (30). However, few studies report ed the selection of mutations 255 targeting the cirA gene that confer significant Cefiderocol resistance in KPC-producing E. 256 coli. In vitro study from Nurjadi et al. also highlighted that production of NDM facilitated the 257 propensity for acquisition of cirA mutations, whereas KPC-2 did not (35). Our results suggest 258 that the production of mutated KPC-2, KPC -3, and KPC -33 by E. coli strains may act 259 synergistically in combination with mutations affecting the cirA gene to reach high 260 cefiderocol MIC. 261 In parallel, WGS revealed the insertion of ISKpn8 within the intergenic region 262 between the fiu and ybiX genes (upstream of the ybiX gene) of KPC-2n2. While the fiu gene 263 is well -characterized and encodes one of the TBDTs essential for the transport of 264 siderophores into the bacterial periplasm, the ybiX gene is described here for the first time 265 as associated with cefiderocol resistance. This gene encodes a putative iron uptake factor 266 and opera tes within an operon alongside fiu, which codes for the outer membrane iron -267 catecholate transporter, both regulated by a common transcriptional regulator : the Ferric 268 Uptake Regulator (Fur) (36–40). ybiX is homologous to piuC in Pseudomonas aeruginosa, a 269 gene implicated in siderophore uptake (41) which has been linked to in vitro resistance 270 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 15, 2025. ; https://doi.org/10.1101/2025.02.14.638246doi: bioRxiv preprint against siderophore-based antibiotics (42, 43). A similar mechanism is like ly responsible for 271 the observed resistance in the E. coli mutant, supporting the hypothesis that ybiX is 272 implicated in the iron acquisition pathway. Recent literature indicates that the in vitro 273 resistance level to cefiderocol is influenced by the contribution of each key TBDT involved in 274 antibiotic substrate transport, and that the combined loss of Fiu function alongside a 275 deletion of the cirA gene in E. coli significantly increase the MIC (32). Here, the KPC -2n2 276 strain exhibited the highest cefiderocol MIC – >32 mg/L and is the one that combined an 277 insertion in both cirA and upstream of ybiX (distancing the promoter and thereby impacting 278 expression) genes alongside with the expression of pBR322 -KPC-2 with the D179Y and 279 D209V mutations. The double deletion likely causes a strong restriction in cefiderocol 280 uptake, explaining the marked increase in MIC. 281 282

In this study, we explored how libraries of mutants of KPC-2, KPC-3, or KPC-33 283 could adapt over a ten -day cefiderocol treatment. The resulting enriched mutants displayed 284 similar phenotypes, including cross -resistance to both ceftazidime -avibactam and 285 cefiderocol but with increased susceptibility to carbapenems and most other beta -lactams. 286 While KPC mutations alone do not lead to high -level cefiderocol resistance in our 287 experiment, we could hypothesize that they facilitate bacterial survival under cefiderocol 288 treatment, creating opportunities for alternative resistance mechanisms, such as 289 chromosomal mutations, to emerge. This intricate interplay between blaKPC mutations and 290 siderophore transport deficiencies underscores the importance of integrated therapeutic 291 and surveillance approaches to mitigate resistance emergence and maintain effective 292 treatment options. These findings provide critical insights into the resistance mechanisms of 293 cefiderocol in KPC -producing E. coli . They suggest that the evolutionary pathway to high -294 level cefiderocol resistance is not straightforward, relying on both enzymat ic mutations and 295 chromosomal adaptations. 296 297

298 299 KPC variants and mutagenesis experiments 300 blaKPC-2, blaKPC-3 (H274Y mutation) and blaKPC-33 (D179Y mutation) cloned into pBR322 301 plasmids were sourced from our existing collection (44) and used as templates for 302 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 15, 2025. ; https://doi.org/10.1101/2025.02.14.638246doi: bioRxiv preprint mutagenesis experiments. To explore KPC overall evolvability and diversity through random 303 point mutations across the blaKPC gene, we employed a PCR protocol using error -prone DNA 304 polymerase (Mutazyme II DNA polymerase, GeneMorph II Random Mutagenesis Kit, Agilent 305 Technologies). The protocol was performed as specified by the manufacturer and optimized 306 to achieve mutation frequency of around 5 nucleotides changes per KPC genes (with a size 307 close to one kb). Primers KPC -PCRmuta-F (5' -308 TAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATG-3') and KPC -PCRmuta-R (5' -309 TAAATCAATCTAAAGTATATGAGTAAACTTGGTCTGACAGTTA-3') were used to amplify the 310 blaKPC gene. The mutated genes were then cloned using Gibson assembly (NEBuilderⓇ HiFi 311 DNA Assembly, New England Biolabs) at a 5:1 ratio into the pBR322 plasmid, which had 312 been pre -amplified with pBR322 -pr-Gibson-F (5' -313 CATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTA-3') and pBR322 -pr-Gibson-R (5'-314 TAACTGTCAGACCAAGTTTACTCATATACTTTAGATTGATTTA-3') primers and purified using the 315 QIAquick Gel Extraction Kit (Qiagen). After assembly, mutated plasmids were transformed 316 by electroporation and expressed in One Shot™ TOP10 Electrocomp™ E. coli (Invitrogen) 317 strains. Selection was p erformed on LB-tetracycline medium to generate the mutant 318 libraries. 319 320 In vitro enrichment experiment 321 The enrichment experiment was designed to select for mutants with high-level 322 resistance to cefiderocol. A 107 CFU/mL initial inoculum of the libraries of m utants were 323 exposed to progressively increasing cefiderocol concentrations (ranging from 0.03 and up to 324 32 mg/L) in an iron-depleted Mueller-Hinton (MH) medium in microdilution plates (with 200 325 µl total volume) . Each well was subcultured daily with an inoculum target of 10⁶ CFU/mL , 326 allowing for a gradual escalation of cefiderocol concentration up to 32 mg/L. At the end of 327 the ten -day period, viable resistant isolates that grew at the highest cefiderocol 328 concentration were plated on LB -tetracycline medium, from which 10 colonies were 329 randomly selected from each enrichment experiment and isolated for phenotypic and 330 genotypic characterization. 331 332 Phenotypic characterization : The beta-lactams antibiotic susceptibility profile of the 333 mutants were assessed both by the disk diffusion method on MH-agar medium and by broth 334 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 15, 2025. ; https://doi.org/10.1101/2025.02.14.638246doi: bioRxiv preprint microdilution plates using sensititre plates (ThermoFisher Scientific) in accordance with the 335 last European Committee on Antimicrobial Susceptibility Te sting guidelines (EUCAST) (31). 336 Additionally, the cefiderocol MIC was determined using the Bruker's UMIC® kit and CZA MIC 337 was determined using the E -test® method (BioMérieux, Marcy l’Etoile, France) on MH -agar 338 medium. 339 340 341 Genotypic characterization of selected mutants 342 Sanger sequencing: for the selected mutants, the blaKPC gene carried by pBR322 343 plasmid was amplified using standard PCR protocol and submitt ed for Sanger sequencing 344 (GenewizⓇ Europe, Azenta Life Sciences) to identify mutations in the blaKPC gene. 345 346 Whole genome sequencing: On ancestral strains as well as on one selected strain per 347 enrichment experiment, genomic DNA extraction was performed using the Genomic 348 DNA|gDNA Isolation Kits (Qiagen) for whole -genome sequencing (WGS). Libraries were 349 prepared us ing the Illumina DNA Flex kit and sequenced on a NextSeq platform, using a 350 NextSeq 500/550 Mid Output Kit v2.5 (300 cycles) (Illumina). 351 Bioinformatic analysis: Raw reads were trimmed using Trim Galore v0.4.4_dev and 352 Trimmomatic v0.38 with a Phred score ≥ 20 and a minimum length of 50 bp. The quality of 353 the trimmed reads was verified using FastQC v 0.11.8 and MultiQC v1.7. First, variant 354 detection was performed based on the K12_DH5_alpha reference genome 355 (GCF_002899475.1 NCBI accession). Alignment was carri ed out using BWA v 0.7.17 -r1188, 356 and variants were called using FreeBayes v 1.3.1 -16. A custom python script was used to 357 merge and generate a matrix of presence and absence of single nucleotide polymorphisms 358 (SNPs). The pangenome was computed by constructi ng genomes of the samples with 359 SPAdes v3.15.4. The presence and absence matrix was generated by Panaroo v1.5.0 based 360 on Prokka v1.14 annotations. Both the SNPs and gene matrices were analyzed using an in -361 house R script to compare variations between cefider ocol resistant evolved strains and 362 ancestral ones. Some insertions escaped detection by FreeBayes, particularly those with 363 minimal overlap. To detect them, PanISa was used to specifically identify such cases and an 364 R script was used to visualize the results (45). 365 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 15, 2025. ; https://doi.org/10.1101/2025.02.14.638246doi: bioRxiv preprint 366 Determination of the role of mutational events in Cefiderocol resistance 367 To differentiate between resistance conferred by the selected KPC variants and 368 potential other chromosomal mechanisms, we extracted mutated pBR322 -KPC plasmids 369 from the evolved strain s, re -electroporated them into fresh One Shot™ TOP10 370 Electrocomp™ E. coli , and reassessed the cefiderocol MICs and antibiotic susceptibility 371 testing. 372 373 Thanks to genomic analysis, we identified genomic modifications in some genes of the TBDT 374 family ( cirA gene and ybiX). To assess the contribution of these genes in Cefiderocol 375 resistance phenotypes, cefiderocol MICs were assessed on strains from the Keio collection 376 as well as on newly constructed deleted mutant (46): Keio WT, ΔcirA, ΔybiX, and ∆cirA/∆ybiX 377 knockout mutants before and after transformation with pBR322 containing the mutated 378 blaKPC. For the construction of double -gene deletion mutant, the DNA fragments 379 ∆ybiX::kanamycin cassette (kmfrt) was amplified from E. coli BW25113∆ybiX::Kmfrt Keio 380 strain. After removing the kanamycin cassette from E. coli BW25113∆cirA::Kmfrt by 381 transforming with the recombinase plasmid pCP20, the PCR products of ∆ ybiX::kmfrt were 382 introduced on the native chromosoma l location under the native promoter in 383 BW25113∆cirA::frt using the λ-red linear recombinase plasmid pKOBEG to generate 384 ∆cirA/∆ybiX construct (47, 48) . The integrity of all cloned fragments and mutations was 385 verified by PCR with specific primers and DNA sequencing (Supplementary table 1). 386 387 388

389 This research received no specific grant from any funding agency in the public, commercial, 390 or not-for-profit sectors 391 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 15, 2025. ; https://doi.org/10.1101/2025.02.14.638246doi: bioRxiv preprint

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The copyright holder for thisthis version posted February 15, 2025. ; https://doi.org/10.1101/2025.02.14.638246doi: bioRxiv preprint 48. Chaveroche MK, Ghigo JM, d’Enfert C. 2000. A rapid method for effici ent gene 563 replacement in the filamentous fungus Aspergillus nidulans. 22. Nucleic Acids Res 564 28:E97. 565 566 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 15, 2025. ; https://doi.org/10.1101/2025.02.14.638246doi: bioRxiv preprint 567 568 569 570 MIC (mg/L) Mutants Ancestral allele Ancestral mutation Acquired mutations AMP CEP FAZ FOX AXO CTX POD FEP CAZ CZA T/C MEM IMP PTZ F/C FDC KPC-2 - - >16 >16 >16 16 32 16 >32 4 16 0.5 16 4 2 >64 2 0.25 KPC-3 - H274Y >16 >16 >16 16 32 16 >32 8 64 1 32 4 2 >64 4 0.12 KPC-33 - D179Y <8 16 <8 8 8 4 8 <1 128 24 2 <1 <0.5 <4 <0.12 2 KPC-2n2 KPC-2 - D179Y D209V 16 <8 8 8 4 16 <1 128 32 2 <1 <0.5 32 KPC-3n4 KPC-3 H274Y L169P 16 16 128 12 2 <1 <0.5 <4 <0.12 2 KPC-33n6 KPC-33 D179Y R6H F20L <8 16 <8 8 8 8 16 128 64 2 <1 <0.5 <4 <0.12 8 plasmid KPC-2n2 in fresh E. coli Top10 KPC-2 - D179Y D209V 16 <8 8 8 4 16 <1 128 32 2 <1 <0.5 <4 0.25 2 plasmid KPC-3n4 in fresh E. coli Top10 KPC-3 H274Y L169P 16 16 128 12 2 <1 <0.5 <4 <0.12 0.5 plasmid KPC-33n6 in fresh E. coli Top10 KPC-33 D179Y R6H F20L <8 16 <8 8 8 8 16 128 64 2 <1 <0.5 <4 <0.12 2 571 Table 1 : MIC determination for selected mutants against different beta-lactams. 572 AMP, Ampicillin ; CEP, Cephalothin, FAZ, Ceftazolin ; FOX, Cefoxitin ; AXO, Ceftriaxone ; CTX, Cefotaxime ; POD, Cefpodoxime ; FEP, Cefepime ; 573 CAZ, Ceftazidime ; CZA; Ceftazidime-avibactam ; T/C, Ceftazdime/clavulanate ; MEM, Meropenem ; IMP, Imipenem ; PTZ, 574 Piperacillin/tazobactam ; F/C, Cefotaxime/clavulanate; FDC, Cefiderocol 575 576 577 578 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 15, 2025. ; https://doi.org/10.1101/2025.02.14.638246doi: bioRxiv preprint 579 Strains Cefiderocol MIC (mg/L) fold-increase in cefiderocol MIC compared to strains without plasmid fold-increase in cefiderocol MIC compared to keio WT keio WT 0.03 - - keio WT + blaKPC-2 (with D179Y-D209V) 0.5 16 16 keio WT + blaKPC-33 (D179Y) 0.5 16 16 keio ΔcirA 0.12 - 4 keio ΔcirA + blaKPC-2 (with D179Y-D209V) 2 16 66 keio ΔcirA + blaKPC-33 (D179Y) 4 32 133 keio ΔybiX 0.03 - - keio ΔybiX + blaKPC-2 (with D179Y-D209V) 1 32 32 keio ΔybiX + blaKPC-33 (D179Y) 0.5 16 16 keio ΔcirA/ΔybiX 0.5 - 16 keio ΔcirA/ΔybiX + blaKPC-2 (with D179Y- D209V) >32 64 1066 keio ΔcirA/ΔybiX + blaKPC-33 (D179Y) >32 64 1066 580 Table 2: Cefiderocol MIC in E. coli keio wild-type strains as well as in ΔcirA, ΔybiX and ΔcirA/ΔybiX strains with and without the blaKPC-2 (with D179Y-581 D209V) or blaKPC-33 (D179Y) plasmids. 582 583 584 585 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 15, 2025. ; https://doi.org/10.1101/2025.02.14.638246doi: bioRxiv preprint 586 Figure 1 : Results of the enrichment experiment of mutant libraries under increasing concentrations of cefiderocol (10 days of evolution) 587 588 589 590 591 592 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 15, 2025. ; https://doi.org/10.1101/2025.02.14.638246doi: bioRxiv preprint 593 594 Figure 2: Kiviat diagram showing the distribution of mean inhibition zone diameters (mm) for the principal antibiotic molecul es tested on 595 selected variants (blue – variants enriched from KPC-2 (D179Y-D209V), orange – variants from KPC-3 (L169P), grey variants from KPC -33 (R6H, 596 F20L +/- G291S) as well as ancestral KPC-2 variant (yellow). 597 AMX, Amoxicillin; AMX/CLAV, amoxicillin/clavulanate; PIP, piperacillin; PTZ, piperacillin/tazobactam; AZT, Aztreonam; FEP, ce fepime; FIX, 598 cefixime; CTX, cefotaxime; FOX, cefoxitin; CAZ, ceftazidime; CZA, ceftazidime-avibactam; IMP, imipenem; MER, meropenem 599 Diameters: millimeters; Blue points: EUCAST Breakpoints 600 601 0 5 10 15 20 25 30 35 40 45 AMX AMX/CLAV PIP PTZ AZT FEP FIXCTX FOX CAZ CZA IMP MER preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 15, 2025. ; https://doi.org/10.1101/2025.02.14.638246doi: bioRxiv preprint 602 603 604 605 606 E. coli strains and plasmids Genotype or description Reference Strains BW25113 E. coli Wild-type strain 1 ∆cirA BW25113∆cirA::Kanamycin cassette (kmfrt) in the Keio collection 1 ∆ybiX BW25113∆ybiX::kmfrt in the Keio collection 1 ∆cirA/∆ybiX Double mutant of ∆cirA and ∆ybiX. BW25113∆cirA, ∆ybiX::kmfrt This study Plasmids pCP20 Rep(Ts) Flp+ 2 pKOBEG oriR101ts araC arabinose-inducible λred γβα operon 3 (1) Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H. 2006. Construction of Escherichia coli K-12 in-frame, gene knockout mutants: the Keio collection. Molecular systems biology 2:2006.0008. (2) Cherepanov PP, Wackernagel W. 1995. Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant. Gene 158:9–14 (3) Chaveroche MK, Ghigo JM, d'Enfert C. 2000. A rapid method for efficient gene replacement in the filamentous fungus Aspergillus nidulans. Nucleic acids research 28:E97 607 Supplementary Table 1: Description of keio strains used and constructed for this study 608 609 610 611 612 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 15, 2025. ; https://doi.org/10.1101/2025.02.14.638246doi: bioRxiv preprint 613 614 615 616 Mean Inhibition zone diameter +/- standard deviation (mm) Ancestral variant Ancestral mutation Selected mutations AMX AMX/CLAV PIP PTZ AZT FEP FIX CTX FOX CAZ CZA IMP MER KPC-2 - - 6 14 6 7 6 22 6 6 17 13 23 18 19 KPC-2 - D179Y-D209V 22 +/-2 23 +/-1 12 +/-0.5 26 +/-1 39 +/-3 34 +/-2 12 +/-0.5 24 +/-3 25 +/-1 6 +/-0 13 +/- 1 45+/-1 37 +/-2 KPC-3 H274Y L169P 9 +/-1 22+/-2 15+/-1 23+/-1 36+/-1 31+/-1 12+/-1 16+/-1 25+/-3 6+/-0 14+/-1 36+/-2 36+/-1 KPC-33 D179Y R6H, F20L +/- G291S 20+/-2 24+/-2 6+/-0 24+/-1 35+/-1 27+/-4 8+/-2 15+/-1 27+/-2 6+/-0 11+/-0.5 41+/-3 35+/-1 617 AMX, Amoxicillin ; AMX/CLAV, amoxicillin/clavulanate; PIP, piperacillin; PTZ, piperacillin/tazobactam; AZT, Aztreonam; FEP, cefepime; FIX, 618 cefixime; CTX, cefotaxime; FOX, cefoxitin; CAZ, ceftazidime; CZA, ceftazidime-avibactam; IMP, imipenem; MER, meropenem 619 620 621 Supplementary Table 2: Results of antibiotic susceptibility tests performed by the disk diffusion method with representative beta-lactams 622 (mean inhibition zone diameter +/- standard deviation in mm performed on 10 isolates) 623 624 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 15, 2025. ; https://doi.org/10.1101/2025.02.14.638246doi: bioRxiv preprint