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. 2024 Oct 9;68(11):e00924-24. doi: 10.1128/aac.00924-24

Assessment of the activity and mechanisms of resistance to cefiderocol and combinations of β-lactams and the novel β-lactamase inhibitors avibactam, taniborbactam, zidebactam, nacubactam, xeruborbactam, and ANT3310 in emerging double-carbapenemase-producing Enterobacterales

Tania Blanco-Martín 1,#, Inmaculada López-Hernández 2,3,#, Belén Aracil 3,4,#, Lucía González-Pinto 1, Pablo Aja-Macaya 1, Isaac Alonso-García 1, Salud Rodríguez-Pallares 1, Lucía Sánchez-Peña 1, Michelle Outeda-García 1, María Pérez-Vázquez 3,4, Juan Carlos Vázquez-Ucha 1,3, Alejandro Beceiro 1,3, Álvaro Pascual 2,3, Germán Bou 1,3,✉, Lorena López-Cerero 2,3,#, Jesús Oteo-Iglesias 3,4,#, Jorge Arca-Suárez 1,3,#; on behalf of the GEMARA-SEIMC/CIBERINFEC Study Group on the activity and resistance mechanisms to new β-lactams and β-lactamase inhibitors (PROTECT)
Editor: Laurent Poirel5
PMCID: PMC11539232  PMID: 39382274

ABSTRACT

We aimed to investigate the activity of and mechanisms of resistance to cefiderocol and innovative β-lactam/β-lactamase inhibitor combinations in a nationwide collection of double-carbapenemase-producing Enterobacterales. In all, 57 clinical isolates co-producing two carbapenemases collected from Spanish hospitals during the period 2017–2022 were analyzed. Minimum inhibitory concentration (MIC) values for ceftazidime, ceftazidime/avibactam, aztreonam, aztreonam/avibactam, aztreonam/nacubactam, cefiderocol, cefepime, cefepime/taniborbactam, cefepime/zidebactam, cefepime/nacubactam, imipenem, imipenem/relebactam, meropenem, meropenem/vaborbactam, meropenem/xeruborbactam, and meropenem/ANT3310 were determined by reference broth microdilution. Genetic drivers of resistance were analyzed by whole-genome sequencing (WGS). The collection covered nine carbapenemase associations: VIM + OXA-48 (21/57), NDM + OXA-48 (11/57), KPC + VIM (10/57), KPC + OXA-48 (6/57), IMP + OXA-48 (3/57), NDM + KPC (2/57), NDM + VIM (2/57), NDM + GES (1/57), and KPC + IMP (1/57). Ceftazidime/avibactam, imipenem/relebactam, and meropenem/vaborbactam were the least active options. Aztreonam/avibactam and aztreonam/nacubactam were active against the whole collection and yielded MIC50/MIC90 values of ≤0.25/0.5 mg/L and 1/2 mg/L, respectively. Cefepime/zidebactam (56/57 susceptible), meropenem/xeruborbactam (56/57 susceptible), cefepime/nacubactam (55/57 susceptible), and cefiderocol (53/57 susceptible) were also highly active, with MIC50/MIC90 values ranging from ≤0.25–2 mg/L to 2–4 mg/L, respectively. Meropenem/ANT3310 (MIC50/MIC90 = 0.5/≥64 mg/L; 47/57 susceptible) and cefepime/taniborbactam (MIC50/MIC90 = 0.5/16 mg/L; 44/57 susceptible) also retained high levels of activity, although they were affected by NDM-type enzymes in combination with porin deficiency. Our findings highlight that cefiderocol and combinations of β-lactams and the novel β-lactamase inhibitors avibactam, nacubactam, taniborbactam, zidebactam, xeruborbactam, and ANT3310 show promising activity against double-carbapenemase-producing Enterobacterales.

KEYWORDS: carbapenemase, double carbapenemase, β-lactamase, antimicrobial resistance, cefiderocol, β-lactamase inhibitor, Enterobacterales

INTRODUCTION

The emergence of infections caused by carbapenemase-producing Enterobacterales (CPE) is a growing clinical threat that severely limits the choice of therapy (1). This threat has been increased in recent years by the emergence of clinical isolates carrying multiple carbapenemases (2). These strains are commonly pan-β-lactam-resistant, as the coexistence in the same clinical isolate of enzymes such as OXA-48 or KPC with metallo-β-lactamases (MBLs) usually compromises the efficacy of all clinically available β-lactams, including recent combinations such as ceftazidime/avibactam, imipenem/relebactam, and meropenem/vaborbactam (3). Thanks to intense drug discovery efforts, a plethora of new β-lactams or β-lactam/β-lactamase inhibitor combinations with promising activity against carbapenemase-producing microorganisms have recently received approval or are currently undergoing evaluation in clinical trials (4). Cefiderocol and aztreonam/avibactam retain high levels of activity against most Gram-negative bacteria collected in surveillance studies, including metallo-β-lactamase-producing strains (5). On the other hand, new generation diazabicyclooctane- (e.g., zidebactam, nacubactam, ANT3310) and boronate-type β-lactamase inhibitors (e.g., taniborbactam, xeruborbactam) have demonstrated to be able to restore in vitro β-lactam susceptibility against CPE (including those carrying MBLs) due to synergistically improved antibacterial activity and/or enhanced β-lactamase inhibition (6, 7). However, the potential activity of most of these newly developed agents against the emerging therapeutic niche of double-carbapenemase-producers has not yet been investigated.

Here, we aimed to investigate the activity and mechanisms of resistance to ceftazidime/avibactam, cefiderocol, imipenem/relebactam, meropenem/vaborbactam, aztreonam/avibactam (recently approved), aztreonam/nacubactam, cefepime/taniborbactam, cefepime/zidebactam, cefepime/nacubactam, meropenem/xeruborbactam, and meropenem/ANT3310 (in phases 1–3 of clinical development; ClinicalTrials.gov identifiers: NCT05905055, NCT06168734, NCT04979806, NCT06079775, and NCT05905913, respectively) in a contemporary multicenter collection of double-carbapenemase-producing Enterobacterales recovered as part of the Spanish PROTECT collaborative network. The PROTECT network is a study group focused on the activity and mechanisms of resistance to new carbapenemase inhibitors, organized under the auspices of the Study Group on the Mechanisms of Action and Resistance to Antimicrobial Agents (GEMARA) of the Spanish Society of Clinical Microbiology and Infectious Diseases (SEIMC).

RESULTS

Integrated minimum inhibitory concentration (MIC) and whole-genome sequencing (WGS) data for each of the 57 clinical isolates evaluated are provided in Table 1. According to Ambler’s molecular classification of β-lactamases, the combination of classes B + D (n = 35; 61.4%) type enzymes was the most common, followed by classes A + B (n = 14; 24.5%), classes A + D (n = 6; 10.5%), and double-class B (n = 2; 3.5%). More specifically, the collection included nine carbapenemase associations: VIM + OXA-48 (n = 21; 36.8%), NDM + OXA-48 (n = 11; 19.3%), KPC + VIM (n = 10; 17.5%), KPC + OXA-48 (n = 6; 10.5%), IMP + OXA-48 (n = 3; 5.3%), NDM + KPC (n = 2; 3.5%), NDM + VIM (n = 2; 3.5%), NDM + GES (n = 1; 1.8%) and KPC + IMP (n = 1; 1.8%). The most frequent carbapenemase was OXA-48-like (n = 41; 71.9%), followed by VIM (n = 33; 57.9%), KPC (n = 19; 33.3%), NDM (n = 16; 28.1%), IMP (n = 4; 7%), and GES-2 (n = 1; 1.8%). Among the 29 isolates of K. pneumoniae included, the ST6347 (n = 4) and the high-risk clones ST147 (n = 6), ST11 (n = 4), ST307 (n = 4), ST15 (n = 3), and ST101 (n = 2) were the most commonly identified, and the other isolates belonged to singleton sequence types (STs).

TABLE 1.

Bacterial species, sample, β-lactamases, sequence type, and susceptibility to cefiderocol and new β-lactam/β-lactamase inhibitor combinations against clinical isolates of double-carbapenemase-producing Enterobacteralesa

Isolate ID Bacterial species Sample Ambler class Carbapenemases ESBLs/AmpCsc MLST MIC (mg/L)b
CAZ
(R > 4)		 C/A
(R > 8)		 ATM (R > 4) A/A
(R > 4)		 A/N
(R > 4)		 FDC
(R > 2)		 FEP (R > 4) F/T (R > 4) F/Z (R > 4) F/N (R > 4) IMP
(R > 4)		 I/R
(R > 2)		 MEM (R > 8) M/V (R > 8) M/X (R > 8) M/ANT (R > 8)
D0308 Klebsiella pneumoniae Urine A, B GES-2, NDM-1 CTX-M-3 ST147 >256 >256 128 ≤0.06 1 1 64 0.5 0.25 2 2 2 2 2 ≤0.06 0.5
AI2642 Citrobacter freundii Urine A, B KPC-2, IMP-22 CMY-79 ST8 256 256 128 0.125 1 ≤0.06 32 8 0.25 2 8 1 32 4 ≤0.06 4
22K0764 Klebsiella pneumoniae Blood A, B KPC-2, NDM-1 CTX-M-15 ST147 >256 >256 >256 0.5 2 2 >256 8 1 4 64 16 128 64 2 64
170 Klebsiella pneumoniae Rectal swab A, B KPC-2, NDM-1 CMY-6 ST147 >256 >256 >256 0.5 2 2 256 16 1 4 64 8 128 64 2 64
157 Klebsiella pneumoniae Ambiental A, B KPC-2, VIM-1 - ST11 >256 256 32 0.125 0.5 ≤0.06 16 ≤0.25 0.25 2 4 4 2 ≤0.25 ≤0.06 0.5
162 Escherichia coli Urine A, B KPC-2, VIM-1 CMY-2 ST453 256 128 32 ≤0.06 1 0.125 16 ≤0.25 0.125 1 4 1 2 ≤0.25 ≤0.06 0.125
173 Citrobacter freundii Blood A, B KPC-2, VIM-1 - ST22 128 32 128 ≤0.06 2 0.25 16 ≤0.25 0.25 2 4 0.5 2 ≤0.25 ≤0.06 0.125
175 Enterobacter cloacae Wound exudate A, B KPC-2, VIM-1 - ST1877 128 128 128 0.25 1 1 64 ≤0.25 0.25 2 8 2 2 1 ≤0.06 1
22Eco0132 Citrobacter freundii Urine A, B KPC-2, VIM-1 CMY-110 ST100 128 128 128 0.25 1 ≤0.06 32 ≤0.25 0.125 2 1 0.5 1 0.5 ≤0.06 ≤0.06
K13122 Klebsiella pneumoniae Urine A, B KPC-3, VIM-1 - ST2388 256 64 256 0.25 2 0.5 32 ≤0.25 0.5 2 8 2 8 0.5 ≤0.06 0.125
K13615 Klebsiella pneumoniae Tracheal aspirate A, B KPC-3, VIM-1 - ST6347 >256 128 >256 0.25 2 0.25 64 ≤0.25 0.5 2 16 0.5 8 0.5 ≤0.06 0.25
22K0062 Klebsiella pneumoniae Urine A, B KPC-3, VIM-1 - ST6347 >256 256 >256 1 4 0.5 >256 4 0.125 4 64 8 128 4 ≤0.06 4
K13624 Klebsiella pneumoniae Sputum A, B KPC-3, VIM-1 - ST6347 >256 128 >256 0.25 2 0.25 64 0.5 0.5 2 16 0.5 16 0.5 ≤0.06 0.25
K13667 Klebsiella pneumoniae Rectal swab A, B KPC-3, VIM-1 - ST6347 >256 256 >256 1 4 1 >256 16 2 2 256 16 256 16 1 8
B0104 Klebsiella pneumoniae Urine A, D KPC-2, OXA-48 - ST392 >256 1 >256 ≤0.06 2 0.25 64 0.5 0.5 1 8 2 8 2 ≤0.06 ≤0.06
A0105 Klebsiella pneumoniae Urine A, D KPC-3, OXA-48 CTX-M-15 ST307 >256 2 >256 0.25 1 0.125 32 0.5 0.5 1 8 0.5 4 1 ≤0.06 ≤0.06
151 Klebsiella pneumoniae Rectal swab A, D KPC-3, OXA-48 - ST512 256 2 >256 ≤0.06 1 0.5 32 ≤0.25 0.5 1 8 ≤0.25 4 ≤0.25 ≤0.06 ≤0.06
AI2888 Klebsiella pneumoniae Urine A, D KPC-3, OXA-48 CTX-M-15, SHV-18 ST15 256 4 >256 0.125 1 0.125 256 ≤0.25 0.25 1 8 ≤0.25 8 ≤0.25 ≤0.06 1
Cit373 Citrobacter freundii Urine A, D KPC-2, OXA-48 - ST22 64 2 128 0.125 2 ≤0.06 32 ≤0.25 0.25 1 4 1 16 2 ≤0.06 ≤0.06
22K0680 Klebsiella pneumoniae Surgical wound A, D KPC-3, OXA-48 CTX-M-15 ST307 128 0.5 128 ≤0.06 1 0.125 64 ≤0.25 0.25 0.5 8 ≤0.25 8 ≤0.25 ≤0.06 ≤0.06
22Entb0065 Enterobacter hormaechei Rectal swab B, B VIM-1, NDM-1 CMY-4, SHV-12 ST88 >256 256 >256 0.5 1 2 128 4 0.25 1 2 2 2 2 ≤0.06 1
H0506 Klebsiella pneumoniae Urine B, B VIM-1, NDM-3 CTX-M-15 ST307 128 128 32 ≤0.06 1 0.5 16 ≤0.25 0.25 2 0.5 0.5 0.5 0.5 ≤0.06 0.125
22Cit0014 Citrobacter freundii Urine B, D IMP-8, OXA-48 CTX-M-210 ST912 256 64 >256 0.125 2 ≤0.06 >256 8 2 2 128 64 128 128 1 2
22Entb0027 Enterobacter hormaechei Urine B, D IMP-13, OXA-48 - ST182 128 128 0.5 0.25 0.5 0.25 4 4 0.25 1 2 2 1 1 ≤0.06 0.125
AI2760 Enterobacter cloacae Urine B, D IMP-13, OXA-48 ACT-16 ST182 64 64 0.5 0.5 0.25 0.25 8 4 0.25 1 2 1 2 2 ≤0.06 0.25
H0510 Klebsiella pneumoniae Catheter B, D NDM-1, OXA-48 CTX-M-15 ST147 >256 >256 256 0.25 1 2 >256 64 0.5 4 32 32 128 128 2 32
165 Klebsiella pneumoniae Rectal swab B, D NDM-1, OXA-48 CTX-M-15, CMY-2 ST307 >256 >256 128 0.125 1 2 128 0.5 0.25 1 2 2 2 2 ≤0.06 1
166 Enterobacter cloacae Rectal swab B, D NDM-1, OXA-48 CTX-M-15 ST136 >256 >256 >256 1 2 1 128 2 1 2 4 2 4 4 0.125 4
169 Klebsiella pneumoniae Blood B, D NDM-1, OXA-48 CTX-M-15 ST101 >256 >256 >256 0.5 2 2 >256 16 1 2 64 32 128 128 8 64
AI3008 Klebsiella pneumoniae Urine B, D NDM-1, OXA-48 CTX-M-15 ST101 >256 >256 >256 0.125 1 1 128 1 0.25 1 2 1 2 1 ≤0.06 0.25
22K0670 Klebsiella pneumoniae Urine B, D NDM-1, OXA-48 CTX-M-15 ST147 >256 >256 >256 0.25 2 2 >256 8 1 2 32 32 64 64 2 32
22K0008 Klebsiella pneumoniae Rectal swab B, D NDM-1, OXA-244 CTX-M-15 ST395 >256 >256 256 0.25 2 2 >256 16 4 >16 32 64 256 256 8 >64
163 Klebsiella pneumoniae Wound exudate B, D NDM-5, OXA-48 CTX-M-15 ST147 >256 >256 256 0.125 1 2 >256 16 1 1 8 4 32 32 1 32
174 Klebsiella pneumoniae Rectal swab B, D NDM-5, OXA-48 CTX-M-15 ST15 >256 >256 256 ≤0.06 1 1 128 0.5 0.25 2 2 2 4 4 ≤0.06 2
22K0732 Klebsiella pneumoniae Rectal swab B, D NDM-5, OXA-181 CTX-M-15 ST16 >256 >256 >256 0.5 2 4 >256 128 4 4 64 64 128 128 64 >64
22 K0727 Klebsiella pneumoniae Wound exudate B, D NDM-5, OXA-181 CTX-M-15 ST16 >256 >256 256 0.25 1 2 >256 32 1 2 64 32 128 128 8 64
AI2799 Enterobacter cloacae Urine B, D VIM-1, OXA-48 ACT-17, CTX-M-9 ST1015 256 256 1 0.125 0.25 0.5 32 2 0.5 2 16 8 4 4 0.125 2
A1502 Klebsiella pneumoniae Bronchoalveolar lavage B, D VIM-1, OXA-48 CTX-M-15 ST11 >256 >256 256 0.5 2 0.125 64 4 2 4 4 4 1 1 ≤0.06 0.5
A1504 Klebsiella pneumoniae Blood B, D VIM-1, OXA-48 CTX-M-15 ST11 >256 >256 256 0.5 2 0.5 >256 16 8 8 128 128 128 128 2 32
K13835 Klebsiella pneumoniae Urine B, D VIM-1, OXA-48 CTX-M-15 ST11 128 64 64 0.125 1 0.125 64 0.5 0.5 2 16 8 16 16 ≤0.06 1
22Entb0055 Enterobacter hormaechei Blood B, D VIM-1, OXA-48 DHA-1, SHV-12 ST114 >256 256 >256 0.5 4 4 64 2 0.25 2 2 2 2 2 ≤0.06 ≤0.06
22Entb0084 Enterobacter hormaechei Sputum B, D VIM-1, OXA-48 CTX-M-9 ST116 >256 >256 2 0.5 0.25 0.25 16 1 0.5 2 1 2 8 8 ≤0.06 0.5
152 Enterobacter cloacae Rectal swab B, D VIM-1, OXA-48 SHV-12 ST8 >256 128 >256 0.25 2 4 16 0.5 0.25 2 2 2 1 1 ≤0.06 0.5
153 Enterobacter cloacae Rectal swab B, D VIM-1, OXA-48 CTX-M-9, SHV-12 ST90 >256 128 >256 0.25 2 4 16 ≤0.25 0.5 2 2 2 1 1 ≤0.06 1
155 Enterobacter asburiae Rectal swab B, D VIM-1, OXA-48 SHV-12 ST563 128 64 128 0.25 2 2 8 ≤0.25 0.25 2 2 1 1 1 ≤0.06 0.25
156 Klebsiella oxytoca Rectal swab B, D VIM-1, OXA-48 CTX-M-9 ST213 >256 256 32 0.25 0.25 0.125 32 ≤0.25 0.5 4 2 2 2 2 ≤0.06 1
158 Enterobacter cloacae Rectal swab B, D VIM-1, OXA-48 SHV-12 ST254 256 64 64 0.125 1 0.5 8 ≤0.25 1 2 8 4 1 0.5 ≤0.06 0.5
159 Citrobacter freundii Rectal swab B, D VIM-1, OXA-48 SHV-12 ST328 128 64 64 ≤0.06 1 ≤0.06 4 ≤0.25 0.25 2 1 1 0.5 0.5 ≤0.06 0.25
160 Citrobacter freundii Urine B, D VIM-1, OXA-48 SHV-12 ST221 256 64 128 0.125 1 0.25 16 ≤0.25 0.25 1 2 1 2 1 ≤0.06 0.125
161 Enterobacter cloacae Rectal swab B, D VIM-1, OXA-48 SHV-12 ST78 256 32 1 0.25 0.5 ≤0.06 4 1 0.25 1 2 1 2 2 ≤0.06 ≤0.06
164 Enterobacter cloacae Urine B, D VIM-1, OXA-48 - ST78 >256 256 256 0.25 2 2 16 ≤0.25 0.5 2 2 1 1 1 ≤0.06 0.125
168 Citrobacter freundii Rectal swab B, D VIM-1, OXA-48 - ST432 128 64 32 ≤0.06 0.5 0.25 8 0.5 0.25 2 2 1 0.5 0.5 ≤0.06 0.125
171 Klebsiella pneumoniae Urine B, D VIM-1, OXA-48 CTX-M-15 ST15 256 256 256 0.125 1 0.5 32 ≤0.25 0.25 2 2 1 1 0.5 ≤0.06 0.25
172 Citrobacter freundii Wound exudate B, D VIM-1, OXA-48 CTX-M-9 ST110 128 64 32 0.125 0.5 ≤0.06 16 ≤0.25 0.25 2 2 1 1 1 ≤0.06 0.125
22Cit0018 Citrobacter freundii Rectal swab B, D VIM-1, OXA-48 - ST603 256 256 256 0.125 2 0.125 16 ≤0.25 2 2 2 2 2 2 ≤0.06 0.25
22Entb0093 Enterobacter cloacae Urine B, D VIM-1, OXA-48 - ST182 >256 >256 32 2 0.5 1 64 2 1 2 4 4 8 8 ≤0.06 0.5
154 Enterobacter cloacae Rectal swab B, D VIM-4, OXA-48 - ST1381 8 4 1 0.25 0.125 1 2 ≤0.25 0.25 1 4 2 1 0.5 ≤0.06 0.5
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a

MLST: multilocus sequence type; CAZ: ceftazidime; C/A: ceftazidime/avibactam; ATM: aztreonam; A/A: aztreonam/avibactam; A/N: aztreonam/nacubactam; FDC: cefiderocol; FEP: cefepime; F/T: cefepime/taniborbactam; F/Z: cefepime/zidebactam; F/N: cefepime/nacubactam; IMP: imipenem; I/R: imipenem/relebactam; MEM: meropenem; M/V: meropenem/vaborbactam; M/X: meropenem/xeruborbactam; M/ANT: meropenem/ANT3310.

b

EUCAST breakpoints indicated.

c

-, no ESBLs/AmpCs were present.

Cumulative MIC data for the whole collection according to the double-carbapenemase content is shown in Table 2. Classic β-lactams showed no relevant activity against the collection: ceftazidime was not active against any of the isolates included (0/57; 0% susceptibility); aztreonam retained its activity against 10.5% of the strains (6/57) and cefepime was only active against four isolates (4/57; 7% susceptibility), all of which yielded MIC50 and MIC90 values of ≥64 mg/L. On the other hand, due to their low baseline MICs, imipenem (31/57; 54.4% susceptible) and meropenem (40/57; 70.2% susceptible) retained activity against a higher proportion of isolates, both with MIC50 and MIC90 values of 4 mg/L and 64 mg/L, respectively. As expected, the recently launched combinations ceftazidime/avibactam, imipenem/relebactam and meropenem/vaborbactam did not significantly enhance the activity of the respective β-lactam when tested alone: ceftazidime/avibactam was the least active β-lactam/β-lactamase inhibitor combination, with MIC50 and MIC90 values of ≥64 mg/L, and only 7/57 strains being susceptible (12.3% susceptibility; mostly isolates that carried combinations of KPC-like plus OXA-48 carbapenemases), followed by imipenem/relebactam and meropenem/vaborbactam, which were active against 38/57 (66.7% susceptibility, MIC50 = 2 mg/L; MIC90 = 32 mg/L) and 44/57 (77.2% susceptibility, MIC50 = 2 mg/L; MIC90 ≥64 mg/L) of the strains respectively.

TABLE 2.

MIC50, MIC90 and cumulative MIC distribution for cefiderocol and new β-lactam/β-lactamase inhibitor combinations against clinical isolates of double-carbapenemase-producing Enterobacteralesa

Carbapenemase content β-lactam or β-lactam/β-lactamase inhibitor Cumulative % of isolates at MIC (mg/L) MIC (mg/L)
≤0.25 0.5 1 2 4 8 16 32 ≥64 MIC50 MIC90
All isolates (n = 57) CAZ 1.8 1.8 1.8 100 ≥64 ≥64
C/A 1.8 3.5 8.8 12.3 12.3 12.3 15.8 100 ≥64 ≥64
ATM 3.5 8.8 10.5 10.5 10.5 10.5 22.8 100 ≥64 ≥64
A/A 75.4 93 98.2 100 ≤0.25 0.5
A/N 8.8 19.3 57.9 94.7 100 1 2
FDC 43.9 57.9 71.9 93 100 0.5 2
FEP 1.8 7 14 33.3 49.1 100 ≥64 ≥64
F/T 40.4 56.1 61.4 68.4 77.2 84.2 94.7 96.5 100 0.5 16
F/Z 49.1 71.9 87.7 94.7 98.2 100 0.5 2
F/N 1.8 28.1 84.2 96.5 98.2 100 2 4
IMP 1.8 7 40.4 54.4 71.9 78.9 84.2 100 4 ≥64
I/R 5.3 15.8 22.8 66.7 75.4 82.5 86 93 100 2 32
MEM 5.3 24.6 49.1 57.9 70.2 75.4 78.9 100 4 ≥64
M/V 10.5 28.1 47.4 64.9 73.7 77.2 80.7 82.5 100 2 ≥64
M/X 78.9 78.9 84.2 93 93 98.2 98.2 98.2 100 ≤0.25 2
M/ANT 43.9 57.9 70.2 75.4 80.7 82.5 82.5 89.5 100 0.5 ≥64
Class A + B (n = 14) CAZ 100 ≥64 ≥64
C/A 100 ≥64 ≥64
ATM 14.3 100 ≥64 ≥64
A/A 71.4 85.7 100 ≤0.25 1
A/N 7.1 42.9 85.7 100 2 4
FDC 50 64.3 85.7 100 ≤0.25 2
FEP 21.4 42.9 100 ≥64 ≥64
F/T 50 64.3 64.3 64.3 71.4 85.7 100 ≤0.25 16
F/Z 57.1 78.6 92.9 100 ≤0.25 1
F/N 7.1 78.6 100 2 4
IMP 7.1 14.3 35.7 57.1 71.4 71.4 100 8 ≥64
I/R 28.6 42.9 64.3 71.4 85.7 100 2 16
MEM 7.1 42.9 42.9 57.1 64.3 71.4 100 8 ≥64
M/V 21.4 50 57.1 64.3 78.6 78.6 85.7 85.7 100 0.5 ≥64
M/X 78.6 78.6 85.7 100 ≤0.25 2
M/ANT 42.9 57.1 64.3 64.3 78.6 85.7 85.7 85.7 100 0.5 ≥64
Class A + D (n = 6) CAZ 100 ≥64 ≥64
C/A 16.1 33.3 83.3 100 2 4
ATM 100 ≥64 ≥64
A/A 100 ≤0.25 ≤0.25
A/N 66.7 100 1 2
FDC 83.3 100 ≤0.25 0.5
FEP 50 100 32 ≥64
F/T 66.7 100 ≤0.25 0.5
F/Z 50 100 ≤0.25 0.5
F/N 16.7 100 1 1
IMP 16.7 100 8 8
I/R 50 66.7 83.3 100 ≤0.25 2
MEM 33.3 83.3 100 16 16
M/V 50 50 66.7 100 0.5 2
M/X 100 ≤0.25 ≤0.25
M/ANT 83.3 83.3 100 ≤0.25 1
Class B + D (n = 35) CAZ 2.9 2.9 2.9 100 ≥64 ≥64
C/A 2.9 2.9 2.9 5.7 100 ≥64 ≥64
ATM 5.7 14.3 17.1 17.1 17.1 17.1 28.6 100 ≥64 ≥64
A/A 74.3 94.3 97.1 100 ≤0.25 0.5
A/N 14.3 28.6 60 97.1 100 1 2
FDC 37.1 48.6 62.9 88.6 100 1 4
FEP 2.9 11.4 22.9 42.9 51.4 100 32 ≥64
F/T 31.4 45.7 54.3 65.7 74.3 80 91.4 94.3 100 1 16
F/Z 42.9 62.9 82.9 91.4 97.1 100 0.5 2
F/N 22.9 82.9 94.3 97.1 100 2 4
IMP 5.7 54.3 65.7 71.4 77.1 85.7 100 2 ≥64
I/R 28.6 60 71.4 77.1 77.1 88.6 100 2 ≥64
MEM 5.7 34.3 57.1 65.7 71.4 74.3 77.1 100 2 ≥64
M/V 14.3 40 57.1 65.7 71.4 74.3 77.1 100 2 ≥64
M/X 74.3 74.3 80 88.6 88.6 97.1 97.1 97.1 100 ≤0.25 8
M/ANT 37.1 54.3 65.7 74.3 77.1 77.1 77.1 88.6 100 0.5 ≥64
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CAZ: ceftazidime; C/A: ceftazidime/avibactam; ATM: aztreonam; A/A: aztreonam/avibactam; A/N: aztreonam/nacubactam; FDC: cefiderocol; FEP: cefepime; F/T: cefepime/taniborbactam; F/Z: cefepime/zidebactam; F/N: cefepime/nacubactam; IMP: imipenem; I/R: imipenem/relebactam; MEM: meropenem; M/V: meropenem/vaborbactam; M/X: meropenem/xeruborbactam; M/ANT: meropenem/ANT3310.

By contrast, aztreonam-based combinations and cefepime/zidebactam retained excellent activity against the whole collection regardless of the combination of carbapenemases produced. The addition of avibactam to aztreonam restored the aztreonam susceptibility against the whole collection (57/57; 100% susceptibility) and displayed the highest activity among the combinations tested in terms of MIC50/MIC90 (≤0.25/0.5 mg/L). Similarly, nacubactam potentiated the activity of aztreonam against the whole collection (57/57; 100% susceptibility), although its activity was slightly lower (MIC50 = 1 mg/L; MIC90 = 2 mg/L) than that observed for aztreonam/avibactam. While CLSI recommends testing nacubactam at a 1:1 ratio when combined with aztreonam, this inhibitor was also evaluated at a fixed concentration of 4 mg/L to directly compare its ability to enhance the activity of aztreonam with that of avibactam, which is also added at 4 mg/L. At this inhibitor concentration, aztreonam/nacubactam was even more potent than aztreonam/avibactam, with MIC50/MIC90 values of ≤0.25/≤0.25 mg/L (comparative data of aztreonam/nacubactam evaluated at different nacubactam concentrations are provided in Tables S1 and S2). Finally, cefepime/zidebactam was also very active, with susceptibility rates of up to 98.3% (56/57 susceptible, MIC50 = 0.5 mg/L; MIC90 = 2 mg/L).

In contrast to that observed for aztreonam/avibactam, aztreonam/nacubactam or cefepime/zidebactam, cefiderocol, and the rest of the β-lactam/β-lactamase inhibitor combinations tested were generally active, but showed important variations in their effectiveness according to the underlying association of carbapenemases produced. Cefiderocol was highly active against the whole collection, with a susceptibility rate of 93% (53/57 susceptible) and MIC50/MIC90 values of 0.5/2 mg/L, respectively. However, a closer look revealed that its activity was reduced to 88.6% against isolates co-producing class B + D carbapenemases (31/35 susceptible; with MIC50/MIC90 values of 1/4 mg/L) or increased to 100% against those isolates carrying combinations of class A + B (14/14 susceptible) and A + D (6/6 susceptible) carbapenemases, with MIC50/MIC90 values of ≤0.25/2 mg/L and ≤0.25/0.5 mg/L, respectively.

Cefepime/taniborbactam and cefepime/nacubactam, respectively, restored the activity of cefepime to 77.2% (44/57 susceptible; MIC50 = 0.5 mg/L and MIC90 = 16 mg/L) and to 96.5% (55/57 susceptible; MIC50 = 2 mg/L and MIC90 = 4 mg/L) of the isolates evaluated. Both combinations demonstrated excellent activity against isolates co-producing class A + D carbapenemases (n = 6), showing rates of activity of 100% and yielding MIC50/MIC90 values of ≤0.25/0.5 mg/L for cefepime/taniborbactam and MIC50/MIC90 values of 1/1 mg/L for cefepime/nacubactam. The activity of cefepime/taniborbactam experienced an important drop against those isolates carrying combinations of class B with A or D carbapenemases, and especially when NDMs were present. Specifically, the percentage of activity and MIC50/MIC90 values of cefepime/taniborbactam decreased to 71.4% (10/14 susceptible) and ≤0.25/16 mg/L for isolates coproducing A + B enzymes, and to 74.3% (26/35 susceptible) and 1/16 mg/L for those isolates coproducing B + D enzymes. The loss of activity was less pronounced for cefepime/nacubactam, which only was inactive against 2 K. pneumoniae isolates with MICs of 8 mg/L and >16 mg/L carrying the carbapenemase combinations VIM-1 + OXA-48 and NDM-1 + OXA-244, respectively.

The MIC of meropenem against the whole collection was significantly decreased by combining this agent with the investigational inhibitor xeruborbactam, yielding susceptibility rates of 98.3% (56/57 susceptible) with MIC50/MIC90 values of ≤0.25/2 mg/L. Moreover, it was extraordinarily active against isolates carrying class A + B enzymes and particularly A + D enzymes, against which this innovative combination demonstrated activity rates of 100% and MIC50/MIC90 values of ≤0.25/2 mg/L and ≤0.25/≤0.25 mg/L, respectively. However, similar to cefiderocol and combinations of cefepime with taniborbactam and nacubactam, its potency was affected by the association of co-produced carbapenemases, particularly when NDM-type enzymes were present. This effect was evident in the group of isolates carrying B + D enzymes, which had MIC50/MIC90 values of ≤0.25/8 mg/L and in which the unique meropenem/xeruborbactam-resistant strain was found. Finally, meropenem/ANT3310 showed 82.5% susceptibility against the whole collection (47/57 susceptible), although the enhancing effect of ANT3310 was not evident against those isolates in which class B enzymes were present (MIC50/MIC90 values of 0.5/≥64 mg/L). However, meropenem/ANT3310 was particularly potent against isolates carrying combinations of OXA-48 plus KPC-like β-lactamases, against which the combination showed MIC50/MIC90 values of ≤0.25/1 mg/L.

Eleven K. pneumoniae isolates included in the collection showed resistance to at least cefiderocol or one of the combinations in clinical development (cefepime/taniborbactam, cefepime/zidebactam, cefepime/nacubactam, meropenem/xeruborbactam, meropenem/ANT3310), and thus their putative horizontally acquired and chromosomally encoded resistance mechanisms were investigated by WGS (Table 3). The isolates included generally showed cross-resistance to cefepime/taniborbactam (11/11-resistant; MIC range 8–128 mg/L), meropenem/ANT3310 (10/11-resistant; MIC range 8 to >64 mg/L), cefepime/nacubactam (2/11-resistant; range 1 to >16 mg/L), cefepime/zidebactam (1/11-resistant; range 0.5–8 mg/L), meropenem/xeruborbactam (1/11-resistant; range 1–64 mg/L), and cefiderocol (1/11-resistant; range 0.5–4 mg/L). The resistance mechanisms most commonly associated with clinical resistance to these agents involved the production of NDM-like β-lactamases (9/11), insertions/disruption in the outer membrane porins OmpK36 (11/11) and OmpK35 (7/11), and inactivation of the acrR-negative regulator of the acrAB-tolC efflux operon (5/11). Missense mutations in PBP-2 (P97H, A100T, Q105S, T127N, S186N, N194D, R204H, E312D, A384V, Y394N, N492D, and Q527L), PBP-3 (A36T, I48V, and V332I), or the siderophores fiuA (A223SS, D387N, and S718R) and cirA (T172A, Y183W, V237I, and D558N) were also common in some strains (not shown), although these mutations were previously found in the genomes of β-lactam susceptible isolates previously sequenced in our laboratory, thus presumptively ruling out their potential role in resistance.

TABLE 3.

Detailed phenotypic and genotypic features of isolates showing resistance to cefiderocol or recent β-lactam/ β-lactamase inhibitor combinationsa

Isolate ID Bacterial species MLST MIC (mg/L)b Resistance mechanismsc,d,e
CAZ
(R > 4)		 C/A
(R > 8)		 ATM (R > 4) A/A
(R > 4)		 A/N
(R > 4)		 FDC
(R > 2)		 FEP (R > 4) F/T (R > 4) F/Z (R > 4) F/N (R > 4) IMP
(R > 4)		 I/R
(R > 2)		 MEM (R > 8) M/V (R > 8) M/X (R > 8) M/ANT (R > 8) Transferable Chromosomalf
Carbapenemases Other β-lactamases OmpK35 OmpK36 AcrAB-TolC (acrR)
22K0764 Klebsiella pneumoniae ST147 >256 >256 >256 0.5 2 2 >256 8 1 4 64 16 128 64 2 64 KPC-2, NDM-1 CTX-M-15, CMY-6 G49fs G134_D135dup T144fs
170 Klebsiella pneumoniae ST147 >256 >256 >256 0.5 2 2 256 16 1 4 64 8 128 64 2 64 KPC-2, NDM-1 CMY-6 G49fs G134_D135dup T144fs
H0510 Klebsiella pneumoniae ST147 >256 >256 256 0.25 1 2 >256 64 0.5 4 32 32 128 128 2 32 NDM-1, OXA-48 CTX-M-15 - G134_D135dup -
22K0727 Klebsiella pneumoniae ST16 >256 >256 256 0.25 1 2 >256 32 1 2 64 32 128 128 8 64 NDM-5, OXA-181 CTX-M-15 - D135_T136dup A47fs
169 Klebsiella pneumoniae ST101 >256 >256 >256 0.5 2 2 >256 16 1 2 64 32 128 128 8 64 NDM-1, OXA-48 CTX-M-15, SHV-12 G62fs G134_D135dup -
22K0732 Klebsiella pneumoniae ST16 >256 >256 >256 0.5 2 4 >256 128 4 4 64 64 128 128 64 >64 NDM-5, OXA-181 CTX-M-15 - D135_T136dup A47fs
22K0008 Klebsiella pneumoniae ST395 >256 >256 256 0.25 2 2 >256 16 4 >16 32 64 256 256 8 >64 NDM-1, OXA-244 CTX-M-15 P255fs G134_D135dup -
22K0670 Klebsiella pneumoniae ST147 >256 >256 >256 0.25 2 2 >256 8 1 2 32 32 64 64 2 32 NDM-1, OXA-48 CTX-M-15 R60fs G134_D135dup -
A1504 Klebsiella pneumoniae ST11 >256 >256 256 0.5 2 0.5 >256 16 8 8 128 128 128 128 2 32 VIM-1, OXA-48 CTX-M-15 - L32fs -
163 Klebsiella pneumoniae ST147 >256 >256 256 0.125 1 2 >256 16 1 1 8 4 32 32 1 32 NDM-5, OXA-48 CTX-M-15 R60fs G134_D135dup, R306del S134fs
K13667 Klebsiella pneumoniae ST6347 >256 256 >256 1 4 1 >256 16 2 2 256 16 256 16 1 8 KPC-3, VIM-1 - T47fs R306del -
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a

MLST: multilocus sequence type; CAZ: ceftazidime; C/A: ceftazidime/avibactam; ATM: aztreonam; A/A: aztreonam/avibactam; A/N: aztreonam/nacubactam; FDC: cefiderocol; FEP: cefepime; F/T: cefepime/taniborbactam; F/Z: cefepime/zidebactam; F/N: cefepime/nacubactam; IMP: imipenem; I/R: imipenem/relebactam; MEM: meropenem; M/V: meropenem/vaborbactam; M/X: meropenem/xeruborbactam; M/ANT: meropenem/ANT3310.

b

EUCAST breakpoints indicated.

c

fs = frameshift change.

d

dup = duplication.

e

del = deletion.

f

-, no mutations related to the studied genes were found.

DISCUSSION

A recent analysis of 79214 Enterobacterales collected globally as part of the ATLAS 2018–2022 surveillance program revealed an increase in the detection of isolates co-carrying multiple carbapenemases in Asia Pacific, Latin America, Middle East-Africa, and North America (8). This type of strain is also on the rise in Europe, particularly in K. pneumoniae, and ST147 blaNDM+blaOXA-48-positive isolates have been detected by WGS-guided surveillance in different countries, such as Germany, Norway, and France (9–11). Our strain collection includes isolates of multiple species recovered as part of three independent studies and thus our data cannot be used to accurately estimate the burden and genomic epidemiology of double-carbapenemase-producing Enterobacterales in Spain. However, it should be noted that among the K. pneumoniae isolates included (n = 29), ST147 (n = 6) and blaNDM+blaOXA-48 (n = 11) were, respectively, the most frequent lineage and association of carbapenemases encountered. Although ST147 and blaNDM+blaOXA-48 -positive K. pneumoniae isolates were already circulating in Spain 5 years ago (12), recent reports have associated the large movement of refugees and migrants from the ongoing Russo-Ukrainian war with a further increase in the transmission of this highly resistant lineage on the European continent (9, 12). In this regard, an ST147 blaNDM+blaOXA-48-positive isolate was detected in a rectal sample from a patient transferred from Marinka (Ukraine) to Madrid, thus suggesting that the Ukraine conflict may have also had implications for the dissemination of this double-carbapenemase-producing clone in Spain (12, 13).

Regarding the antimicrobial susceptibility profiles, the recently marketed β-lactam/β-lactamase inhibitor combinations such as ceftazidime/avibactam, meropenem/vaborbactam, and imipenem/relebactam were the least active options against the collection. This is explained by the high proportion of isolates carrying class B-type carbapenemases, which were present in 51 of the 57 isolates included and against which avibactam, vaborbactam, and relebactam poorly potentiated the activity of the β-lactam partners. By contrast, aztreonam/avibactam, which has very recently received FDA and EMA approval (in 2024), proved to be very active against the collection. These data are consistent with findings commonly reported for aztreonam/avibactam and large collections of carbapenemase-producing Enterobacterales, against which this combination usually shows percentages of activity to nearly 100% (12, 14). In Spain, aztreonam/avibactam resistance has scarcely been reported, although NDM-producing E. coli strains with elevated aztreonam/avibactam MICs due to co-carriage of CMY-like enzymes and PBP-3 inserts were recently documented by Vázquez-Ucha et al. (12, 15). On the other hand, aztreonam/nacubactam, which has now entered clinical phase 3, also yielded 100% susceptibility rates. Data on the activity of aztreonam/nacubactam against Gram-negative bacteria are scarce, as most of the previous studies with the nacubactam inhibitor involved combining this agent with meropenem (16, 17). Mushtaq et al. demonstrated that aztreonam/nacubactam was universally active against a panel of 210 Enterobacterales isolates with NDM or VIM MBLs isolated in diagnostic laboratories from the United Kingdom (16, 18). Our results confirm that such activity is maintained in isolates with an additional carbapenemase, probably related to the PBP-2 binding effect of nacubactam, which usually yields MICs between 1 and >16 mg/L (not shown) against Enterobacterales when tested alone, regardless of the enzyme produced (19).

Cefiderocol proved to be a stable option with 93% of the strain collection showing MICs below the susceptibility breakpoint. Cefiderocol usually shows activity rates higher than 95% against Enterobacterales, including meropenem-non-susceptible strains, although elevated MIC values are commonly encountered in blaNDM-positive isolates (6, 20). Our findings are consistent with these previous data, as the subgroup of isolates carrying class A + D carbapenemases showed lower MIC50/MIC90 values, whereas those that carried NDM-type enzymes usually yielded decreased cefiderocol susceptibility (MIC = 2 mg/L) or cefiderocol resistance (MIC >2 mg/L).

Promising results were also obtained for cefepime combinations. Considering the cefepime clinical breakpoint (4 mg/L), cefepime/taniborbactam, which has recently proved superiority compared to meropenem for treating complicated urinary tract infections (21) was the least active cefepime combination evaluated. However, a closer look revealed that the addition of taniborbactam increased the cefepime susceptibility rates from 7% to 77.2% in the whole collection, thus highlighting the previously observed efficacy of this new ultra-broad spectrum boronate-type inhibitor against carbapenemase-producing Enterobacterales, including those carrying MBLs (22). Potential resistance mechanisms affecting taniborbactam include the following: (i) the IMP and NDM-9 β-lactamase variants, which are recalcitrant to taniborbactam inhibition (23, 24); (ii) the combination of NDM enzymes with low outer membrane permeability, which confers very high cefepime MIC values which cannot be totally restored by the addition of taniborbactam (6); or (iii) the presence of amino acid insertions in PBP-3 (-YRIK- and -YRIN-) together with the production of NDM-like enzymes in association with some AmpC variants (CMY-42), which has also been associated with decreased aztreonam/avibactam activity (25). Interestingly, analysis of the subset of the 11 K. pneumoniae isolates showing resistance to at least one of these new agents revealed that nine showed cefepime/taniborbactam resistance due to this latter combined mechanism. This finding demonstrates that preexisting cefepime/taniborbactam-resistant double-carbapenemase-producing K. pneumoniae strains are already circulating in Spain and encourages close monitoring of its activity once this combination becomes available for clinical use. The positive finding is that both cefepime/zidebactam and cefepime/nacubactam, which are currently undergoing phase 3 clinical trials, were less affected by these mechanisms and maintained their activity against 10 and 9 of these sets of isolates, respectively. As previously mentioned for aztreonam/nacubactam, the high in vitro activity of both cefepime/zidebactam and cefepime/nacubactam relies on their intrinsic anti-PBP-2 activity, and they thus yield high susceptibility rates in the isolates tested, regardless of the conventional β-lactam resistance mechanism involved (26, 27). This rationale supports further progression in the clinical development of these drugs to expand our armamentarium against strains equipped with two carbapenemases.

Regarding meropenem combinations with xeruborbactam and ANT3310, both potentiated the bacterial susceptibility rates further than those obtained with vaborbactam, reaching percentages of susceptibility of respectively 98.3% and 82.5%. Xeruborbactam has been extensively tested in combination with meropenem in previous research (28, 29). On the other hand, ANT3310, a new broad-spectrum diazabicyclooctane with a fluoro substituent on the piperidine ring, is currently in the clinical phase in combination with meropenem and has been demonstrated to possess activity against class D carbapenemases that are resistant to other inhibitors, such as the widespread OXA-48 or those class D enzymes scattered among Acinetobacter species (e.g., OXA-23, OXA-24/40, OXA-58) (7). In previous studies, both combinations have displayed a broader spectrum of inhibition than other already approved boronates (e.g., vaborbactam) or diazabicyclooctanes (e.g., avibactam, relebactam), with both even being able to inhibit class D carbapenemases in the nM range (7), and also MBLs in the case of xeruborbactam (29–31). Xeruborbactam restored meropenem susceptibility against most of the isolates evaluated in the collection, including eight of the nine isolates that showed cefepime/taniborbactam resistance due to a combination of NDMs with truncated outer membrane porins. This highlights the role of xeruborbactam as one of the most promising β-lactamase inhibitors to counter carbapenem resistance in Enterobacterales, and reinforces the need to continue with its clinical development. On the other hand, both meropenem/xeruborbactam and meropenem/ANT3310 yielded the lowest MIC values against the strains carrying KPCs plus OXA-48 enzymes, with all isolates inhibited at ≤0.06 mg/L for meropenem/xeruborbactam and at 1 mg/L for meropenem/ANT3310, adding further evidence of their strong potency against serine-type enzymes.

The main limitations of this work include the following: (i) The absence of strains carrying other less prevalent but problematic carbapenemases in certain epidemiological settings and bacterial species, such as IMI-, SME-, NmcA-, and FRI-like enzymes. Fortunately, previous work has shown that these carbapenemase variants are well inhibited by already approved β-lactam/β-lactamase inhibitor combinations, such as ceftazidime/avibactam and meropenem/vaborbactam (32). (ii) The absence of strains carrying emerging class B carbapenemases that pose a threat to the newly developed boronates, such as NDM-9, which has been demonstrated to confer resistance to taniborbactam (24), or IMP-10, which has recently been associated with xeruborbactam resistance (31). However, our findings conclusively demonstrate that while diazabicyclooctanes do not inhibit the MBLs present in most of the double-carbapenemase-producing strains, their intrinsic activity (e.g., zidebactam, nacubactam) or combination with MBL-stable partners (e.g., aztreonam, at the cost of sacrificing antipseudomonal coverage) results in almost universal activity against these strains. On the other hand, boronates show very promising activity, although their potent and broad inhibitory action may be neutralized in some strains when NDM enzymes are combined with lesions in porins or upregulated efflux, which trigger at very high levels the MICs of nearly all β-lactam partners. It should be noted, however, that the association of carbapenemases in the same strain does not necessarily have a synergistic effect on the activity of newly developed β-lactam/β-lactamase inhibitor combinations as the combination would retain activity if each of the individual β-lactamases falls within the spectrum of the β-lactam or β-lactam/β-lactamase inhibitor combination. This fact should be considered for the future development of new combinations. Altogether, our data indicate that cefiderocol, aztreonam/avibactam, aztreonam/nacubactam, cefepime/taniborbactam, cefepime/zidebactam, cefepime/nacubactam, meropenem/xeruborbactam, and meropenem/ANT3310 show promising activity against double-carbapenemase-producing Enterobacterales.

MATERIALS AND METHODS

Clinical isolates

This study included a representative collection of 57 non-duplicated clinical isolates of double-carbapenemase-producing Enterobacterales recovered from patients admitted to 29 Spanish hospitals from 18 different regions during the period 2017–2022. The strains were collected by researchers in three reference laboratories in Northern, Central, and Southern Spain, each of which is involved in a different large-scale surveillance initiative: (i) the Carbapenem-Resistant Enterobacterales (CRE) Genome Database Project (Microbiology Department, Complexo Hospitalario Universitario A Coruña, A Coruña) (33); (ii) the CARB-ES-19 Spanish nationwide survey on carbapenemase-producing Enterobacterales (Spanish Reference Laboratory for Research in Antibiotic Resistance and Healthcare-associated Infections, Centro Nacional de Microbiología, Madrid) (34); and (iii) the Andalusian Reference PIRASOA surveillance Programme for the Prevention and Control of Healthcare-associated Infections (PIRASOA Reference Laboratory, Hospital Universitario Virgen Macarena, Seville) (35). The collection of isolates included 29 Klebsiella pneumoniae, 16 Enterobacter cloacae complex, 10 Citrobacter freundii, 1 Klebsiella oxytoca, and 1 Escherichia coli. In each laboratory, preliminary characterization and detection of double carbapenemase production was performed by phenotypic (such as the mCIM or the Carba NP tests) and molecular methods (PCR + Sanger sequencing) and further confirmed by WGS. The clinical strains and fastq files obtained in each laboratory were finally centralized in one reference center (Microbiology Department, Complexo Hospitalario Universitario A Coruña) for antimicrobial susceptibility testing, WGS resistome analysis and characterization of resistance mechanisms.

Antimicrobial susceptibility testing

MICs of ceftazidime, ceftazidime/avibactam, aztreonam, aztreonam/avibactam, aztreonam/nacubactam, cefiderocol, cefepime, cefepime/taniborbactam, cefepime/zidebactam, cefepime/nacubactam, imipenem, imipenem/relebactam, meropenem, meropenem/vaborbactam, meropenem/xeruborbactam, and meropenem/ANT3310 were determined in triplicate by reference broth microdilution assays with cation-adjusted Müeller-Hinton (MH) broth, and for cefiderocol, iron-depleted cation-adjusted MH broth, prepared according to CLSI M100 guidelines. MIC values were interpreted following EUCAST guidelines (v. 14.0) for Enterobacterales (https://www.eucast.org/clinical_breakpoints). Clinical breakpoints for combinations that have not yet been approved were interpreted using the β-lactam partner. Avibactam, relebactam, and taniborbactam were tested at a fixed concentration of 4 mg/L, while vaborbactam, xeruborbactam, and ANT3310 were determined at 8 mg/L. Zidebactam was fixed at a 1:1 ratio. Nacubactam was tested at a fixed concentration of 4 mg/L with aztreonam and a 1:1 ratio with aztreonam and cefepime. Reference strains Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 700603, Escherichia coli NCTC 13353 and Acinetobacter baumannii NCTC 13304 (for cephalosporin/β-lactamase inhibitor combinations), and K. pneumoniae ATCC BAA-2814 (for carbapenem/β-lactamase inhibitor combinations) were used as controls.

Resistance genomics

The quality control of all paired-end Illumina reads received by the reference laboratory was carried out with fastp (v.0.32.2) (36) and BMTagger (v.3.1) (37). Genome assembly was performed with Unicycler (v.0.5.0) (38). To ascertain high quality, completeness, and the absence of contamination, the assembled genomes were subsequently analyzed with CheckM (v.1.1.3) (39). Identification to species level was performed with Kmerfinder (v.3.2) (40) and MLST (Center for Genomic Epidemiology, v2.0). Putative open reading frames were annotated with Bakta (v.1.7.0) (41), and horizontally acquired resistance determinants were characterized using RGI (v.5.2.0) and CARD (v.3.2.8) webtools. In addition, for those isolates showing resistance to cefiderocol or novel combinations, the presence of chromosomal mutations in genes coding for PBPs, porins, efflux pump regulators, and iron-uptake-related genes was further analyzed via variant calling with Snippy (v.4.6.0) (42) and using the genome of the β-lactam susceptible Klebsiella pneumoniae ATCC 10031 as reference. The natural missense mutations identified in classical chromosomal genes previously related to β-lactam resistance (e.g., mrdA, ftsI, ramA, marA, acrR, tolC) were reviewed and filtered out after screening a representative collection of carbapenemase-producing Enterobacterales with a wild-type antimicrobial susceptibility profile toward new β-lactam/β-lactamase inhibitor combinations (33).

ACKNOWLEDGMENTS

This study was made under the auspices of the Study Group of Mechanisms of Antimicrobial Action and Resistance (GEMARA) from the Spanish Society of Clinical Microbiology and Infectious Diseases (SEIMC) and the Area of Infectious Diseases (CIBERINFEC) from the Centers for Network Biomedical Research (CIBER) of the Spanish National Institute of Health ISCIII.

This work was supported by the Instituto de Salud Carlos III (ISCIII projects: PI20/01212, PI21/00704, PI22/01212 and PI23/00851) and co-funded by the European Union. This work was also supported by Merck Sharp & Dohme (MSD) through the Investigator Initiated Studies Program. The research was also funded by Centro de Investigación Biomédica en Red de Enfermedades Infecciosas (CIBERINFEC: CB21/13/00012, CB21/13/00055, and CB21/13/00095). The study was also funded by the Axencia Galega de Innovación (GAIN), Consellería de Innovación, Consellería de Emprego e Industria (IN607A 2016/22 to G.B., IN607D 2021/12 to A.B. and IN607D 2024/008 to J.A.-S.). This research was also supported by Personalized and precision medicine grant from the Instituto de Salud Carlos III (MePRAM Project, PMP22/00092), Instituto de Salud Carlos III, and Ministerio de Ciencia e Innovación. T.B.-M. was financially supported by the ISCIII project PI20/00686 and by the Río Hortega program (ISCIII, CM23/00095). L.G.-P. was financially supported by the ISCIII project PI21/00704 and by the PFIS program (ISCIII, FI23/00074). I.A.-G. was financially supported by the Río Hortega program (ISCIII, CM21/00076) and by the Juan Rodés program (ISCIII, JR23/00036). S.R.-P. was financially supported by the Río Hortega program (ISCIII, CM23/00104). L.S.-P. was financially supported by Fundación Pública Galega de Investigación Biomédica INIBIC through “Programa de apoyo a la Innovación en Áreas Clínicas 2022–2023" and by Xunta de Galicia (IN606A 2024/022). M.O.-G. was financially supported by Xunta de Galicia (IN606A 2023/023). J.C.V.-U. was financially supported by Xunta de Galicia (IN606B 2022/009). J.A.-S. was financially supported by the Juan Rodés program (ISCIII, JR21/00026).

T.B.-M., I.L.-H., B.A., L.G.-P., P.A.-M., I.A.-G., S.R.-P., L.S.-P., and M.O.-G. performed the research and analyzed the data. M.P.-V., J.C.V.-U., A.P., A.B., G.B., L.L.-C., J.O.-I., and J.A.-S. designed the research, analyzed the data, and wrote the manuscript. All authors read and revised the manuscript.

Contributor Information

Germán Bou, Email: german.bou.arevalo@sergas.es.

Laurent Poirel, University of Fribourg, Fribourg, Switzerland.

DATA AVAILABILITY

The WGS data of the double-carbapenemase-producing strains included in this work have been deposited in GenBank under accession number PRJNA1104021.

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/aac.00924-24.

Table S1. aac.00924-24-s0001.docx.

Bacterial species, sample, β-lactamases, sequence type, and susceptibility to aztreonam and aztreonam-based combinations (at different nacubactam concentrations; 1:1 and 4 mg/L) against clinical isolates of double-carbapenemase-producing Enterobacterales.

aac.00924-24-s0001.docx (33.3KB, docx)
DOI: 10.1128/aac.00924-24.SuF1
Table S2. aac.00924-24-s0002.docx.

MIC50, MIC90, and cumulative MIC distribution for aztreonam and aztreonam-based combinations against clinical isolates of double-carbapenemase-producing Enterobacterales.

aac.00924-24-s0002.docx (19.4KB, docx)
DOI: 10.1128/aac.00924-24.SuF2

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Table S1. aac.00924-24-s0001.docx.

Bacterial species, sample, β-lactamases, sequence type, and susceptibility to aztreonam and aztreonam-based combinations (at different nacubactam concentrations; 1:1 and 4 mg/L) against clinical isolates of double-carbapenemase-producing Enterobacterales.

aac.00924-24-s0001.docx (33.3KB, docx)
DOI: 10.1128/aac.00924-24.SuF1
Table S2. aac.00924-24-s0002.docx.

MIC50, MIC90, and cumulative MIC distribution for aztreonam and aztreonam-based combinations against clinical isolates of double-carbapenemase-producing Enterobacterales.

aac.00924-24-s0002.docx (19.4KB, docx)
DOI: 10.1128/aac.00924-24.SuF2

Data Availability Statement

The WGS data of the double-carbapenemase-producing strains included in this work have been deposited in GenBank under accession number PRJNA1104021.

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