# Antimicrobial Activity of Cefiderocol against the Carbapenemase-Producing Enterobacter cloacae Complex and Characterization of Reduced Susceptibility Associated with Metallo-β-Lactamase VIM-1

Cristina Lasarte-Monterrubio, Paula Guijarro-Sánchez, Juan Carlos Vázquez-Ucha, Isaac Alonso-Garcia, Laura Alvarez-Fraga, Michelle Outeda, Marta Martinez-Guitian, Andrea Peña-Escolano, Romina Maceiras, Emilio Lence, et al.

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Cristina Lasarte-Monterrubio, Paula Guijarro-Sánchez, Juan Carlos Vázquez-Ucha, Isaac Alonso-Garcia, Laura Alvarez-Fraga, et al. Antimicrobial Activity of Cefiderocol against the Carbapenemase-Producing Enterobacter cloacae Complex and Characterization of Reduced Susceptibility Associated with Metallo-β-Lactamase VIM-1. Antimicrobial Agents and Chemotherapy, 2023, 67, 10.1128/aac.01505-22. hal-04986279

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# Antimicrobial Activity of Cefiderocol against the Carbapenemase-Producing *Enterobacter cloacae* Complex and Characterization of Reduced Susceptibility Associated with Metallo-β-Lactamase VIM-1

Cristina Lasarte-Monterrubio,ª Paula Guijarro-Sánchez,ª Juan Carlos Vázquez-Ucha,ª Isaac Alonso-García,ª Laura Alvarez-Fraga,ª Michelle Outeda,ª Marta Martinez-Guitian,ª,b Andrea Peña-Escolano,ª Romina Maceiras,ª Emilio Lence,ª Concepción González-Bello,ª

Jorge Arca-Suárez,ª German Bou,ª Alejandro Beceiro,ª on behalf of the GEMARA-SEIMC/REIPI Enterobacterales Study Group

Cristina Lasarte-Monterrubio and Paula Guijarro-Sánchez contributed equally to the study. Author order was determined by the corresponding author after negotiation. Alejandro Beceiro and German Bou contributed equally to the study.

**ABSTRACT** Emergence of cefiderocol resistance among carbapenemase-producing Enterobacterales, particularly those in the *Enterobacter cloacae* complex (ECC), is becoming of alarming concern; however, the mechanistic basis of this phenomenon remains poorly understood. We describe the acquisition of VIM-1-mediated reduced cefiderocol susceptibility (MICs 0.5 to 4 mg/L) in a collection of 54 carbapenemase-producing isolates belonging to the ECC. MICs were determined by reference methodologies. Antimicrobial resistance genomic analysis was performed through hybrid WGS. The impact of VIM-1 production on cefiderocol resistance in the ECC background was examined at microbiological, molecular, biochemical, and atomic levels. Antimicrobial susceptibility testing yielded 83.3% susceptible isolates and MIC₅₀/₉₀ values of 1/4 mg/L. Decreased susceptibility to cefiderocol was mainly associated with isolates producing VIM-1, with cefiderocol MICs 2- to 4-fold higher than for isolates carrying other types of carbapenemases. *E. cloacae* and *Escherichia coli* VIM-1 transformants displayed significantly enhanced cefiderocol MICs. Biochemical assays with purified VIM-1 protein revealed low but detectable cefiderocol hydrolysis. Simulation studies revealed how cefiderocol is anchored to the VIM-1 active site. Additional molecular assays and WGS data analysis highlighted the implication of SHV-12 coproduction and suggested the inactivation of the FcuA-like siderophore receptor as further contributors to the higher cefiderocol MICs. Our findings warn of the potential of the VIM-1 carbapenemase to at least partly limit the activity of cefiderocol in the ECC. This effect is probably enhanced due to combination with additional mechanisms, such as ESBL production and siderophore inactivation, and indicates the need for active surveillance to extend the life span of this promising cephalosporin.

**KEYWORDS** *Enterobacter cloacae* complex, cefiderocol, VIM-1, carbapenemase, WGS, resistance mechanisms

Enterobacter species belong to the ESKAPE group (WHO, 2017), which comprises highly virulent and antimicrobial resistant pathogens that pose an important threat to human health. Within this genus, the *Enterobacter cloacae* complex (ECC) includes the most frequently isolated species in clinical infections: *Enterobacter cloacae*, *Enterobacter*

Copyright © 2023 American Society for Microbiology. All Rights Reserved.

Address correspondence to Jorge Arca-Suárez, Jorge.Arca.Suarez@sergas.es.

The authors declare no conflict of interest.

Received 11 November 2022

Returned for modification 28 November 2022

Accepted 16 March 2023

Published 13 April 2023

<footnote>ªMicrobiology Department, A Coruña University Hospital Complex (CHUAC), A Coruña Institute for Biomedical Research (INIBIC), CIBER de Enfermedades Infecciosas (CIBERINFEC), A Coruña, Spain</footnote>

<footnote>ᵇNANOBIOFAR, Centre for Research in Molecular Medicine and Chronic Diseases (CiMUS), Universidad de Santiago de Compostela, Santiago de Compostela, Spain</footnote>

<footnote>ᶜCentre for Research in Biological Chemistry and Molecular Materials (CiQUS), Department of Organic Chemistry, Universidade de Santiago de Compostela, Santiago de Compostela, Spain</footnote>
*asburiae*, *Enterobacter hormaechei*, *Enterobacter kobei*, *Enterobacter ludwigii*, *Enterobacter mori*, *Enterobacter roggenkampii*, and *Enterobacter nimipressuralis* (1, 2).

Treatment of ECC infections is often challenging, partly due to the ability of these
species to develop β-lactam resistance through overexpression of AmpC, inactivation
of the outer membrane proteins Omp35 and Omp36, or the increasing association
with horizontally acquired ESBLs and carbapenemases, and also to the increasing prev-
alence of these species in life-threatening hospital-acquired infections (3). Reports of
carbapenemase-producing ECC in Europe have risen during the last decade, with the
metallo-β-lactamase VIM-1 and the class D carbapenemase OXA-48 being two of the
most frequently detected (4–6). In this context, studies aimed at exploring the treat-
ment potential of novel agents against CPE, including carbapenemase-producing ECC,
are essential for the medical community.

One of the most promising new options is cefiderocol (formerly S-649266), a new siderophore-cephalosporin (Fig. S1) with improved stability against ESBL, AmpC and carbapenemase-mediated hydrolysis. The compound has a catechol moiety on the R2 group that enhances bacterial internalization through iron uptake pathways (7). It has also demonstrated high *in vitro* activity against Enterobacterales (including carbapenemase producing isolates), yielding MIC<sub>50</sub> and MIC<sub>90</sub> values of 0.12 to 0.25 mg/L and 1 mg/L, respectively (7, 8).

Although there is still some controversy surrounding the specific mechanisms that
may limit the effectiveness of cefiderocol against Enterobacterales, resistance among
carbapenemase-producing strains, such as those belonging to the ECC, is increasingly
reported (9). Production of metallo-β-lactamases, such as NDM, together with inactiva-
tion/decreased expression of siderophore-cephalosporin receptors (e.g., CirA, TonB), is
suggested to cause cefiderocol resistance (10).

Studies involving detailed analysis of the mechanistic basis of cefiderocol resistance in carbapenemase-producing Enterobacterales, which increasingly involves ECC species, are scarce. We therefore evaluated a representative multicentre collection of carbapenemase-producing ECC in order to examine the activity and identify the resistance mechanisms that are potentially involved and that may affect this innovative siderophore-cephalosporin.

RESULTS AND DISCUSSION

Activity of cefiderocol against carbapenemase-producing *E. cloacae* complex.

The most prevalent carbapenemases in the ECC collection were OXA-48 and VIM-1, with up to 27 (50%) and 23 (42.6%) isolates, respectively. Two isolates coproduced two carbapenemases (OXA-48 + VIM-1 and OXA-48 + IMP-13) and two isolates (3.7%) produced IMP-type carbapenemases (IMP-8 or IMP-13). Cefiderocol displayed high activity against the ECC strains, with MIC₅₀ and MIC₉₀ values of 1 and 4 mg/L, respectively, while carbapenems yielded higher MIC₅₀ and MIC₉₀ values (2 and 8 mg/L for imipenem and 2 and 16 mg/L for meropenem). Considering the EUCAST breakpoints, 83.3% of all isolates displayed susceptibility to cefiderocol (resistance breakpoint >2 mg/L). However, major differences were observed depending on the underlying carbapenemase. While 100% of the OXA-48-producing isolates were susceptible to cefiderocol (MIC₅₀/₉₀ of 0.5/1 mg/L), only 60.9% of VIM-producing isolates were susceptible to this agent (MIC₅₀/₉₀ of 2/4 mg/L) (Fig. 1 and Table 1). Comparative MIC data are shown in Table 2.

Thus, based on the results for the cefiderocol MICs, we propose the existence of an
association between the MBL VIM-1 and decreased susceptibility to cefiderocol in the *E.
cloacae* complex, and we resolve to evaluate the underlying mechanisms in greater depth.

**Cloning and expression of bla**~VIM-1~ **in susceptible backgrounds.** For precise determination of the impact of VIM-1 production in cefiderocol resistance, the *bla*~VIM-1~ gene was cloned into plasmid pBGS18 and then transformed into *E. coli* strain MG1655 and *E. cloacae* strains ATCC 13047 and JC194 WT. Comparative MIC data of the different transformants are highlighted in Table 3. As shown, production of VIM-1 conferred 16- and 8-fold increases in cefiderocol MICs for the *E. coli* and *E. cloacae* transformants, respectively, thus providing good evidence that production of VIM-1 has a major impact on the cefiderocol MIC.
<figure><img src="image_3.png" /><figcaption>**FIG 1** Distribution of cefiderocol MICs and carbapenemases among the 54 isolates of *E. cloacae* complex included in the study.</figcaption></figure>

**Kinetic parameters of VIM-1 against cefiderocol.** In order to confirm the involvement of VIM-1 in the MICs of cefiderocol, the VIM-1 protein was purified and its steady-state parameters for cefiderocol were determined relative to meropenem (Table 4). VIM-1 exhibited a catalytic efficiency ($k_{cat}/K_m$) of 0.004 $\mu M^{-1} s^{-1}$. Although lower than that of meropenem, this was sufficient to confer a phenotype with increased cefiderocol MICs, as mentioned above. More specifically, VIM-1 showed affinity for cefiderocol in the $\mu M$ range ($K_m$ = 295.52 $\pm$ 25.43 $\mu M$), which is 3.4-fold lower than for meropenem. However, it displayed an intermediate rate of hydrolysis ($k_{cat}$ = 1.17 $\pm$ 0.17 $s^{-1}$), 13.5-fold lower than for the carbapenem. These data are consistent with those recently reported by Ito-Horiyama et al., who found that VIM-2 and IMP-1 enzymes exhibit some activity against cefiderocol, but did not find detectable levels of hydrolysis in other well-known class A or D carbapenemases such as KPC-3 and OXA-23 (7). Thus, although the specific contribution of $\beta$-lactamases to cefiderocol resistance has yet to be determined, there is growing evidence that some $\beta$-lactamases with high catalytic efficiency against cephalosporins may limit the activity of cefiderocol to some extent. Recently, a new *E. cloacae* AmpC variant with an alanine-proline deletion increased the cefiderocol MIC from 0.03 to 2 mg/L (64-fold) in an *E. coli* TOP10 background (11). The purified AmpC variant showed increased hydrolysis of ceftazidime and cefiderocol, displaying the following kinetics for cefiderocol: $K_m$ = 49.8 $\mu M$; $k_{cat}$ = 0.14 $s^{-1}$; and $k_{cat}/K_m$ = 0.0028 $\mu M^{-1} s^{-1}$. Thus, this new AmpC showed a lower hydrolysis rate and a higher affinity than VIM-1, reaching a $k_{cat}/K_m$ similar to that observed in our study for VIM-1.

**Molecular basis of the interaction between cefiderocol and the VIM-1 enzyme.** To understand the molecular basis underlying the microbiological and kinetic data for the activity of the VIM-1 enzyme against cefiderocol, as well as the differences from meropenem, the binding mode of both antibiotics in the enzyme active site was explored by molecular docking and was further validated by Molecular Dynamics (MD) simulation studies. The active site of the VIM-1 enzyme is characterized by (i) a hydroxide anion bridged by two Zn(II) ions, which are in close proximity to one another and with distinct coordination patterns (Fig. 2A); (ii) a large loop, namely, L10, which maximizes the interactions with the

<table><caption>**TABLE 1** Cumulative distribution of the MIC, $MIC_{50}$, $MIC_{90}$ and % of isolates susceptible and resistant to cefiderocol in 54 carbapenemase-producing *E. cloacae* complex strains<sup>a</sup></caption><thead><tr><th rowspan="2">Isolates and type of<br>carbapenemase</th><th colspan="6">Cumulative % of isolates at MIC in mg/L (num. of total isolates)</th><th rowspan="2">MIC<sub>50</sub></th><th rowspan="2">MIC<sub>90</sub></th><th colspan="2">EUCAST<br>category</th></tr><tr><th>≤0.12</th><th>0.25</th><th>0.5</th><th>1</th><th>2</th><th>4</th><th>% S</th><th>% R</th></tr></thead><tbody><tr><td>Isolates producing OXA-48 (n = 27)</td><td>3.7 (1)</td><td>33.3 (9)</td><td>77.8 (21)</td><td>92.6 (25)</td><td>100 (27)</td><td>100 (27)</td><td>0.5</td><td>1</td><td>100</td><td>0</td></tr><tr><td>Isolates producing VIM-1 (n = 23)</td><td>0</td><td>0</td><td>8.7 (2)</td><td>30.4 (7)</td><td>60.9 (14)</td><td>100 (23)</td><td>2</td><td>4</td><td>60.9</td><td>39.1</td></tr><tr><td>Isolates producing IMP-8/13 (n = 2)</td><td>0</td><td>0</td><td>0</td><td>50 (1)</td><td>100 (2)</td><td>100 (2)</td><td>-</td><td>-</td><td>-</td><td>-</td></tr></tbody></table>

<footnote><sup>a</sup>Two strains produced two carbapenemases: OXA-48 + VIM-1 (MIC = 2 mg/L) and OXA-48 + IMP-13 (MIC = 1 mg/L), which are not included in the table.</footnote>
<table><caption>**TABLE 2** Antimicrobial susceptibility (MICs in mg/L) to imipenem, meropenem, and cefiderocol of carbapenem-producing *E. cloacae* complex isolates, according to the carbapenemase present, description of β-lactamases produced, and possible cefiderocol resistance mechanismsª</caption><thead><tr><th>Set of isolates</th><th>Strains</th><th>ST</th><th>Species</th><th>Carbapenemase</th><th>Sample</th><th>FDC</th><th>IMI</th><th>MPM</th><th>Presence of ESBLs</th><th>Presence of other β-lactamases</th><th>Other possible resistance mechanisms</th></tr></thead><tbody><tr><td rowspan="24">Non-VIM-producing isolates</td><td>AH0328</td><td>171</td><td>E. hormaechei</td><td>OXA-48</td><td>Urine</td><td>≤0.12</td><td>2</td><td>4</td><td></td><td>ACT-like, OXA-1</td><td></td></tr><tr><td>A12624</td><td>114</td><td>E. hormaechei</td><td>OXA-48</td><td>Blood culture</td><td>0.25</td><td>0.5</td><td>1</td><td></td><td>ACT-like</td><td></td></tr><tr><td>A12695</td><td>78</td><td>E. hormaechei</td><td>OXA-48</td><td>Sputum</td><td>0.25</td><td>1</td><td>1</td><td>CTX-M-9</td><td>ACT-24</td><td></td></tr><tr><td>A12688</td><td>78</td><td>E. hormaechei</td><td>OXA-48</td><td>Wound</td><td>0.25</td><td>1</td><td>2</td><td>CTX-M-15</td><td>ACT-24, ACT-24</td><td></td></tr><tr><td>A12689</td><td>171</td><td>E. hormaechei</td><td>OXA-48</td><td>Urine</td><td>0.25</td><td>1</td><td>1</td><td></td><td>ACT-like, OXA-1</td><td></td></tr><tr><td>A12774</td><td>78</td><td>E. hormaechei</td><td>OXA-48</td><td>Pleural fluid</td><td>0.25</td><td>0.25</td><td>0.5</td><td></td><td>ACT-24</td><td></td></tr><tr><td>A12915</td><td>114</td><td>E. hormaechei</td><td>OXA-48</td><td>Urine</td><td>0.25</td><td>8</td><td>16</td><td></td><td>ACT-16, OXA-1</td><td></td></tr><tr><td>A12999</td><td>1382</td><td>E. cloacae</td><td>OXA-48</td><td>Blood culture</td><td>0.25</td><td>0.25</td><td>0.25</td><td></td><td>CMH class C</td><td></td></tr><tr><td>AN2364</td><td>168</td><td>E. hormaechei</td><td>OXA-48</td><td>Urine</td><td>0.25</td><td>0.25</td><td>1</td><td></td><td>ACT-23, DHA-1</td><td></td></tr><tr><td>A12672</td><td>1379</td><td>E. hormaechei</td><td>OXA-48</td><td>Sputum</td><td>0.5</td><td>1</td><td>1</td><td>CTX-M-15</td><td>ACT-like, OXA-1, TEM-1</td><td></td></tr><tr><td>A12673</td><td>1379</td><td>E. hormaechei</td><td>OXA-48</td><td>Sputum</td><td>0.5</td><td>1</td><td>2</td><td>CTX-M-15</td><td>ACT-like, OXA-1, TEM-1</td><td></td></tr><tr><td>A12674</td><td>23</td><td>E. asburiae</td><td>OXA-48</td><td>Blood culture</td><td>0.5</td><td>2</td><td>2</td><td></td><td>ACT-like</td><td></td></tr><tr><td>A12679</td><td>23</td><td>E. asburiae</td><td>OXA-48</td><td>Blood culture</td><td>0.5</td><td>2</td><td>1</td><td></td><td>ACT-like</td><td></td></tr><tr><td>A12683</td><td>66</td><td>E. hormaechei</td><td>OXA-48</td><td>Sputum</td><td>0.5</td><td>1</td><td>2</td><td>CTX-M-15</td><td>ACT-like, OXA-1, TEM-1</td><td></td></tr><tr><td>A12694</td><td>23</td><td>E. asburiae</td><td>OXA-48</td><td>Wound</td><td>0.5</td><td>0.5</td><td>0.5</td><td></td><td>ACT-like</td><td></td></tr><tr><td>A12696</td><td>1379</td><td>E. hormaechei</td><td>OXA-48</td><td>Wound</td><td>0.5</td><td>1</td><td>1</td><td>CTX-M-15</td><td>ACT-like, OXA-1, TEM-1</td><td></td></tr><tr><td>A12796</td><td>1380</td><td>E. hormaechei</td><td>OXA-48</td><td>Urine</td><td>0.5</td><td>0.25</td><td>0.5</td><td></td><td>ACT-16</td><td></td></tr><tr><td>A12866</td><td>90</td><td>E. hormaechei</td><td>OXA-48</td><td>Blood culture</td><td>0.5</td><td>2</td><td>4</td><td></td><td>ACT-like</td><td></td></tr><tr><td>A12904</td><td>662</td><td>E. hormaechei</td><td>OXA-48</td><td>Urine</td><td>0.5</td><td>2</td><td>0.25</td><td>CTX-M-15</td><td>ACT-17, TEM-1</td><td></td></tr><tr><td>A12916</td><td>732</td><td>E. roggenkampii</td><td>OXA-48</td><td>Wound</td><td>0.5</td><td>1</td><td>2</td><td>CTX-M-15</td><td>MIR-like, OXA-1, TEM-1</td><td></td></tr><tr><td>A12924</td><td>114</td><td>E. hormaechei</td><td>OXA-48</td><td>Urine</td><td>0.5</td><td>2</td><td>2</td><td>CTX-M-15</td><td>ACT-16, OXA-1, TEM-1</td><td></td></tr><tr><td>A12950</td><td>1379</td><td>E. hormaechei</td><td>OXA-48</td><td>Wound</td><td>0.5</td><td>0.5</td><td>2</td><td>CTX-M-15</td><td>ACT-like, OXA-1, TEM-1</td><td></td></tr><tr><td>A12662</td><td>171</td><td>E. hormaechei</td><td>OXA-48</td><td>Urine</td><td>1</td><td>≥256</td><td>128</td><td>CTX-M-15(×2), CTX-M-9</td><td>ACT-25, OXA-1(x2)</td><td></td></tr><tr><td>A12697</td><td>171</td><td>E. hormaechei</td><td>OXA-48</td><td>Wound</td><td>1</td><td>1</td><td>1</td><td>CTX-M-15</td><td>ACT-like, OXA-1</td><td></td></tr><tr><td>A12905</td><td>732</td><td>E. roggenkampii</td><td>OXA-48</td><td>Abscess</td><td>1</td><td>1</td><td>2</td><td>CTX-M-15</td><td>MIR-like, OXA-1, TEM-1</td><td></td></tr><tr><td>A12760</td><td>182</td><td>E. hormaechei</td><td>OXA-48 + IMP-13</td><td>Urine</td><td>1</td><td>1</td><td>1</td><td>CTX-M-15</td><td>ACT-16, OXA-10, OXA-2</td><td></td></tr><tr><td>A12964</td><td>171</td><td>E. hormaechei</td><td>OXA-48</td><td>Blood culture</td><td>2</td><td>1</td><td>1</td><td>CTX-M-15(×2)</td><td>ACT-like, OXA-1(x2)</td><td></td></tr><tr><td>A13038</td><td>171</td><td>E. hormaechei</td><td>OXA-48</td><td>Wound</td><td>2</td><td>1</td><td>1</td><td>CTX-M-15(×2)</td><td>ACT-like, OXA-1(x2)</td><td></td></tr><tr><td>A12992</td><td>96</td><td>E. roggenkampii</td><td>IMP-8</td><td>Feces</td><td>2</td><td>0.25</td><td>0.25</td><td></td><td>MIR-like, OXA-101</td><td></td></tr><tr><td>A12747</td><td>182</td><td>E. hormaechei</td><td>IMP-13</td><td>Urine</td><td>1</td><td>0.25</td><td>0.25</td><td>CTX-M-15</td><td>ACT-16, OXA-1, TEM-1(x2)</td><td></td></tr><tr><td rowspan="24">VIM-producing isolates</td><td>A12613</td><td>93</td><td>E. hormaechei</td><td>VIM-1</td><td>Abscess</td><td>0.5</td><td>4</td><td>4</td><td></td><td>ACT-17, ACC-1(x2), OXA-1</td><td></td></tr><tr><td>AN2360</td><td>93</td><td>E. hormaechei</td><td>VIM-1</td><td>Abscess</td><td>0.5</td><td>2</td><td>0.5</td><td></td><td>ACT-17, ACC-1(x2), OXA-1</td><td></td></tr><tr><td>A12615</td><td>78</td><td>E. hormaechei</td><td>VIM-1</td><td>Peritoneal fluid</td><td>1</td><td>0.5</td><td>1</td><td>CTX-M-9</td><td>ACT-24</td><td></td></tr><tr><td>A12656</td><td>133</td><td>E. hormaechei</td><td>VIM-1</td><td>BAS</td><td>1</td><td>8</td><td>16</td><td>CTX-M-9</td><td>ACT-17</td><td></td></tr><tr><td>A12657</td><td>133</td><td>E. hormaechei</td><td>VIM-1</td><td>Urine</td><td>1</td><td>16</td><td>64</td><td>CTX-M-9</td><td>ACT-17</td><td></td></tr><tr><td>A12772</td><td>110</td><td>E. hormaechei</td><td>VIM-1</td><td>Urine</td><td>1</td><td>2</td><td>0.5</td><td>CTX-M-15</td><td>ACT-24, DHA-1</td><td></td></tr><tr><td>A12972</td><td>114</td><td>E. hormaechei</td><td>VIM-1</td><td>Wound</td><td>1</td><td>8</td><td>1</td><td>CTX-M-15</td><td>ACT-16, OXA-1, TEM-1</td><td></td></tr><tr><td>A12619</td><td>78</td><td>E. hormaechei</td><td>VIM-1</td><td>Blood culture</td><td>2</td><td>1</td><td>1</td><td></td><td>ACT-24, ACC-1</td><td></td></tr><tr><td>A12620</td><td>78</td><td>E. hormaechei</td><td>VIM-1</td><td>Wound</td><td>2</td><td>0.25</td><td>0.25</td><td>CTX-M-9</td><td>ACT-24, DHA-1</td><td></td></tr><tr><td>A12658</td><td>133</td><td>E. hormaechei</td><td>VIM-1</td><td>Blood culture</td><td>2</td><td>8</td><td>8</td><td>CTX-M-9</td><td>ACT-17</td><td></td></tr><tr><td>A12659</td><td>78</td><td>E. hormaechei</td><td>VIM-1</td><td>Sputum</td><td>2</td><td>8</td><td>8</td><td>CTX-M-9</td><td>ACT-24, DHA-1</td><td></td></tr><tr><td>A12859</td><td>133</td><td>E. hormaechei</td><td>VIM-1</td><td>BAS</td><td>2</td><td>4</td><td>2</td><td></td><td>ACT-17, TEM-1</td><td></td></tr><tr><td>A12936</td><td>78</td><td>E. hormaechei</td><td>VIM-1</td><td>BAS</td><td>2</td><td>8</td><td>8</td><td>CTX-M-9</td><td>ACT-17, TEM-1</td><td></td></tr></tbody></table>

(Continued on next page)
<table><caption>TABLE 2 (Continued)</caption><thead><tr><th>Set of isolates</th><th>Strains</th><th>ST</th><th>Species</th><th>Carbapenemase</th><th>Sample</th><th>FDC</th><th>IMI</th><th>MPM</th><th>Presence of ESBLs</th><th>Presence of other β-lactamases</th><th>Other possible resistance mechanisms</th></tr></thead><tbody><tr><td>A12939</td><td>133</td><td>E. hormaechei</td><td>VIM-1</td><td>Catheter</td><td>2</td><td>8</td><td>8</td><td>CTX-M-9</td><td>CTX-M-9</td><td>ACT-17</td><td></td></tr><tr><td>A12799</td><td>1015</td><td>E. hormaechei</td><td>VIM-1 + OXA-48</td><td>Urine</td><td>2</td><td>8</td><td>8</td><td>CTX-M-9</td><td>CTX-M-9</td><td>ACT-like, OXA-1</td><td></td></tr><tr><td>A12759</td><td>114</td><td>E. hormaechei</td><td>VIM-1</td><td>Urine</td><td>4</td><td>4</td><td>4</td><td>CTX-M-15</td><td>CTX-M-15</td><td>ACT-16, OXA-1, TEM-1</td><td></td></tr><tr><td>A12797</td><td>24</td><td>E. asburiae</td><td>VIM-1</td><td>Wound</td><td>4</td><td>2</td><td>1</td><td>CTX-M-9</td><td>CTX-M-9</td><td>ACT-1, OXA-1</td><td></td></tr><tr><td>A12798</td><td>78</td><td>E. hormaechei</td><td>VIM-1</td><td>Sputum</td><td>4</td><td>2</td><td>2</td><td>CTX-M-9, SHV-12</td><td>CTX-M-9, SHV-12</td><td>ACT-24, TEM-1</td><td>FcuA-like NF (Gln70fs)<sup>b</sup></td></tr><tr><td>A12802</td><td>24</td><td>E. asburiae</td><td>VIM-1</td><td>Urine</td><td>4</td><td>2</td><td>2</td><td>CTX-M-9</td><td>CTX-M-9</td><td>ACT-1, OXA-1</td><td></td></tr><tr><td>A12804</td><td>78</td><td>E. hormaechei</td><td>VIM-1</td><td>Wound</td><td>4</td><td>2</td><td>4</td><td>CTX-M-9, SHV-12</td><td>CTX-M-9, SHV-12</td><td>ACT-24, TEM-1(x2)</td><td>FcuA-like NF (Gln70fs)<sup>b</sup></td></tr><tr><td>A12839</td><td>413</td><td>E. sichuanensis</td><td>VIM-1</td><td>Peritoneal fluid</td><td>4</td><td>1</td><td>0.5</td><td></td><td></td><td>ACT-like, OXA-1</td><td></td></tr><tr><td>A12865</td><td>108</td><td>E. hormaechei</td><td>VIM-1</td><td>BAS</td><td>4</td><td>2</td><td>1</td><td></td><td></td><td>ACT-like</td><td></td></tr><tr><td>A12941</td><td>1074</td><td>E. roggenkampii</td><td>VIM-1</td><td>Blood culture</td><td>4</td><td>16</td><td>16</td><td></td><td></td><td>MIR-like, OXA-1</td><td></td></tr><tr><td>A13013</td><td>764</td><td>E. hormaechei</td><td>VIM-1</td><td>Urine</td><td>4</td><td>1</td><td>0.5</td><td>CTX-M-9</td><td>CTX-M-9</td><td>ACT-like, OXA-1</td><td></td></tr></tbody></table>

<footnote>aFDC, cefiderocol; IMI, imipenem; MPM, meropenem.</footnote>

<footnote>bNF, nonfunctional; fs, frameshift;*, premature stop codon.</footnote>
<table><caption>**TABLE 3** MICs (mg/L) for the *E. coli* MG1655 and *E. cloacae* JC194 WT and ATCC 13047 strains expressing the *bla*<sub>VIM-1</sub> gene<sup>a</sup></caption><thead><tr><th rowspan="2">Strains and plasmids</th><th colspan="6">MIC (mg/L)</th></tr><tr><th>FDC</th><th>PIP</th><th>CAZ</th><th>FEP</th><th>IMI</th><th>MPM</th></tr></thead><tbody><tr><td><i>E. coli</i> MG1655</td><td>0.03</td><td>1</td><td>≤0.5</td><td>≤0.5</td><td>0.125</td><td>≤0.03</td></tr><tr><td><i>E. coli</i> MG1655 (pBGS18/VIM-1)</td><td>0.5</td><td>128</td><td>128</td><td>8</td><td>1</td><td>0.125</td></tr><tr><td><i>E. cloacae</i> JC194 WT</td><td>0.5</td><td>4</td><td>1</td><td>≤0.5</td><td>0.25</td><td>0.064</td></tr><tr><td><i>E. cloacae</i> JC194 WT (pBGS18/VIM-1)</td><td>4</td><td>256</td><td>256</td><td>16</td><td>1</td><td>1</td></tr><tr><td><i>E. cloacae</i> ATCC 13047</td><td>0.5</td><td>4</td><td>1</td><td>≤0.5</td><td>0.25</td><td>0.064</td></tr><tr><td><i>E. cloacae</i> ATCC 13047 (pBGS18/VIM-1)</td><td>4</td><td>128</td><td>256</td><td>16</td><td>1</td><td>0.5</td></tr></tbody></table>

substrate for hydrolysis, particularly by modulating the position of residue Asn210, which has an important role in recognition; and (iii) an apolar pocket created by one of the faces of the β2 and β3 sheets, involving residues Trp87 and Tyr67, and the L3 loop. The plasticity of the L3 and L10 loops seems to be crucial for substrate recognition.

We found that cefiderocol would be anchored to the VIM-1 active site by a set of hydrogen bonding interactions involving three distinct moieties of the antibiotic, specifically the cephalosporine core and the R1 and R2 groups (Fig. 2B). Thus, the carboxylate group of the cephalosporine scaffold would establish a hydrogen bonding interaction with the main NH group of Asn210. The latter contact would locate the β-lactam moiety of cefiderocol on top of the hydroxide anion and in a suitable position for hydrolysis, with an average distance throughout the simulation of 3.4 Å. For the R1 group, an interaction between its amide carbonyl group with the aromatic NH group of Tyr67 was identified. The siderophore group, which is located on the terminal part of the R2 group, would be neatly trapped in the pocket adjacent to the catalytic site by diverse hydrogen bonding interactions with residues Val200, Glu202 and His240. The cefiderocol@VIM-1 enzyme complex proved to be very stable during the whole simulation (Fig. S2).

Our studies showed that meropenem would be more effectively fixed in the catalytic site for hydrolysis than cefiderocol, because the latter would promote a hydrogen bonding with the side chain of Asn210, which was not observed for cefiderocol (Fig. 2C and D). As a result, the β-lactam moiety would be located closer to the hydroxide anion (Fig. 2E). This extra interaction would also enhance the β-lactam moiety for hydrolysis by acting as a Lewis acid and would explain the 3.4-fold decrease in $K_m$, relative to cefiderocol, as well as the experimentally observed 13.5-fold increase in $k_{cat}$.

**Mechanisms involved in reduced susceptibility to cefiderocol.** Currently available data suggest that acquisition of cefiderocol resistance requires the accumulation of multiple mechanisms (12). Recent studies propose that some β-lactamases, such as NDM, PER and some KPC variants conferring resistance to ceftazidime-avibactam, can contribute to cefiderocol nonsusceptibility. However, very few of them have precisely determined the impact of the production of the β-lactamase in cefiderocol resistance (13, 14). In recent studies involving a collection of laboratory strains expressing the most widespread β-lactamases in Enterobacterales, Acinetobacter baumannii, and Pseudomonas aeruginosa, transformants harboring NDM-like carbapenemases or some ESBLs (such as VEB, PER-1 and SHV-12) showed decreased susceptibility to cefiderocol (2 to 8 mg/L), although the VIM-1 enzyme was not evaluated (15–17). Nonetheless, other underlying mechanisms must also be involved in the resistance to this antibiotic, as these β-lactamases alone will not yield high MIC values.

To assess the involvement of the main ESBLs found in this collection, SHV-12, CTX-M-9 and CTX-M-15 were cloned into the ATCC 13047 reference strain in the presence

<table><caption>**TABLE 4** Kinetic parameters of VIM-1 toward the indicated antibiotics</caption><thead><tr><th>β-Lactam</th><th>k<sub>cat</sub> (s<sup>-1</sup>)</th><th>K<sub>m</sub> (μM)</th><th>k<sub>cat</sub>/K<sub>m</sub> (μM<sup>-1</sup> s<sup>-1</sup>)</th></tr></thead><tbody><tr><td>Cefiderocol</td><td>1.17 ± 0.17</td><td>295.52 ± 25.43</td><td>0.004</td></tr><tr><td>Meropenem</td><td>15.81 ± 1.65</td><td>87.63 ± 9.37</td><td>0.18</td></tr></tbody></table>

<footnote><sup>a</sup>FDC, cefiderocol; PIP, piperacillin; CAZ, ceftazidime; FEP, cefepime; IMI, imipenem; MPM, meropenem.</footnote>
<figure><img src="image_4.png" /><figcaption>**FIG 2** (A) Three-dimensional structure of VIM-1 in the wild-type form, highlighting the relevant sites. An overall view of the motion of the L3 and L10 loops involved in substrate recognition obtained by examination of the vibrational modes is shown. Close view of the dinuclear center that has a hydroxide anion bridging the Zn1 (tetrahedral) and Zn2 (bipyramidal) sites. (B) Binding mode of cefiderocol in the active site of VIM-1 enzyme obtained by MD simulation studies. Overall and detailed views are provided. Relevant hydrogen bonding interactions and the distance between the oxygen atom of the hydroxide group and the carbonyl group in the antibiotic are shown as dashed blue lines. Relevant residues are shown and labeled. (C) Main contacts of meropenem in the active site of VIM-1 enzyme. Note how a dual interaction between Asn210 and the penem core of this compound (carbonyl and carboxylate groups) fixes the antibiotic in a suitable position for hydrolysis. This interaction is less effective for cefiderocol (carboxylate group only). (D) Variation in the relative distance between the oxygen atom of the β-lactam carbonyl group in meropenem and side chain amide group of Asn210 (red line) and the carboxylate group in meropenem and the main NH group of Asn210 (green line) in the meropenem@VIM-1 Michaelis complex during 100 ns of simulation. (E) Variation in the relative distance between the carbon atom of the β-lactam carbonyl group and the oxygen atom of the hydroxide group in the corresponding antibiotic/VIM-1 Michaelis complexes during the whole simulation. Snapshots taken after 90 ns of simulation are given.</figcaption></figure>

and absence of VIM-1, and were further cloned into an *E. cloacae* complex clinical isolate carrying VIM-1. As can be observed in Table 5, SHV-12 increased the cefiderocol MICs, while both CTX-M enzymes showed no effect, which confirms the recently published results (15). SHV-12 and VIM-1 also showed increased cefiderocol MICs when coexpressed in both ATCC 13047 reference strain and AI2615 clinical isolate.

On the other hand, *ampD*, *ampG*, and *ampR* genes were also analyzed in the ECC isolates, and no mutations were identified, thus suggesting to exclude the involvement of changes in AmpC expression in the reduced susceptibility to cefiderocol. In order to confirm this observation, *bla*<sub>ampC</sub> expression of all VIM-1-producing isolates was evaluated. As reflected in Table S1 all but one of the VIM-1-producing isolates showed *bla*<sub>ampC</sub> basal
<table><caption>**TABLE 5** Cefiderocol MICs (mg/L) for the transformants of ATCC 13047 *E. cloacae* reference strain and AI2615 *E. hormaechei* clinical isolate expressing the *bla*<sub>VIM-1</sub>, *bla*<sub>SHV-12</sub>, *bla*<sub>CTX-M-9</sub> and *bla*<sub>CTX-M-15</sub> genes</caption><thead><tr><th><i>E. cloacae</i> complex strains and plasmids</th><th>FDC<sup>a</sup> (mg/L)</th></tr></thead><tbody><tr><td>ATCC 13047</td><td>0.5</td></tr><tr><td>ATCC 13047 (pBGS18/VIM-1)</td><td>4</td></tr><tr><td>ATCC 13047 (pUCP24/SHV-12)</td><td>8</td></tr><tr><td>ATCC 13047 (pUCP24/CTX-M-15)</td><td>0.5</td></tr><tr><td>ATCC 13047 (pUCP24/CTX-M-9)</td><td>0.25</td></tr><tr><td>ATCC 13047 (pUCP24/SHV-12 + pBGS18-VIM-1)</td><td>16</td></tr><tr><td>ATCC 13047 (pUCP24/CTX-M-15 + pBGS18/VIM-1)</td><td>4</td></tr><tr><td>ATCC 13047 (pUCP24/CTX-M-9 + pBGS18/VIM-1)</td><td>4</td></tr><tr><td>AI2615</td><td>1</td></tr><tr><td>AI2615 (pUCP24/SHV-12)</td><td>16</td></tr><tr><td>AI2615 (pUCP24/CTX-M-15)</td><td>1</td></tr><tr><td>AI2615 (pUCP24/CTX-M-9)</td><td>1</td></tr></tbody></table>

expression, showing minor expression differences, which were not related with the cefiderocol MICs obtained (AI2657 isolate exhibited the highest *bla*<sub>ampC</sub> expression; however, displayed a low cefiderocol MIC, 1 mg/L). In addition, *bla*<sub>ampC</sub> expression analysis among three OXA-48-producing isolates showed that AI2624 displayed overexpression of this gene while presenting very low cefiderocol MICs, the same as for the other two strains (AH0328 and AI2695), with considerably lower *bla*<sub>ampC</sub> expression. Taken together, these analyses demonstrate that the chromosomal β-lactamase AmpC is not implicated in the decreased susceptibility to cefiderocol of these *E. cloacae* isolates.

Likewise, decreased cefiderocol internalization due to inactivation of siderophore receptors such as *cirA* or *fiu* is increasingly recognized as a major cause of cefiderocol resistance (18–20). Thus, we explored *in silico* the presence of mutational changes in genes previously related to iron uptake in the genomes of the whole ECC isolates included in the study. Interestingly, we found inactivating changes in siderophore receptors in three isolates that yielded cefiderocol MICs of 4 mg/L and exhibited frame-shifts (Gln70fs) or a premature stop codon (Trp441*) in FcuA-like, a TonB-dependent ferrichrome receptor (21). The specific role of this receptor in cefiderocol resistance should be precisely determined in future studies, although it is tempting to suggest, on the basis of our findings, that its inactivation may contribute to cefiderocol resistance in the *E. cloacae* background (Table 2 and S2).

**Determination of bla<sub>VIM-1</sub> expression.** High levels of β-lactamase expression are a well-known cause of β-lactam resistance, even to new antimicrobial combinations such as ceftazidime-avibactam and meropenem-vaborbactam (22, 23). We analyzed the potential *bla*<sub>VIM-1</sub> expression in cefiderocol resistance, and although we observed a possible trend, no major differences were observed in *bla*<sub>VIM-1</sub> expression among the ECC isolates (Table S2). Thus, in the strains yielding a cefiderocol MIC of 1 mg/L, *bla*<sub>VIM-1</sub> expression was equal to or lower than in controls (AI2613 and AN2360 isolates, MIC = 0.5 mg/L). Among the eight strains with a cefiderocol MIC of 2 mg/L, most displayed equal or higher expression than controls, while two isolates showed slightly lower expression (one of them produced a nonfunctional Omp35 porin). Finally, among the nine strains yielding a cefiderocol MIC of 4 mg/L (resistant strains following EUCAST criteria), all but one (which presented the nonfunctional FcuA-like siderophore receptor) showed equal or slightly higher expression of *bla*<sub>VIM-1</sub> than controls.

**Epidemiology and distribution of β-lactamases in the *E. cloacae* complex.** The genetic context of all VIM carbapenemases was evaluated in order to gain an in-depth perspective of the genetic drivers that may contribute to the spread of cefiderocol resistance among ECC. The methods used for molecular typing and phylogenetic analysis are included in the supplemental material. Among the 54 carbapenemase-producing ECC analyzed, VIM-1 was present in 24 ECC (44.4%) isolates and in all cases it was located in plasmids (Fig. 3). Most of the strains with VIM-1 yielded MICs ≥ 1 mg/L and in most cases belonged to *E. hormaechei* (79.6% of the whole ECC collection). Fig. 3 also

<footnote><sup>a</sup>FDC, cefiderocol.</footnote>
Tree scale: 0.01

<figure><img src="image_5.png" /><figcaption>**FIG 3** Phylogenetic tree of clinical *E. cloacae* complex (ECC) isolates showing relationships between VIM-1 presence, MIC ≥ 1 mg/L FDC and clonal relatedness. The first column represents the species of each ECC isolate, the second column represents the Sequence Type (ST) and the carbapenemase is indicated in the third column. Isolates yielding a cefiderocol MIC ≥1 mg/L are indicated in dark purple in the fourth column. The incompatibility group is included in the final column. Clonal groups of isolates carrying VIM-1 with a clonal relatedness of ≤ 24 SNPs are marked in gray.</figcaption></figure>

shows the core genome phylogenetic relatedness among the isolates. Phylogenetic analysis revealed 4 clusters and 10 distantly related isolates in *E. hormaechei*. An additional cluster was found in *E. asburiae* strains. MLST analysis of the ECC isolates revealed up to 23 different sequence types, with ST78 (18.5%), ST171 (11.1%) and ST114 and ST133 (9.3% each) being the most prevalent. These findings highlight that VIM-1 enzymes are scattered among different clones.

The VIM-1 enzymes were located in all cases in plasmids from different incompatibility groups. Of note, the IncHI2/IncHI2A incompatibility group plasmids were the most common (n = 11), followed by IncL (n = 4), IncFIB(pECLA)/IncFII(pECLA)/IncHI2/IncHI2A (n = 2), IncHI2/IncHI2A/IncR (n = 2), IncU (n = 2), IncM2 (n = 1), IncN2 (n = 1), and IncX3 (n = 1). Finally, a detailed analysis of the region surrounding the *bla*<sub>VIM-1</sub> in plasmids
revealed that in all cases they were embedded within class-1 integrons, together with aminoglycoside modifying enzymes, as part of large transposition modules (Fig. S3).

The diversity of conjugative plasmids with transposable platforms carrying *bla*<sub>VIM</sub> is involved in increasing the propagation and persistence of the VIM carbapenemase among the different ECC species. Different plasmids have also been implicated in the dispersion of *bla*<sub>VIM</sub> genes in other European countries (24) Altogether, these findings highlight that VIM-1 emergence and expansion in the *E. cloacae* complex occurs through both plasmid-borne and polyclonal spread.

**Conclusion** The factors limiting cefiderocol activity in Gram-negative pathogens are still largely unexplored and probably partake in a complex resistome that involves β-lactamase production and inactivation of siderophores, together with other mechanisms such as porin loss and PBP modifications. In this respect, most published studies focus exclusively on the susceptibility profiling of large clinical and laboratory bacterial collections, the mechanisms of siderophore inactivation, and the modification of β-lactamases after prolonged treatment with this new agent.

Here, we provide a more detailed perspective, weighing the impact that the widespread VIM-1 carbapenemase may have on the acquisition of borderline resistance to cefiderocol in the ECC background. Detailed investigation involving WGS, molecular, biochemical, and structural analyses of a multicentre Spanish collection of clinical isolates revealed that VIM-1 confers a low but detectable hydrolysis of cefiderocol that is sufficient to increase the MICs of this innovative agent to borderline levels. More specifically, our structural analysis revealed that VIM-1 and cefiderocol form a stable complex that undergoes hydrolysis thanks to the key interaction between the Asn210 residue of the metalloenzyme and the carboxylate group of the cephalosporine core. Our work also provides an insight into other mechanisms that probably further contribute to decreasing cefiderocol susceptibility and that should be considered in future evaluations of cefiderocol resistance in ECC. These include inactivation of the FcuA-like siderophore receptor, levels of carbapenemase expression and concomitant production of ESBLs (e.g., SHV-12). Finally, this study also warns of the expansion of the VIM-1 carbapenemase among ECC in Spain, a phenomenon that should hence be cautiously monitored as it is driven by an interplay between clonal expansion and plasmid propagation. Altogether, our findings call for active surveillance to be maintained on emerging cefiderocol resistance in VIM-1-producing ECC.

## MATERIALS AND METHODS

**Bacterial strains and susceptibility testing.** The bacterial isolates were recovered during a multi-centre Spanish survey conducted to evaluate the epidemiology and resistance mechanisms in carbapenemase-producing Enterobacterales (CPEs). A representative collection of 443 nonduplicate clinical isolates (6) from 24 participating hospitals was included. Among those, 54 clinical isolates of *E. cloacae* complex were identified by MALDI-TOF. Imipenem, meropenem, and cefiderocol MICs were determined by reference broth microdilution. Iron-depleted cation-adjusted Mueller-Hinton broth was used for cefiderocol microdilution assays. EUCAST v12.0 clinical breakpoints and guidelines were used for interpretation.

**WGS.** All ECC isolates were sequenced in parallel using short- (Illumina MiSeq, Illumina) and long-read (MinION, Oxford Nanopore Technologies) approaches involving previously described methodologies (25). The resulting short and long reads from each isolate were assembled using the Unicycer v0.4.8 hybrid assembler (26). Genomes were annotated using Prokka v1.14.6 (27).

**Genomic analysis of antimicrobial resistance.** For analysis of possible cefiderocol resistance mechanisms, a search for amino acid substitutions or major inactivating changes was made via variant calling, which was performed using Snippy v4.6.0 (https://github.com/tseeman/snippy). Horizontally acquired resistance mechanisms were also explored using the Comprehensive Antibiotic Resistance Database (CARD) webtool (https://card.mcmaster.ca/home) and the ResFinder server (https://cge.food.dtu.dk/services/ResFinder/). The studied genes involved in resistance and the details of reference genomes are in the supplemental material.

**Cloning of *bla*<sub>VIM-1</sub> in susceptible backgrounds.** The *bla*<sub>VIM-1</sub> gene was amplified in parallel by PCR, with primers 5'-GGATCCATGTTAAAAGTTATTAGTAGTTTATTG-3' and 5'-GAATT CCTACTCGGCGACTGAGC-3'. The PCR product was ligated to plasmid pBGS18 using EcoRI and BamHI restriction sites (28) to obtain plasmid pBGS18-VIM-1, which was electroporated in parallel into *E. coli* strain MG1655 and into two carbapenem-susceptible *E. cloacae* strains, ATCC 13047 and JC194 WT (a reference strain and a clinical isolate with basal efflux pump expression, respectively) (29). Transformants were selected in 50 mg/L kanamycin LB agar plates. The piperacillin, ceftazidime, cefepime, cefiderocol, imipenem, and meropenem MICs for transformants were determined using the above-described methodology.
**Steady-state kinetics.** Purified VIM-1 carbapenemase was used in biochemical studies with cefiderocol and meropenem as the substrates. Purification of VIM-1 enzyme, hydrolysis measurements, and determination of K_m and k_cat parameters are described in detail in the supplemental material.

**Access to data.** The raw data (Illumina and Oxford Nanopore Technologies sequence reads) and annotated assemblies corresponding to the CPE genomes have been submitted to the European Nucleotide Archive under BioProject accessions PRJEB42440 and PRJEB39112.

## SUPPLEMENTAL MATERIAL

Supplemental material is available online only.

**SUPPLEMENTAL FILE 1**, PDF file, 0.6 MB.

## ACKNOWLEDGMENTS

This research was made possible by the helpful collaboration of the following researchers from the GEMARA-SEIMC/REIPI Enterobacterales Study Group: Irene Merino, Emilia Cercenado, Rosa Gómez, Tamara Soler, Irene Gracia-Ahufinger, Lina Martín, Fátima Galán, Nuria Tormo, Juan Carlos Rodríguez, Silvia Capilla, Francesc Marco, María Dolores Quesada, Emma Padilla, Fe Tubau, Juanjo González, Ana Isabel López-Calleja, José Luis del Pozo, María Inmaculada García, Mariela Martinez, Jorge Calvo, Xavier Mulet, Fernanda Peña, Ana Isabel Rodríguez, María José Gude, Ana Fernández, Javier Fernández, Felipe Fernandez-Cuenca, and Alvaro Pascual. We thank the Centro de Supercomputación de Galicia (CESGA) for use of the Finis Terrae III computer.

This research was supported by Projects PI17/01482 and PI20/01212 awarded to AB, PI22/01212 to JAS and PI18/00501 to GB, within in the National Plan for Scientific Research, Development and Technological Innovation 2017 to 2020 and funded by the ISCIII-General Subdirection of Assessment and Promotion of the Research-European Regional Development Fund (FEDER) "A way of making Europe." The work was also supported by CIBERINFEC (CIBER de Enfermedades Infecciosas, Instituto de Salud Carlos III). The study was also funded by project IN607A 2020/05 (GAIN-Agencia Gallega de Innovación-Consellería de Economía, Emprego e Industria) awarded to GB and project IN607D 2021/12 awarded to AB. It was also supported by Planes Nacionales de I+D+i2017 to 2020 and Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía y Competitividad, Spanish Network for Research in Infectious Diseases (REIPI RD16/0016/006) cofinanced by European Development Regional Fund "A way to achieve Europe" and operative program Intelligent Growth2014 to 2020. J.A.-S. was financially supported by the Juan Rodés program (ISCIII, JR21/00026), J.C.V.-U. was financially supported by IN606B-2022/009 Grant (Xunta de Galicia), C.L.-M. was financially supported by IN606A-2019/029 Grant (Xunta de Galicia) and PGS was financially supported by IN606A 2021/021 Grant (Xunta de Galicia). M.O. was financially supported by IN607D 2021/12 (Xunta de Galicia). I.A.-G. was financially supported by the Rio Hortega program (ISCIII, CM21/00076). C.G.-B. acknowledges financial support from the Spanish Ministry of Science and Innovation (PID2019-105512RB-I00), the Xunta de Galicia (ED431C 2021/29 and the Centro singular de investigación de Galicia accreditation2019 to 2022 [ED431G 2019/03]), and the European Regional Development Fund (ERDF).

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