TROJAN-MDR: In vitro activity of cefiderocol and comparators against multidrug-resistant Enterobacterales and Pseudomonas aeruginosa strains in Southern France, evaluation of available testing methods performances

doi:10.21203/rs.3.rs-5326846/v1

Background

Cefiderocol, a newly introduced siderophore cephalosporin, exhibits activity against various multidrug-resistant (MDR) Gram-negative bacilli (GNB), including producers of Ambler class A, B and D carbapenemases. The TROJAN-MDR study aimed to i) compare the in vitro activity of cefiderocol with other last-resort antibiotics against a well-characterized collection of Enterobacterales and Pseudomonas aeruginosa strains from Southern France, and ii) assess the performance of available cefiderocol antimicrobial susceptibility testing (AST) methods.

Methods

The collection comprised 127 Enterobacterales from various clones, including 119 carbapenemase producers (93.7%), and 53 MDR P. aeruginosa. The minimum inhibitory concentrations (MICs) of cefiderocol were determined using the UMIC® broth microdilution method (BMD) as the reference. Comparators MICs were measured using Sensititre™ EUMDRXXF plates and Liofilchem strips for aztreonam-avibactam. Results were interpreted according to EUCAST breakpoints, with CLSI breakpoints also used for cefiderocol. The performance of the ComASP® BMD and disk diffusion on two different Mueller-Hinton media (Bio-Rad and BD) were evaluated according to ISO 20776-2:2007 and 2021.

Results

Cefiderocol demonstrated potent activity on Enterobacterales (81.9% susceptible) and P. aeruginosa (84.9%) using EUCAST breakpoints. Among Enterobacterales, the most effective comparators were colistin, aztreonam-avibactam, meropenem-vaborbactam, and amikacin, with susceptibility rates of 99.2%, 98.4%, 85%, and 76.4%, respectively. For P. aeruginosa, only colistin exhibited better activity (100%). The disk diffusion method showed superior performance on BD medium compared to Bio-Rad. The ComASP® method did not provide sufficient performance to be considered reliable.

Conclusions

Cefiderocol was highly active against a large collection of MDR GNB, including high-risk clones. It is crucial to assess susceptibility to this last-resort antibiotic using a validated method when considering clinical use.

Cefiderocol

multi-drug resistance

Enterobacterales

Pseudomonas aeruginosa

difficult-to-treat

carbapenemase

aztreonam-avibactam

antimicrobial susceptibility testing

UMIC®

ComASP®

Carbapenemase- and/or extended spectrum β-lactamase (ESBL)-producing Enterobacterales, carbapenem-resistant Pseudomonas aeruginosa, and Acinetobacter baumannii are on the WHO's list of priority bacteria for which new active antibiotics are urgently needed [1]. The incidence of infections caused by these bacteria is increasing, and with treatment options rapidly depleting, they are associated with increased morbidity, mortality, and associated costs [2].

Cefiderocol, a recently developed siderophore β-lactam, is used as a last-resort antibiotic in the treatment of infections caused by aerobic Gram-negative bacteria in adult patients with limited therapeutic options [3]. Its innovative structure, a hybrid of cefepime and ceftazidime combined with a siderophore, enables it to penetrate bacterial cells like a Trojan horse, via both porins- and siderophores-mediated pathways [4]. Additionally, this unique structure increases its stability against β-lactamases and efflux pumps, enhancing its antibacterial activity [4].

Consequently, cefiderocol has a broad spectrum of activity against Gram-negative bacilli (GNB) such as Enterobacterales, P. aeruginosa, and A. baumannii, including multidrug-resistant (MDR) strains. It remains active in cases of porin deficiency, upregulation of efflux pumps, and production of various β-lactamases, including active serine carbapenemases and metallo-enzymes [5]. This molecule joins the list of last-resort antibiotics that can be used in the treatment of MDR-GNB infections, including ceftolozane-tazobactam, ceftazidime-avibactam, imipenem-relebactam, meropenem-vaborbactam, aztreonam-avibactam, colistin, tigecycline, and eravacycline [3].

To ensure treatment efficacy and increase the chances of clinical success, it is crucial to assess the in vitro susceptibility of cefiderocol using a reliable method [5]. Broth microdilution (BMD) for MIC determination is the commonly accepted reference method for antimicrobial susceptibility testing (AST) [6, 7]. Given the importance of iron in the mechanism of action of cefiderocol, iron-depleted Mueller-Hinton broth is required to mimic in vivo conditions. The classical agar diffusion method using disks has also been proposed, but some studies have reported that its performances did not meet the recommendations of the International Organization for Standardization (ISO) [8, 9].

In this study, we aimed to i) compare the activity of cefiderocol with that of last-resort antibiotics on a panel of various clones of MDR-GNB strains isolated in the South of France and ii) evaluate the performance of different AST methods to assess the in vitro activity of cefiderocol.

Bacterial isolates

We selected 180 MDR-GNB isolates from the Regional Reference Laboratory of Occitania in the South of France (LBMR BHRe, Nîmes University Hospital). These isolates belong to various clones, and their resistome had been previously characterized. The panel comprised i) 127 Enterobacterales, including 119 carbapenemase-producing Enterobacterales (CPE) of classes A (n = 13), B (n = 32), D (n = 63), B + D (n = 10) or A + D (n = 1), as well as 8 non-CPE isolates (ESBL-producing Enterobacterales (ESBL-E), derepressed AmpC ± decreased permeability), and ii) 53 MDR P. aeruginosa strains, including 6 metallo-β-lactamase (MBL) producers (Table 1 and Table 2).

Table 1

Characteristics of the Enterobacterales isolates.
Resistance mechanism (n. of strains)		Species (n. of strains)	Associated β-lactamases (n. of strains)	STs (n. of strains)
Class A (13)	KPC-2 (9)	K. pneumoniae (1)	KLUY-1 (1)	258 (1)
		E. coli (1)	None	744 (1)
		C. freundii (3)	CMY-48 (3), CTX-M-15 (1)	22 (3)
		E. cloacae complex (4)	ACT-15 (1), ACT-23 (1), ACT-59 (1), ACT-67 (1)	106 (1), 286 (1), 873 (1), new ST (1)
	KPC-3 (4)	K. pneumoniae (4)	CTX-M-15 (1)	11 (1), 147 (3)
Class A + D (1)	KPC-2 + OXA-48 (1)	C. freundii (1)	CMY-48 (1)	22 (1)
Class B (32)	NDM-1 (20)	K. pneumoniae (16)	CTX-M-15 (10)	11 (1), 17 (1), 147 (6), 247 (5), 273 (1), 307 (1), 2084 (1)
		C. freundii (1)	CMY-65 (1)	91 (1)
		E. cloacae complex (3)	ACT-23 (2), CMY-4 (1), CTX-M-15 (1)	102 (2), 749 (1)
	NDM-4 (1)	K. pneumoniae (1)	CTX-M-15 (1)	16 (1)
	NDM-5 (5)	K. pneumoniae (2)	CTX-M-15 (1)	219 (1), 785 (1)
		C. farmeri (1)	CTX-M-15 (1)	NA
		E. coli (2)	CTX-M-15 (2)	167 (1), 361 (1)
	NDM-14 (2)	K. pneumoniae (2)	CTX-M-15 (2)	147 (2)
	NDM-19 (1)	E. coli (1)	None	38 (1)
	VIM-1 (1)	E. cloacae complex (1)	ACT-24 (1), CTX-M-9 (1)	118 (1)
	VIM-4 (2)	E. cloacae complex (2)	ACT-23 (2)	78 (1), 168 (1)
Class B + D (10)	NDM-1 + OXA-48 (4)	K. pneumoniae (4)	CTX-M-15 (3)	147 (2), 2084 (2)
	NDM-5 + OXA-48 (2)	K. pneumoniae (2)	CTX-M-15 (1)	383 (2)
	NDM-5 + OXA-181 (3)	K. pneumoniae (2)	CTX-M-15 (2)	147 (1), new ST (1)
	NDM-5 + OXA-181 (3)	E. cloacae complex (1)	ACT-73 (1), CTX-M-15 (1)	116 (1)
	NDM-7 + OXA-48 (1)	E. cloacae complex (1)	ACT-25 (1), CTX-M-15 (1)	121 (1)
Class D (63)	OXA-48 (59)	K. pneumoniae (18)	CTX-M-15 (6), DHA-1 (1)	12 (1), 13 (3), 15 (1), 29 (1), 37 (1), 147 (1), 307 (4), 405 (1), 2074 (1), 2084 (2), 2674 (1), 3167 (1)
		E. coli (2)	None	38 (1), 648 (1)
		C. freundii (11)	ACT-25 (1), CMY-48 (8), CMY-79 (1), CTX-M-15 (8), DHA-7 (1), SHV-12 (1)	8 (1), 22 (4), 107 (1), 216 (4), 261 (1)
		C. farmeri (1)	None (1)	NA
		E. cloacae complex (27)	ACT-15 (6), ACT-16 (1), ACT-23 (1), ACT-24 (6), ACT-25 (2), ACT-45 (4), ACT-46 (1), ACT-59 (3), ACT-72 (2), ACT-73 (1), DHA-7 (1), MIR-6 (1), CTX-M-15 (12), KLUB-1 (4)	66 (1), 78 (6), 90 (2), 106 (3), 109 (1), 113 (1), 114 (1), 116 (1), 121 (1), 168 (1), 171 (3), 279 (1), 418 (1), 595 (1), 873 (3)
	OXA-181 (4)	K. pneumoniae (3)	CTX-M-15 (2)	16 (1), 4988 (2)
	OXA-181 (4)	E. cloacae complex (1)	ACT-25 (1)	418 (1)
Non-CPE (8)		K. pneumoniae (4)	CTX-M-15 (3), outer membrane decreased permeability (1)	11 (1), 13 (1), 247 (1), new ST (1)
		C. freundii (1)	CMY-109 (1), CTX-M-15 (1), outer membrane decreased permeability (1)	98 (1)
		E. cloacae complex (2)	ACT-3 (1), ACT-59 (1), CTX-M-15 (1), KLUB-1 (1), outer membrane decreased permeability (2)	252 (1), new ST (1)
		K. aerogenes (1)	AmpC (1), outer membrane decreased permeability (1)	137 (1)
CPE, Carbapenemase-Producing Enterobacterales; ST, Sequence Type.

Table 2

Characteristics of the *Pseudomonas aeruginosa* isolates.
Resistance mechanism (n. of strains)		Associated β-lactamases (n. of strains)	STs (n. of strains)
Class B CP (6)	IMP-1 (2)	OXA-35 (1), OXA-488 (1), OXA-904 (1), PDC-35 (1), PDC-119 (1)	235 (1)
	IMP-26 (1)	OXA-488 (1), PDC-35 (1)	235 (1)
	VIM-2 (1)	OXA-488 (1), PDC-35 (1)	35 (1)
	VIM-4 (1)	OXA-10 (1), OXA-488 (1), PDC-19a (1)	308 (1)
	NDM-1 (1)	OXA-488 (1), PDC-19a (1)	308 (1)
Non-CP (47)		OXA-35 (1), OXA-50 (6), OXA-395 (6), OXA-396 (9), OXA-488 (9), OXA-494 (1), OXA-846 (1), OXA-847 (9), OXA-902 (1), OXA-904 (3), OXA-913 (2), PDC-3 (2), PDC-15 (1), PDC-16 (2), PDC-19a (3), PDC-19b (1), PDC-24 (1), PDC-25 (1), PDC-30 (1), PDC-34 (1), PDC-35 (5), PDC-37 (2), PDC-43 (3), PDC-58 (9), PDC-60 (2), PDC-80 (1), PDC-109 (1), PDC-114 (1), PDC-120 (1), PDC-172 (1), PDC-189 (1), PDC-198 (1), PDC-303 (2), PDC-321 (3), PDC-394 (1)	27 (1), 175 (3), 207 (1), 235 (5), 244 (2), 253 (1), 267 (1), 308 (2), 309 (1), 313 (2), 446 (2), 609 (1), 611 (2), 654 (9), 679 (1), 1182 (1), 1184 (1), 1330 (1), 1613 (1), 1616(1), 1755 (1), 2128 (2), 2475 (1), 2844 (1), 2996 (1), new ST (2)
CP, Carbapenemase-Producers; ST, Sequence Type.

The vast majority of strains (n = 128, 71.1%) were isolated from diagnostic samples: respiratory samples (n = 42, 23.3%), urines (n = 33, 18.3%), blood (n = 26, 14.4%), abscesses (n = 21, 11.7%), bone (n = 4, 2.2%), catheter (n = 1, 0.6%), and cerebrospinal fluid (n = 1, 0.6%) (Table 3). Among the Enterobacterales, the most represented species were K. pneumoniae (n = 59, 46.5%), followed by the Enterobacter cloacae complex (n = 42, 33.1%), Citrobacter freundii (n = 17, 13.4%), Escherichia coli (n = 6, 4.7%), Citrobacter farmeri (n = 2, 1.6%), and Klebsiella aerogenes (n = 1, 0.8%).

Table 3

Origin of the strains included in the study.
Specimen	Enterobacterales							Pseudomonas aeruginosa				Total
Specimen	KPC	KPC + OXA-48-like	NDM	VIM	NDM + OXA-48-like	OXA-48-like	Non-CP	NDM	VIM	IMP	Non-CP	Total
Rectal swab	6	1	12	2	4	24	2			1		52
Respiratory samples			4			6	3				29	42
Urine	3		7	1	2	20						33
Blood	2		4		2	6	3		1	1	7	26
Abcess	2		2		2	5		1	1	1	7	21
Bone						1					3	4
Cerebrospinal fluid											1	1
Catheter						1						1
Total	13	1	29	3	10	63	8	1	2	3	47	180
CP: Carbapenemase-producers.

Control strains were associated with each set of AST: E. coli from American Type Culture Collection (ATCC) 25922 for Enterobacterales and P. aeruginosa ATCC 27853 for P. aeruginosa.

Comparators susceptibility testing

The susceptibility of the strains to various antibiotics was determined by the classic agar diffusion method on Mueller-Hinton (MH) medium (Bio-Rad, Marne-la-Coquette, France), in accordance with EUCAST recommendations. The MICs of comparators (ceftolozane-tazobactam, ceftazidime-avibactam, imipenem-relebactam, meropenem-vaborbactam, tigecycline, eravacycline, amikacin, fosfomycin and colistin) were measured by BMD using the Sensititre™ EUMDRXXF plate (ThermoFisher, Les Ulys, France), following the manufacturer's recommendations. The MICs of aztreonam-avibactam were determined with the recently marketed strips (Liofilchem, Roseto degli Abruzzi, Italia).

Results were interpreted according to EUCAST breakpoints [10].

Cefiderocol susceptibility testing

The MICs of cefiderocol were determined by BMD using two different commercial tests, with the same 0.5 McFarland bacterial inoculum prepared for agar diffusion. For the UMIC® test (Bruker), 25 µL of the prepared suspension were added to the iron-depleted, cation-adjusted Mueller-Hinton broth medium provided (ID-CAMHB, 5 mL), and then 100 µL was distributed to each well of the strip. For the ComASP® BMD (Liofilchem), the 0.5 McFarland suspension was diluted to 1:20 in sterile 0.9% NaCl, then 400 µL of this dilution was added to the ID-CAMHB medium provided (3.6 mL), and 100 µL was dispensed into each well of the plate. In both cases, the MIC value was read by two different readers after 18–20 hours of incubation at 35°C. Results were interpreted according to both EUCAST and CLSI breakpoints [7, 10].

Furthermore, susceptibility to cefiderocol was assessed by the diffusion method using 30 µg cefiderocol disks (Mast group) on two different MH media: Bio-Rad MH Agar and BD BBL™ MH II Agar.

The UMIC® testing, approved by the French national reference centers, was considered as the gold standard [11–13].

Clinical performance of cefiderocol AST

The performance of the ComASP® BMD method and disk diffusion, using Bio-Rad and BD BBL™ MH agar media, was evaluated according to EUCAST 2024 breakpoints [10]. This evaluation involved the calculation of the Categorical Agreement (CA), Essential Agreement (EA), Major Errors (ME), Very Major Errors (VME), inferior and superior bias.

Acceptable performance was defined by the following criteria, as recommended by the ISO 20776-2:2007 and 2021 standards: CA and EA ≥ 90%, VME ≤ 1.5%, ME ≤ 3%, and the difference for bias within ± 30% [14, 15].

Overall in vitro activity of cefiderocol against MDR-GNB

Cefiderocol showed potent in vitro activity when tested using the BMD approach, which is approved by the French reference centers (UMIC®) as the reference AST method. When interpreted using the EUCAST breakpoints, susceptibility was observed in 81.9% (104/127) of Enterobacterales and 84.9% (45/53) of P. aeruginosa MDR strains. Using the CLSI breakpoints, the susceptibility increased to 87.4% (111/127) for Enterobacterales and 94.3% (51/53) for P. aeruginosa (Table 4).

Table 4

Overall *in vitro* activity of cefiderocol against MDR Enterobacterales and *P. aeruginosa*.
Strains (n. of isolates)	MICs range (mg/L)	MIC₅₀ (mg/L)	MIC₉₀ (mg/L)	Clinical categorization
				Breakpoints		S	I	R
Enterobacterales (127)	≤ 0,03 - > 32	1	8	N. of isolates (%)	EUCAST	104 (81.9)	NA	23 (18.1)
					CLSI	111 (87.4)	8 (6.3)	8 (6.3)
P. aeruginosa (53)	0.125–32	1	4	N. of isolates (%)	EUCAST	45 (84.9)	NA	8 (15.1)
					CLSI	50 (94.3)	1 (1.9)	2 (3.8)
S, Susceptible; I, susceptible, Increased exposure; R, Resistant. NA, Non-Applicable

In vitro activity of cefiderocol and comparators against Enterobacterales

Table 5 presents the in vitro activity of cefiderocol and its comparators against the 127 strains of Enterobacterales included in this study. Table 6 details the characteristics of cefiderocol-resistant strains.

Table 5

**Activity of cefiderocol and its comparators against MDR Enterobacterales and** **P. aeruginosa** **from South of France**.
Resistance mechanism (Number of strains)		Antibiotic	MIC range (mg/L)	MIC₅₀ (mg/L)	MIC₉₀ (mg/L)	Clinical interpretation (%)
Resistance mechanism (Number of strains)		Antibiotic	MIC range (mg/L)	MIC₅₀ (mg/L)	MIC₉₀ (mg/L)		S	I	R
Enterobacterales (n = 127)
Total (127)		Cefiderocol	≤ 0.03 - >32	1	8	EUCAST	81.9	NA	18.1
		Cefiderocol	≤ 0.03 - >32	1	8	CLSI	87.4	6.3	6.3
		Piperacillin-tazobactam	> 32	> 32	> 32		0	NA	100
		Cefepime	≤ 1 - >16	16	> 16		19.7	12.6	67.7
		Ceftolozane-tazobactam	0.5 - >8	> 8	> 8		15.7	NA	84.3
		Ceftazidime-avibactam	≤ 0.25 - >16	1	> 16		65.4	NA	34.6
		Aztreonam	≤ 1 - > 32	> 32	> 32		25.2	1.6	73.2
		Aztreonam-avibactam	0.023–24	0.125	0.75		98.4	NA	1.6
		Imipenem	≤ 1 - >8	2	> 8		59.8	11.8	28.4
		Imipenem-relebactam	0.12 - >8	1	> 8		68.5	NA	31.5
		Meropenem	≤ 0.12 - >16	2	> 16		54.3	29.9	15.8
		Meropenem-vaborbactam	≤ 0.06 - >16	1	16		85	NA	15
		Amikacin	≤ 2 - >32	2	> 32		76.4	NA	23.6
		Tobramycin	≤ 0.5 - >4	> 4	> 4		34.6	NA	65.4
		Fosfomycin^a	≤ 16 - >64	32	> 64		69.3	NA	30.7
		Tigecycline^b	≤ 0.5 - >1	≤ 0.5	> 1		63.8	NA	36.4
		Eravacycline^c	0.06 - >0.5	0.5	> 0.5		66.1	NA	33.9
		Colistin	0.25 - >16	≤ 0.5	≤ 0.5		99.2	NA	0.8
Class A	KPC (13)	Cefiderocol	0.125–2	1	2	EUCAST	100	NA	0
		Cefiderocol	0.125–2	1	2	CLSI	100	0	0
		Piperacillin-tazobactam	> 32	> 32	> 32		0	NA	100
		Cefepime	2 - >16	8	> 16		0	23.1	76.9
		Ceftolozane-tazobactam	4 - >8	> 8	> 8		0	NA	100
		Ceftazidime-avibactam	≤ 0.25–2	1	1		100	NA	0
		Aztreonam	16 - >32	> 32	> 32		0	0	100
		Aztreonam-avibactam	0.032–0.75	0.19	0.5		100	0	0
		Imipenem	≤ 1 - >8	4	8		38.4	30.8	30.8
		Imipenem-relebactam	0.12–0.5	0.25	0.5		100	NA	0
		Meropenem	1 - >16	4	16		38.4	38.4	23.2
		Meropenem-vaborbactam	≤ 0.06–0.5	≤ 0.06	0.12		100	NA	0
		Amikacin	≤ 2 - >32	8	> 32		69.2	NA	30.8
		Tobramycin	≤ 0.5 - >4	> 4	> 4		23.1	NA	76.9
		Fosfomycin^a	≤ 16 - >64	≤ 16	64		69.2	NA	30.8
		Tigecycline^b	≤ 0.5 - >1	≤ 0.5	1		76.9	NA	23.1
		Eravacycline^c	0.25 - >0.5	0.5	> 0.5		76.9	NA	23.1
		Colistin	≤ 0.5 - >16	≤ 0.5	≤ 0.5		92.3	NA	7.7
Class A + D	KPC + OXA-48 (1)	Cefiderocol	NC	NC	NC	EUCAST	100	NA	0
		Cefiderocol	NC	NC	NC	CLSI	100	0	0
		Piperacillin-tazobactam	NC	NC	NC		0	NA	100
		Cefepime	NC	NC	NC		0	0	100
		Ceftolozane-tazobactam	NC	NC	NC		0	NA	100
		Ceftazidime-avibactam	NC	NC	NC		100	NA	0
		Aztreonam	NC	NC	NC		0	0	100
		Aztreonam-avibactam	NC	NC	NC		100	0	0
		Imipenem	NC	NC	NC		100	0	0
		Imipenem-relebactam	NC	NC	NC		100	NA	0
		Meropenem	NC	NC	NC		100	0	0
		Meropenem-vaborbactam	NC	NC	NC		100	NA	0
		Amikacin	NC	NC	NC		100	NA	0
		Tobramycin	NC	NC	NC		0	NA	100
		Fosfomycin^a	NC	NC	NC		100	NA	0
		Tigecycline^b	NC	NC	NC		0	NA	100
		Eravacycline^c	NC	NC	NC		0	NA	100
		Colistin	NC	NC	NC		100	NA	0
Class B	NDM (29)	Cefiderocol	0.5 - >32	2	8	EUCAST	79.3	NA	20.7
		Cefiderocol	0.5 - >32	2	8	CLSI	86.2	6.9	6.9
		Piperacillin-tazobactam	> 32	> 32	> 32		0	NA	100
		Cefepime	2 - >16	> 16	> 16		0	6.9	93.1
		Ceftolozane-tazobactam	> 8	> 8	> 8		0	NA	100
		Ceftazidime-avibactam	> 16	> 16	> 16		0	NA	100
		Aztreonam	≤ 1 - >32	> 32	> 32		34.5	0	65.5
		Aztreonam-avibactam	0.032–1.5	0.25	0.75		100	NA	0
		Imipenem	≤ 1 - >8	8	> 8		17.2	20.7	62.1
		Imipenem-relebactam	0.5 - >8	8	> 8		17.2	NA	82.8
		Meropenem	0.25 - >32	8	> 16		17.3	37.9	44.8
		Meropenem-vaborbactam	0.25 - >16	8	> 16		58.6	NA	41.4
		Amikacin	≤ 2 - >32	16	> 32		44.8	NA	55.2
		Tobramycin	≤ 0.5 - >4	> 4	> 4		13.8	NA	86.2
		Fosfomycin^a	≤ 16 - >64	32	64		65.5	NA	34.5
		Tigecycline^b	≤ 0.5 - >1	≤ 0.5	> 1		72.4	NA	27.6
		Eravacycline^c	0.06 - >0.5	0.5	> 0.5		82.8	NA	17.2
		Colistin	≤ 0.5–2	≤ 0.5	1		100	NA	0
	VIM (3)	Cefiderocol	1–2	NC	NC	EUCAST	100	NA	0
		Cefiderocol	1–2	NC	NC	CLSI	100	0	0
		Piperacillin-tazobactam	> 32	NC	NC		0	NA	100
		Cefepime	4 - >16	NC	NC		0	33.3	66.7
		Ceftolozane-tazobactam	> 8	NC	NC		0	NA	100
		Ceftazidime-avibactam	16 - >16	NC	NC		0	NA	100
		Aztreonam	≤ 1–16	NC	NC		33.3	33.3	33.3
		Aztreonam-avibactam	0.125 - ≤1	NC	NC		100	0	0
		Imipenem	2–4	NC	NC		66.7	33.3	0
		Imipenem-relebactam	2–4	NC	NC		33.3	NA	66.7
		Meropenem	1–2	NC	NC		100	0	0
		Meropenem-vaborbactam	1–2	NC	NC		100	NA	0
		Amikacin	≤ 2–16	NC	NC		66.7	NA	33.3
		Tobramycin	> 4	NC	NC		0	NA	100
		Fosfomycin^a	≤ 16–32	NC	NC		100	NA	0
		Tigecycline^b	≤ 0.5	NC	NC		100	NA	0
		Eravacycline^c	0.25 - >0.5	NC	NC		66.7	NA	33.3
		Colistin	≤ 0.5	NC	NC		100	NA	0
	Overall class B (32)	Cefiderocol	0.5 - >32	1	8	EUCAST	81.3	NA	18.7
		Cefiderocol	0.5 - >32	1	8	CLSI	87.6	6.2	6.2
		Piperacillin-tazobactam	> 32	> 32	> 32		0	NA	100
		Cefepime	2 - >16	> 16	> 16		0	9.4	90.6
		Ceftolozane-tazobactam	8 - >8	> 8	> 8		0	NA	100
		Ceftazidime-avibactam	16 - >16	> 16	> 16		0	NA	100
		Aztreonam	> 32	> 32	> 32		34.4	3.1	62.5
		Aztreonam-avibactam	0.032–1.5	0.125	0.75		100	NA	0
		Imipenem	≤ 1 - >8	8	> 8		21.9	21.9	56.2
		Imipenem-relebactam	0.5 - >8	8	> 8		18.7	NA	81.3
		Meropenem	0.25–32	8	> 16		28.1	40.6	31.2
		Meropenem-vaborbactam	0.25 - >16	4	> 16		62.5	NA	37.5
		Amikacin	≤ 2 - >32	16	> 32		46.9	NA	53.1
		Tobramycin	≤ 0.5 - >4	> 4	> 4		12.5	NA	87.5
		Fosfomycin^a	≤ 16 - >64	32	64		68.8	NA	31.2
		Tigecycline^b	≤ 0.5 - >1	≤ 0.5	1		75	NA	25
		Eravacycline^c	0.06 - >0.5	0.5	> 0.5		81.2	NA	18.8
		Colistin	≤ 0.5–2	≤ 0.5	0.5		100	NA	0
Class B + D	NDM + OXA-48-like (10)	Cefiderocol	0.125–4	2	2	EUCAST	90	NA	10
		Cefiderocol	0.125–4	2	2	CLSI	100	0	0
		Piperacillin-tazobactam	> 32	> 32	> 32		0	NA	100
		Cefepime	8 - >16	> 16	> 16		0	0	100
		Ceftolozane-tazobactam	> 8	> 8	> 8		0	NA	100
		Ceftazidime-avibactam	> 16	> 16	> 16		0	NA	100
		Aztreonam	≤ 1 - >32	> 32	> 32		20	0	80
		Aztreonam-avibactam	0.064–2	0.125	0.5		100	NA	0
		Imipenem	2 - >8	8	> 8		10	10	80
		Imipenem-relebactam	2 - >8	8	> 8		10	NA	90
		Meropenem	4 - >32	16	> 32		0	40	60
		Meropenem-vaborbactam	2 - >16	16	> 16		40	NA	60
		Amikacin	≤ 2 - >32	4	> 32		60	NA	40
		Tobramycin	≤ 0.5 - >4	> 4	> 4		10	NA	90
		Fosfomycin^a	≤ 16 - >64	32	> 64		60	NA	40
		Tigecycline^b	≤ 0.5 - >1	1	1		20	NA	80
		Eravacycline^c	0.12 - >0.5	> 0.5	> 0.5		30	NA	70
		Colistin	≤ 0.5	≤ 0.5	≤ 0.5		100	NA	0
Class D	OXA-48-like (63)	Cefiderocol	≤ 0.03–16	1	8	EUCAST	84.1	NA	15.9
		Cefiderocol	≤ 0.03–16	1	8	CLSI	87.2	6.4	6.4
		Piperacillin-tazobactam	> 32	> 32	> 32		0	NA	100
		Cefepime	≤ 1 - >16	4	> 16		34.9	19.1	46
		Ceftolozane-tazobactam	0.5 - >8	> 8	> 8		28.6	NA	71.4
		Ceftazidime-avibactam	≤ 0.25–16	0.5	2		98.4	NA	1.6
		Aztreonam	≤ 1 - >32	32	> 32		30.2	1.6	68.2
		Aztreonam-avibactam	0.023–24	0.125	0.75		98.4	NA	1.6
		Imipenem	≤ 1 - >8	≤ 1	4		87.3	4.8	7.9
		Imipenem-relebactam	0.12 - >8	1	2		92.1	NA	7.9
		Meropenem	0.5 - >16	1	4		76.2	22.2	1.6
		Meropenem-vaborbactam	0.25 - >16	1	4		98.4	NA	1.6
		Amikacin	≤ 2 - >32	≤ 2	8		92.1	NA	7.9
		Tobramycin	≤ 0.5 - >4	2	> 4		52.4	NA	47.6
		Fosfomycin^a	≤ 16 - >64	≤ 16	> 64		76.2	NA	23.8
		Tigecycline^b	≤ 0.5 - >1	≤ 0.5	> 1		63.5	NA	36.5
		Eravacycline^c	0.12 - >0.5	0.5	> 0.5		63.5	NA	36.5
		Colistin	≤ 0.25–1	≤ 0.5	≤ 0.5		100	NA	0
Non CPE (8)		Cefiderocol	0.5 - >32	NC	NC	EUCAST	25	NA	75
		Cefiderocol	0.5 - >32	NC	NC	CLSI	50	25	25
		Piperacillin-tazobactam	> 32	NC	NC		0	NA	100
		Cefepime	4 - >16	NC	NC		0	12.5	87.5
		Ceftolozane-tazobactam	0.5 - >8	NC	NC		25	NA	75
		Ceftazidime-avibactam	≤ 0.25 - >16	NC	NC		87.5	NA	12.5
		Aztreonam	8 - >32	NC	NC		0	0	100
		Aztreonam-avibactam	0.032–16	NC	NC		87.5	NA	12.5
		Imipenem	≤ 1–8	NC	NC		87.5	0	12.5
		Imipenem-relebactam	0.25–2	NC	NC		100	NA	0
		Meropenem	≤ 0.12–8	NC	NC		75	25	0
		Meropenem-vaborbactam	≤ 0.06–4	NC	NC		100	NA	0
		Amikacin	≤ 2–8	NC	NC		100	NA	0
		Tobramycin	≤ 0.5 - >4	NC	NC		37.5	NA	62.5
		Fosfomycin^a	≤ 16 - >64	NC	NC		37.5	NA	62.5
		Tigecycline^b	≤ 0.5 - > 1	NC	NC		62.5	NA	37.5
		Eravacycline^c	0.25 - >0.5	NC	NC		62.5	NA	37.5
		Colistin	≤ 0.5	NC	NC		100	NA	0
Pseudomonas aeruginosa (n = 53)
Overall (53)		Cefiderocol	0.125–32	1	4	EUCAST	84.9	NA	15.1
		Cefiderocol	0.125–32	1	4	CLSI	94.3	1.9	3.8
		Piperacillin-tazobactam	16 - >32	> 32	> 32		0	7.5	92.5
		Cefepime	4 - >16	> 16	> 16		0	15.1	84.9
		Ceftolozane-tazobactam	0.5 - >8	4	> 8		56.6	NA	43.4
		Ceftazidime-avibactam	1 - >16	8	> 16		52.8	NA	47.2
		Aztreonam	2 - >32	32	> 32		0	18.9	81.1
		Aztreonam-avibactam^d	2 - >256	16	> 256		0	52.8	47.2
		Imipenem	≤ 1 - >8	> 8	> 8		0	30.2	69.8
		Imipenem-relebactam	0.25 - >8	2	> 8		56.6	NA	43.4
		Meropenem	0.5 - >32	16	> 16		13.2	35.8	51
		Meropenem-vaborbactam	0.5 - >16	16	> 16		47.2	NA	52.8
		Amikacin	≤ 2 - >32	16	> 32		67.9	NA	32.1
		Tobramycin	≤ 0.5 - >4	2	> 4		52.8	NA	47.2
		Fosfomycin^e	≤ 16 - >64	> 64	> 64		NA	NA	NA
		Colistin	≤ 0.5–2	2	2		100	NA	0
Class B carbapenemase producers (6)		Cefiderocol	0.5–16	NC	NC	EUCAST	66.7	NA	33.3
		Cefiderocol	0.5–16	NC	NC	CLSI	83.3	0	16.7
		Piperacillin-tazobactam	32 - >32	NC	NC		0	0	100
		Cefepime	> 16	NC	NC		0	0	100
		Ceftolozane-tazobactam	> 8	NC	NC		0	NA	100
		Ceftazidime-avibactam	> 16	NC	NC		0	NA	100
		Aztreonam	4 - >32	NC	NC		0	50	50
		Aztreonam-avibactam^d	3–256	NC	NC		0	66.7	33.3
		Imipenem	> 8	NC	NC		0	0	100
		Imipenem-relebactam	> 8	NC	NC		0	NA	100
		Meropenem	> 16	NC	NC		0	0	100
		Meropenem-vaborbactam	> 16	NC	NC		0	NA	100
		Amikacin	16 - >32	NC	NC		16.7	NA	83.3
		Tobramycin	> 4	NC	NC		0	NA	100
		Fosfomycin^e	32 - >64	NC	NC		NA	NA	NA
		Colistin	1–2	NC	NC		100	NA	0
S, Susceptible; I, susceptible, Increased exposure; R, Resistant. NA, Non-Applicable; NC, Not Calculated because the number of strains was less than 10.
^aIntravenous breakpoints. ^bE. coli and Citrobacter koseri breakpoints. ^cE. coli breakpoints. ^dAztreonam breakpoints. ^eNo clinical breakpoints.

Table 6

Characteristics of the resistant strains according to EUCAST breakpoints.
Species (number of strains)	Resistance mechanism	Strain	β-lactamases content and other β-lactams resistance proteins	ST	Cefiderocol MIC (mg/L)
K. pneumoniae (9)	Class B	1866	NDM-5, SHV-77, LAP-2, TEM-1, OmpK36 mutations	25	16
	Class B	1976	NDM-5, CTX-M-15, SHV-223, TEM-1, OmpK36 mutations	219	8
	Class B + D	1724	NDM-1, OXA-48, CTX-M-15, OXA-1, SHV-11, TEM-1, OmpK36 mutations	2084	4
	Class D	1966	OXA-181, CTX-M-15, SHV-28, OmpK36 mutations	4988	16
		2069	OXA-181, CTX-M-15, SHV-28, OmpK36 mutations	4988	16
		2205	OXA-48, SHV-40, OmpK36 mutations	2074	4
	Non-CP	1802	CTX-M-15, OXA-1, SHV-101, OmpK36 mutations	13	4
		1957	SHV-1, OmpK36 mutations	New ST	4
		2081	CTX-M-15, OXA-1, SHV-11, TEM-1	11	8
E. cloacae complex (7)	Class B	1248	NDM-1, ACT-23, CTX-M-15, OXA-1, OXA-10	102	4
	Class B	1742	NDM-1, ACT-23, CMY-4, OXA-1, SHV-12, TEM-1	102	8
	Class D	1618	OXA-48, ACT-15, CTX-M-15	106	16
		1787	OXA-48, ACT-43	90	8
		1967	OXA-48, ACT-59, DHA-7	873	4
	Non-CP	1585	ACT-3, KLUB-1, TEM-1, OmpE35 and OmpE36 mutations	252	16
	Non-CP	2082	ACT-59, CTX-M-15, OXA-1, OmpE35 and OmpE36 mutations	New ST	8
C. freundii (5)	Class B	1926	NDM-1, CMY-65, TEM-1	91	4
	Class D	1545	OXA-48, CMY-48, OXA-4, TEM-1	216	8
		1583	OXA-48, CMY-48, CTX-M-15, OXA-1, TEM-1	216	8
		1668	OXA-48, CMY-48, DHA-7, CTX-M-15, SHV-12, OXA-1, TEM-1	216	16
		1928	OXA-48, CMY-48, OXA-13	216	8
K. aerogenes (1)	Non-CP	2265	AmpC, Omp36 mutations	137	> 32
E. coli (1)	Class B	2280	NDM-5, EC-15, CTX-M-15, TEM-1	167	> 32
P. aeruginosa (8)	Class B	1113	IMP-26, PDC-35, OXA-488, OprD mutations	235	16
	Class B	2309	NDM-1, PDC-19a, OXA-488, OprD mutations	308	4
	Non-CP	1764	PDC-120, OXA-50, OprD mutations	2996	8
		1820	PDC-394, OXA-847, OprD mutations	1613	4
		1869	PDC-35, OXA-488, OXA-35, OprD mutations	235	4
		2010	PDC-321, OXA-50, OprD mutations	175	4
		2111	PDC-321, OXA-50, OprD mutations	175	4
		2124	PDC-43, OXA-488	267	32
CP, Carbapenemase-producers; ST, Sequence Type

Overall, cefiderocol demonstrated high activity against Enterobacterales, with 81.9% (104 isolates) and 87.4% (111 isolates) of the strains deemed susceptible according to the EUCAST and CLSI breakpoints, respectively. The MIC₅₀/MIC₉₀ was 1/8 mg/L. Among the comparators, colistin, aztreonam-avibactam, and meropenem-vaborbactam were the most effective agents, with susceptibility rates of 99.2%, 98.4%, and 85%, respectively. The MIC₅₀/MIC₉₀ values were ≤ 0.5/≤0.5 mg/L, 0.125/0.75 mg/L, and 1/16 mg/L, respectively. Amikacin's activity was comparable to that of cefiderocol, with 76.4% of the strains being susceptible and a MIC₅₀/MIC₉₀ of 2/>32 mg/L. Fosfomycin, imipenem-relebactam, eravacycline, ceftazidime-avibactam, tigecycline, imipenem, meropenem, exhibited lower activity, with susceptibility rates of 69.3%, 68.5%, 66.1%, 65.4%, 63.8%, 59.8%, and 54.3%, respectively. Their MIC₅₀/MIC₉₀ values were 32/>64 mg/L, 1/>8 mg/L, 0.5/>0.5 mg/L, 1/>16 mg/L, ≤ 0.5/>1 mg/L 2/>8 mg/L, and 2/>16 mg/L, respectively.

The distribution of cefiderocol MICs varied with the resistance mechanism of Enterobacterales strains (Fig. 1A). The nine KPC-2 producers and four KPC-3 producers were all susceptible to cefiderocol, with MIC₅₀/MIC₉₀ of 1/2 mg/L. These strains were also all susceptible to ceftazidime-avibactam, aztreonam-avibactam, imipenem-relebactam, and meropenem-vaborbactam, with MIC₅₀/MIC₉₀ of 1/1 mg/L, 0/19/0.5 mg/L, 0.25/0.5 mg/L, and ≤ 0.06/0.12 mg/L, respectively. Colistin, tigecycline, eravacycline, amikacin, and fosfomycin were active against 92.3%, 76.9%, 76.9%, 69.2%, and 69.2% of the isolates, respectively.

Cefiderocol was active against 79.3% of the 29 NDM-producing isolates, with MIC₅₀/MIC₉₀ values of 2/8 mg/L. In more detail, 15 of the NDM-1 producers (n = 20) were susceptible, two of the NDM-5 producers (n = 5) were susceptible, and the NDM-4 (n = 1), NDM-14 (n = 2), and NDM-19 producers (n = 1) were susceptible. When cefiderocol MICs were interpreted using CLSI breakpoints, 86.2% of the isolates were susceptible, 6.9% were intermediate, and 6.9% were resistant (two NDM-5 producers). The only potent comparator agents were colistin (100% of susceptibility; MIC₅₀/MIC₉₀ ≤ 0.5/1 mg/L), aztreonam-avibactam (100%; 0.25/0.75 mg/L), eravacycline (82.8%; 0.5/>0.5 mg/L), tigecycline (72.4%; ≤0,5/>1 mg/L), and fosfomycin (65.5%; 32/64 mg/L). The VIM-1 and the two VIM-4 producing isolates were susceptible to cefiderocol regardless of the breakpoints used. Among the 10 NDM + OXA-48-like producers, only cefiderocol (90%), aztreonam-avibactam (100%), and colistin (100%) demonstrated efficacy greater than 75% with MIC₅₀/MIC₉₀ of 2/2 mg/L, 0.125/0.5 mg/L, and ≤ 0.5/≤.0,5 mg/L, respectively. The only isolate of C. freundii co-producing KPC-2 + OXA-48 had a low cefiderocol MIC of 0.5 mg/L.

Concerning the main resistance mechanism of the strains included, i.e. OXA-48-like production, 84.1% and 87.2% of the 63 isolates were susceptible to cefiderocol according to EUCAST and CLSI breakpoints, respectively. The MICs distribution was wide, ranging from ≤ 0.03 to 16 mg/L, with MIC₅₀/MIC₉₀ of 1/8 mg/L. Other comparators with high activity rates included colistin (100% susceptible), ceftazidime-avibactam (98.4%), aztreonam-avibactam (98.4%), imipenem ± relebactam (87.3% and 92.1%, respectively), meropenem ± vaborbactam (76.2% and 92.4%, respectively), amikacin (92.1%), and fosfomycin (76.2%).

Within the eight non-CPE isolates, 25% and 50% were susceptible to cefiderocol according to EUCAST and CLSI breakpoints, respectively, with MIC values ranging between 0.5 and > 32 mg/L. The six resistant isolates included three K. pneumoniae showing OmpK36 mutation (associated with CTX-M-15 production for two of them), two E. cloacae complex with decreased permeability of the outer membrane (associated with KLUB-1 or CTX-M-15 ESBL production in addition to the constitutive ACT-type AmpC), and one K. aerogenes with overproduction of AmpC associated with decreased permeability of the outer membrane. Many of the comparators had superior activity, with susceptibility rates of 100% to colistin and amikacin, 75% to meropenem alone and 100% when combined with vaborbactam, 87.5% to imipenem alone and 100% when combined to relebactam, 87.5% to ceftazidime-avibactam and aztreonam-avibactam, and 62.5% to tigecycline and eravacycline.

In vitro activity of cefiderocol and comparators against P. aeruginosa

Table 5 shows the in vitro activity of cefiderocol and its comparators against the 53 strains of MDR P. aeruginosa included in this study. Table 6 details the characteristics of cefiderocol-resistant strains.

Cefiderocol was active against 84.9% (n = 45) and 96.2% (n = 51) of the 53 P. aeruginosa isolates, according to EUCAST and CLSI breakpoints, respectively. The MIC₅₀/MIC₉₀ ratio was 1/4 mg/L. The distribution of cefiderocol MICs is illustrated in Fig. 1B. Among the comparators, only colistin had better rates of susceptibility (100%; MIC₅₀/MIC₉₀ 2/2 mg/L). The susceptibility rates and MIC₅₀/MIC₉₀ for other antibiotics tested were as follows: 67.9% and 16/>32 mg/L for amikacin, 56.6% and 4/>8 mg/L for ceftolozane-tazobactam, 56.6% and 2/>8 for imipenem-relebactam, 52.8% and 8/>16 mg/L for ceftazidime-avibactam, 52.8% and 16/>256 mg/L for aztreonam-avibactam, 52.8% and 2/>4 mg/L for tobramycin. The remaining antibiotics tested (piperacillin-tazobactam, cefepime, aztreonam, imipenem, and meropenem) had susceptibility rates below 50%.

Among the six MBL producers, 66.7% and 83.3% were susceptible to cefiderocol according to EUCAST and CLSI breakpoints, respectively, with MIC values between 0.5 and 16 mg/L. Only aztreonam (high exposure), aztreonam-avibactam (high exposure), and colistin had susceptibility rates above 50% (50%, 66.7%, and 100%, respectively).

Comparison of ComASP® and UMIC® BMD to evaluate cefiderocol MIC

The MICs of cefiderocol for the 127 Enterobacterales and 53 P. aeruginosa strains ranged between ≤ 0.03 and > 32 mg/L. Overall, the ComASP® method had an EA of 63.3% (95% CI: 56.2–70.3%), a CA of 86.1% (95% CI: 81–91.2%), and a bias of -50%, with 22 VME and three ME compared to the UMIC® method. Figure 2 shows the distribution of cefiderocol MICs using the UMIC® and ComASP® methods. Table 7 shows the performance of ComASP® BMD method in comparison with UMIC® reference method.

Table 7

Performance of cefiderocol AST methods in comparison with UMIC^® reference method.
AST method	Enterobacterales (n = 127)					P. aeruginosa (n = 53)
AST method	CA (%)	EA (%)	VME (n.)	ME (n.)	Difference of bias (%)	CA (%)	EA (%)	VME (n.)	ME (n.)	Difference of bias (%)
ComASP® BMD	85.8	65.4	15	3	-49.6	86.8	58.5	7	0	-60.2
BD BBL™ MH II agar DD	89 (73.2)	NA	4 (4)	7 (30)	NA	96 (94.3)	NA	2 (2)	0 (1)	NA
Bio-Rad MH agar DD	87 (74)	NA	6 (6)	7 (27)	NA	88.2 (88.7)	NA	6 (6)	0 (0)	NA
CA, Categorical Agreement; EA, Essential Agreement; VME, Very Major Errors; ME, Major Errors; BMD, Broth Microdilution; DD, Disk Diffusion; NA, Non-Applicable. EUCAST 2024 clinical breakpoints were used for the interpretation of the results. Values in brackets are the performance when the ATU is ignored.

Focusing on Enterobacterales, the EA and CA rates of the ComASP® method were 65.4% (95% CI: 57.1–73.6%) and 85.8% (95% CI: 79.7–91.9%), respectively, with a bias of -49.6%, with 15 VME and three ME. Among the 15 VME, six isolates had a MIC of 4 mg/L with the reference method, and the obtained MICs using ComASP® were 1 mg/L for three of them and 2 mg/L for three of them.

Among the P. aeruginosa isolates, the ComASP® method had an EA of 58.5% (95% CI: 45.2–71.8%), a CA of 86.8% (95% CI: 77.8–96%), and a bias of -60.2%. Seven VME were noticed with five isolates displaying a reference MIC of 4 mg/L, and no ME were identified.

Comparison of disk diffusion to UMIC® BMD to assess cefiderocol susceptibility

Figure 3 and Fig. 4 illustrate the distribution of inhibition zone diameter of cefiderocol on BD BBL™ MH II and Bio-Rad MH agar compared to the MICs determined using the UMIC® method. The performance of disk diffusion using BD BBL™ MH II and Bio-Rad MH agar, in comparison with the MICs obtained using UMIC® method, is presented in Table 7.

For Enterobacterales, when using the BD BBL™ MH medium, the CA rate was 89% (95% CI: 82.9–95.1%), with four VME and seven ME, excluding isolates in the Area of Technical Uncertainty (ATU, 21.3% of the isolates, n = 27). When the EUCAST ATU (21–23 mm) was not considered, the CA decreased to 73.2% (95% CI: 65.5–90.9%), with four VME and 30 ME. For P. aeruginosa, CA rates were 96% (95% CI: 90.5%-100%) with two VME and no ME, excluding isolates in the ATU (5.7% of the isolates, n = 3), and 94.3% (95% CI: 88–100%) with two VME and one ME when the ATU (20–21 mm) was not considered.

When applying the Bio-Rad MH, the CA for Enterobacterales was 87% (95% CI: 80.4–93.6%) with six VME and seven ME, excluding the 27 strains with an inhibition zone diameter in the ATU. Ignoring the ATU, the CA was 74% (95% CI: 66.4–81.6%) with six VME and 27 ME among the 127 Enterobacterales isolates. For P. aeruginosa isolates, the CA rate was 88.2% (95% CI: 79.3–97.1%) when the isolates with an inhibition zone diameter in the ATU were excluded, with six VME and no ME. The CA rate remained at 88.7% (95% CI: 80.2–97.1%) when the ATU was ignored, also with six VME and no ME.

The escalating challenge of antibiotic resistance has expedited the development of new drugs and novel β-lactam/β-lactamase inhibitor combinations. In the TROJAN-MDR study, we assessed the in vitro susceptibility of cefiderocol and other last-resort antibiotics against a variety of well-characterized MDR Enterobacterales and P. aeruginosa isolates from the South of France. These isolates were primarily collected from clinical samples.

Our findings revealed that cefiderocol exhibited significant activity against MDR Enterobacterales and P. aeruginosa, with 82.8% and 89.4% of the isolates respectively being categorized as susceptible according to EUCAST and CLSI breakpoints. Compared to other tested compounds, cefiderocol emerged as one of the most potent antibiotics. These findings align with those from similar studies [16–22].

When focusing on the KPC producers, the 100% susceptibility rate obtained is higher than those reported by other authors [23]. This could be attributed to the low number of KPC-producing isolates (n = 13) in our study, and the absence of variants responsible for resistance to ceftazidime-avibactam in our panel [4]. Indeed, recent reports have indicated higher cefiderocol MICs among ceftazidime-avibactam resistant strains harboring KPC-3 variants [4].

The cefiderocol MIC₅₀ for NDM-producing Enterobacterales was found to be 2 mg/L, which is the value of the EUCAST breakpoint. This aligns with the MICs₅₀ measured in other studies [18, 24, 25]. As described by Mushtaq et al., the addition of the MBL inhibitor dipicolinic acid reduced cefiderocol MICs in NDM-producing Enterobacterales indicating that these carbapenemases have the capacity to hydrolyze cefiderocol [23]. This finding may explain why MICs₅₀ values for NDM-producers are higher than to those of other resistance mechanisms. Although NDM production alone does not seem to be sufficient to cause cefiderocol resistance, its overproduction or the association with mutations of siderophore receptors are some of the described mechanisms that can lead to resistance [26–28]. The susceptibility rates among NDM-producing Enterobacterales vary across studies. When considering only studies that have applied the EUCAST breakpoints, susceptibility rates of 41% (n = 61 NDM-producing Enterobacterales), 48% (n = 21), 48.1% (n = 27), 51.4% (n = 37), 53.1% (n = 96), 70% (n = 118), 82.5% (n = 97) and 90.6% (n = 53) have been reported by various authors [8, 18, 20, 21, 23–25, 29]. The susceptibility rates obtained in our study (79.3% for NDM-producing isolates and 90% of NDM + OXA-48 producers susceptible) are closer to those of Bonnin et al., Malisova et al. and Delgado-Valverde et al. [8, 20, 29] Seven strains were resistant to cefiderocol according to EUCAST breakpoints. Four isolates were NDM-1 producers (one ST2084 K. pneumoniae coproducing OXA-48, two ST102 E. cloacae complex, one ST91 C. freundii) and three isolates were NDM-5 producers (one ST25 and one ST219 K. pneumoniae, one E. coli belonging to ST167 international high-risk clone for which cefiderocol resistance has already been reported, due to modified PBP3 and overexpression of NDM-5 and mutation of CirA iron transporter [28]). Finally, concerning NDM-producing Enterobacterales, colistin, aztreonam-avibactam, and eravacycline were the only comparators with susceptibility rates superior to cefiderocol (100%, 100%, and 82.8% respectively).

A majority of the OXA-48-like enzymes do not hydrolyze extended-spectrum cephalosporins [30], but in cases of ESBL production or AmpC overexpression, the isolates become resistant to these drugs. Ceftazidime-avibactam, which is the treatment of choice for infections involving this type of isolates, had a very high susceptibility rate in our study (98.4%), as expected [3, 31]. The selection pressure following the use of ceftazidime-avibactam has led to the emergence of OXA-48 variants resistant to this combination [32]. Given that OXA-48-like are the most common carbapenemases in France [13], we cannot rule out the future emergence and diffusion of ceftazidime-avibactam resistant isolates. Cefiderocol, with a susceptibility rate of 84.1% on OXA-48-like CPE, which is globally comparable to other studies, may be an interesting alternative to ceftazidime-avibactam [18, 25]. The resistant isolates in this study were eight OXA-48 producers (one ST2074 K. pneumoniae, one ST90, one ST106 and one ST873 E. cloacae complex, four ST216 C. freundii) and two OXA-181 producers (ST4988 K. pneumoniae), also producers of diverse β-lactamases and in some cases with outer membrane porin mutations (Table 6). The bla_SHV−12 gene has been detected in one of the C. freundii resistant isolates. The production of SHV-12 could explain the elevated MIC (16 mg/L) as Poirel et al. showed that production of this “old” EBSL can lead to cefiderocol resistance [33]. Since OXA-48 does not significantly hydrolyze cefiderocol [34], further investigations such as sequence analysis of PBP3 or iron transport encoding genes would be necessary to explain the mechanism of resistance to cefiderocol in the other isolates. Despite good susceptibility rates related to the weaker hydrolysis of carbapenems by OXA-48-like enzymes compared with class A or B carbapenemases, imipenem ± relebactam and meropenem ± vaborbactam are not recommended for the treatment of OXA-48 CPE infections [31].

For the non-CPE isolates included in this study, six (75%) were resistant to cefiderocol (MICs range 4 to > 32 mg/L). The β-lactamase genes harbored by these isolates (bla_SHV−1, bla_SHV−11, bla_SHV−101, bla_OXA−1, bla_TEM−1 and bla_CTX−M−15 for the three K. pneumoniae strains; bla_ACT−3, bla_ACT−59, bla_OXA−1, bla_TEM−1, bla_CTX−M−15, bla_KLUB−1 for the two E. cloacae complex strains and only bla_AmpC for the K. aerogenes isolate) have not been described as related to cefiderocol resistance [4]. The exhibited phenotype may result from decreased membrane permeability, as observed for five isolates, or from alterations in PBP3 or iron transport systems such as CirA. Mushtaq et al., described fewer susceptibility rates among Enterobacterales isolates expressing ESBL + porin loss phenotype [23].

The high susceptibility rate of aztreonam-avibactam (98.4% susceptible) among Enterobacterales of this study highlights the potential contribution of this new combination in the treatment arsenal, regardless of the resistance mechanism of MDR strains. Other studies reported similar susceptibility rates (94 to 100%) [35, 36].

Even though the susceptibility rate of meropenem-vaborbactam on Enterobacterales was high (85%), this combination is almost exclusively reserved for the treatment of infections involving KPC-producing isolates [3]. The high susceptibility rate can be explained by its breakpoint being 8 mg/L, which is higher than that of meropenem (2 mg/L). If the breakpoints were identical, meropenem-vaborbactam would have the same susceptibility rate as meropenem alone (54.3%). This underlines the importance of interpreting antimicrobial susceptibility results by the clinical microbiologist according to the genotypic profile to avoid inappropriate use, which can compromise clinical success [37].

Concerning MDR P. aeruginosa isolates, cefiderocol was the second most potent agent in vitro (84.9% and 96.2% of susceptibility rate by applying EUCAST and CLSI breakpoints, respectively), following colistin (100%) and preceding other β-lactams of last resort. These results are consistent with those observed in other studies [18, 20, 23], and highlight the key role of cefiderocol in overcoming severe MDR P. aeruginosa infections [38]. Notably, two of the eight cefiderocol resistant isolates produced a MBL (one ST235 IMP-26 producer and one ST308 NDM-1 producer). Aztreonam-avibactam inhibited only 52.8% of P. aeruginosa isolates in this study, which is lower compared with Enterobacterales. This may be in link with the diversity of resistance mechanisms in P. aeruginosa (efflux, production of diverse β-lactamases) [39].

An interesting finding of our study was that cefiderocol was active against 93% of the 57 isolates belonging to high-risk clones spreading worldwide (25 K. pneumoniae from ST11, ST15, ST147, ST258 and ST307; 12 E. cloacae complex from ST66, ST78, ST114 and ST171; eight C. freundii from ST22 and ST98; and 12 P. aeruginosa from ST175, ST235, ST244 and ST253) [19, 30, 39–41].

Different AST methods have been developed to assess cefiderocol in vitro activity [8, 9, 11, 12, 42]. The BMD method using Iron Depleted Cation Adjusted MH Broth (ID-CAMHB) is considered as the reference method [43]. UMIC® (Bruker) is a commercial unitary BMD test in which cefiderocol is dried into the wells (concentration range: 0.03–32 mg/L). A matching ID-CAMHB is available separately from the same manufacturer. This method was considered as the reference method in our study as it has been validated for both Enterobacterales and non-fermenting GNB [11, 12]. The ComASP® (Liofilchem) cefiderocol BMD test is another commercial assay on which two isolates are tested on every plate (concentration range: 0.008–128 mg/L), an ID-CAMHB is provided with the kit. Our results showed that ComASP® BMD method did not fulfill ISO 20776-2:2007 and 2021 criteria (CA and EA ≥ 90%, VME ≤ 1.5%, ≤ 3% and − 30% ≤ bias ≤ + 30%) on Enterobacterales and P. aeruginosa isolates, as EA rate was below 90% (90% was outside of 95% CI), VME rate was above 1.5% and biais below − 30%. This low rate of EA and the bias lower than − 30% illustrate the tendency of this method to underestimate MICs. Forty percent of the 15 VME identified among Enterobacterales and 71.4% of the seven VME on P. aeruginosa concerned isolates with a MIC of 4 mg/L. Bianco et al. compared ComASP® method with a reference BMD method on 50 MDR-GNB (Enterobacterales, P. aeruginosa and A. baumannii) that had an inhibition zone diameter in the former EUCAST ATU [44]. They obtained CA and EA rates of 94% and 84% respectively, with one VME and two ME. Emeraud et al. compared ComASP® and UMIC® tests with a reference BMD method on 60 carbapenem-resistant Enterobacterales [12]. ComASP® had 76.7% of EA and 83.3% of CA with 34.5% of VME and no ME, while UMIC® method had an EA of 91.7% and a CA of 83.3% with 24.1% of VME and 9.7% of ME. The findings in our study are in line with those of Emeraud et al. who conclude that ComASP® BMD assay is not a reliable method to assess cefiderocol susceptibility [12].

Easy-to-perform diffusion techniques with MIC strips and disks have also been developed on conventional MH agar. However, the cefiderocol-impregnated MIC strips initially drawn up for P. aeruginosa are no longer recommended, regardless of the species, due to the major underestimation of MICs that lead to a high number of VME [8, 9]. Matuschek et al. concluded that disk diffusion on conventional MH agar is a robust method to assess cefiderocol susceptibility in Enterobacterales and P. aeruginosa [43]. In addition to the breakpoint of 23 mm (Enterobacterales) and 22 mm (P. aeruginosa), an ATU has been proposed by EUCAST for Enterobacterales (21–23 mm) and P. aeruginosa (21–22 mm) to increase the performance of disk diffusion method [10]. Devoos et al. compared the performance of disk diffusion on 150 MDR P. aeruginosa isolates using disks from three manufacturers on MH agar plates from six manufacturers [9]. When isolates with an inhibition zone diameter in the ATU of EUCAST 2023 (14–22 mm) were excluded, CA rates were ≥ 90% for MH agar from BD, bioMérieux, and Mast group regardless of the disk manufacturer, while Bio-Rad did not reach the target. Nevertheless, in the same study, the authors demonstrated that the iron concentration was lower for Bio-Rad MH agar. Moreover, Bonnin et al. evaluated the performance of disk diffusion on Bio-Rad MH-agar compared to BMD and concluded that this method did not meet the CA of 90% [8]. It would therefore suggest that the different iron concentrations alone do not explain the differences in performance between the different MH media, since the agar medium mimics an iron-depleted medium (the iron being bound to the agar) [9]. Currently, in France, the CA-SFM and the Antimicrobial Resistance National Reference Center do not recommend disk diffusion to assess cefiderocol susceptibility in Enterobacterales, while it can still be used on P. aeruginosa and A. baumannii to screen susceptible isolates when inhibition diameter is ≥ 27 mm and ≥ 22 mm respectively [13].

In our study, we evaluated the cefiderocol inhibition zone diameter on two MH agar from two manufacturers (BD and Bio-Rad) using Mast disks in comparison with UMIC® BMD. The inhibition diameters were on average 1.39 mm (95% CI: 1.11–1.67) smaller on BD BBL™ MH II agar than on Bio-Rad. On Enterobacterales, the BD BBL™ MH II agar (CA 89%, four VME) was more reliable than Bio-Rad MH medium (CA 87%, six VME). Similar results were found for P. aeruginosa isolates with CA of 96% and two VME for BD BBL™ MH II agar while Bio-Rad’s MH agar had CA of 88.2% and six VME. It is noteworthy that none of the VME strains had an inhibition diameter ≥ 27 mm on BD BBL™ MH II agar, but these results must be interpreted with caution given the lower number of P. aeruginosa strains.

The TROJAN-MDR study demonstrated the potent activity of cefiderocol against a large collection of Enterobacterales and P. aeruginosa, both harboring various resistance mechanisms. This reaffirms the importance of cefiderocol in the treatment of difficult-to-treat infections. Constant surveillance of antimicrobial resistance and further investigation into resistant strains are keys to understanding the various mechanisms that may contribute to cefiderocol resistance. AST according to validated methods is crucial to ensure the efficacy of cefiderocol. Furthermore, antimicrobial stewardship is essential to ensure the appropriate use of this siderophore cephalosporin and to preserve its activity.

AST

Antimicrobial Susceptibility Testing

ATU

Area of Technical Uncertainty

BMD

Broth Microdilution

CA

Categorical Agreement

CA-SFM

Comité de l’Antibiogramme de la Société Française de Microbiologie

CLSI

Clinical & Laboratory Standards Institute

CPE

Carbapenemase-producing Enterobacterales

EA

Essential Agreement

EUCAST

European Committee on Antimicrobial Susceptibility Testing

ID-CAMHB

Iron Depleted Cation Adjusted MH Broth

MDR

Multi-Drug Resistance

ME

Major Error

mE

minor Error

MIC

Minimal Inhibitory Concentration

MH

Mueller Hinton

ST

Sequence Type

VME

Verry Major Error

Ethics approval and consent to participate
Not applicable

Consent for publication
Not applicable

Competing Interests

This research was partially supported by SHIONOGI & Co. which provided supplies for antimicrobial susceptibility testing (disk, medium and commercial BMD assays) and for open access fees. AP received fees as Advisory Board member from SHIONOGI & Co., Ltd., Osaka, Japan. However, the funder did not influence the study design, data collection and analysis, decision to publish, or the preparation of the manuscript in any way.

Funding

This research was partially supported by SHIONOGI & Co. which provided supplies for antimicrobial susceptibility testing (disk, medium and commercial BMD assays) and for open access fees. We thank the Nîmes University hospital for its structural, human and financial support through the award obtained by our team during the internal call for tenders « Thématiques phares ». However, the funders did not influence the study design, data collection and analysis, decision to publish, or the preparation of the manuscript in any way.

Author Contribution

Concept and design: AP, MBAcquisition, analysis, or interpretation of data: MB, AP, SL, ABD, MG, CM, MMDrafting of the manuscript: MB, APRevision of the manuscript: JPL, LR, HM, VJP, ABD

Acknowledgement

The authors wish to thank Dr. Florian Salipante for his assistance with the statistical analysis and Dr Matthieu Curtil Dit Galin and Romain Durello for technical assistance.

Availability of data and materials

Not applicable

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Competing interest reported. This research was partially supported by SHIONOGI & Co. which provided supplies for antimicrobial susceptibility testing (disk, medium and commercial BMD assays) and for open access fees. AP received fees as Advisory Board member from SHIONOGI & Co., Ltd., Osaka, Japan. However, the funder did not influence the study design, data collection and analysis, decision to publish, or the preparation of the manuscript in any way.

TROJAN-MDR: In vitro activity of cefiderocol and comparators against multidrug-resistant Enterobacterales and Pseudomonas aeruginosa strains in Southern France, evaluation of available testing methods performances

Status:

Journal Publication

Version 1

Abstract

Background

Methods

Results

Conclusions

Figures

Background

Material and methods

Bacterial isolates

Comparators susceptibility testing

Cefiderocol susceptibility testing

Clinical performance of cefiderocol AST

Results

Overall in vitro activity of cefiderocol against MDR-GNB

In vitro activity of cefiderocol and comparators against Enterobacterales

In vitro activity of cefiderocol and comparators against P. aeruginosa

Comparison of ComASP® and UMIC® BMD to evaluate cefiderocol MIC

Comparison of disk diffusion to UMIC® BMD to assess cefiderocol susceptibility

Discussion

Conclusions

Abbreviations

Declarations

Ethics approval and consent to participate
Not applicable

Consent for publication
Not applicable

Competing Interests

Funding

Author Contribution

Acknowledgement

Availability of data and materials

References

Additional Declarations

Status:

Journal Publication

Version 1

TROJAN-MDR: In vitro activity of cefiderocol and comparators against multidrug-resistant Enterobacterales and Pseudomonas aeruginosa strains in Southern France, evaluation of available testing methods performances

Status:

Journal Publication

Version 1

Abstract

Background

Methods

Results

Conclusions

Figures

Background

Material and methods

Bacterial isolates

Comparators susceptibility testing

Cefiderocol susceptibility testing

Clinical performance of cefiderocol AST

Results

Overall in vitro activity of cefiderocol against MDR-GNB

In vitro activity of cefiderocol and comparators against Enterobacterales

In vitro activity of cefiderocol and comparators against P. aeruginosa

Comparison of ComASP® and UMIC® BMD to evaluate cefiderocol MIC

Comparison of disk diffusion to UMIC® BMD to assess cefiderocol susceptibility

Discussion

Conclusions

Abbreviations

Declarations

Ethics approval and consent to participate Not applicable

Consent for publication Not applicable

Competing Interests

Funding

Author Contribution

Acknowledgement

Availability of data and materials

References

Additional Declarations

Status:

Journal Publication

Version 1

Ethics approval and consent to participate
Not applicable

Consent for publication
Not applicable