Evaluation of Antimicrobial Effects of a New Polymyxin Molecule (SPR741) When Tested in Combination with a Series of β-Lactam Agents Against a Challenge Set of Gram-Negative Pathogens

Abstract

The antimicrobial activities of several β-lactam agents were tested by broth microdilution alone and in combination with a new polymyxin analog, SPR741 (at a fixed concentration of 8 mg/L), against a challenge set of clinical isolates (202 Escherichia coli and 221 Klebsiella pneumoniae isolates). Using Clinical and Laboratory Standards Institute (CLSI) or European Committee on Antimicrobial Susceptibility Testing (EUCAST) susceptibility criteria for each partner antibiotic, mecillinam–SPR741, temocillin–SPR741, and piperacillin–tazobactam–SPR741 combinations had susceptibility rates higher (85.6–100.0%) than the respective agents tested alone (47.5–88.7%) against extended-spectrum β-lactamase (ESBL)-producing E. coli and K. pneumoniae. Temocillin–SPR741 (97.8% susceptible) had MIC₅₀ (minimum inhibitory concentration) and MIC₉₀ results of 0.5 and 2 mg/L, respectively, against K. pneumoniae carbapenemase (KPC)-producing E. coli, 8- to 16-fold lower than temocillin alone (MIC_50/90, 8/16 mg/L; 65.2% susceptible). The mecillinam MIC₅₀/MIC₉₀ results dropped to 1/4 mg/L (from 128/>256 mg/L when tested alone) against metallo-β-lactamase (MBL)-producing E. coli. These MICs for mecillinam–SPR741 resulted in a susceptibility rate of 96.9% versus 9.4% for mecillinam. In general, a decrease in MICs for β-lactams (MIC₉₀, >32 mg/L) in the presence of SPR741 was not observed against KPC-, MBL- or OXA-48-like-producing K. pneumoniae. These study results indicate that some agents had a significant increase in in vitro activity in the presence of SPR741 and could become potential strategic options for treating serious infections caused by multidrug-resistant Enterobacteriaceae.

Introduction

Antibiotic resistance has been well established as being associated with significant adverse impact on clinical outcomes and increased consumption of health care resources, leading to higher costs.¹ The increased rate of Enterobacteriaceae resistant to extended-spectrum β-lactam agents appears to be mostly due to the presence of plasmid-carrying bla_CTX-M variants. More specifically, the literature reports that the vast majority of Escherichia coli and Klebsiella pneumoniae displaying nonsusceptibility to extended-spectrum β-lactams carry bla_CTX-M-15 and related variants.^2–4 In addition, although overall less prevalent, the dissemination of carbapenem-resistant isolates due to the production of carbapenemases, such as K. pneumoniae carbapenemase (KPC) and OXA-48-like have become a serious threat in several regions worldwide.^5–7

An increasing number of patients with underlying comorbid conditions and escalating antimicrobial resistance call for new treatment strategies to effectively manage the growing population of patients.⁸ SPR741 (formerly NAB741) is a novel cationic peptide derived from polymyxin that interacts with the outer membrane of Gram-negative bacteria and compromises its integrity.^9–11 This compound has minimal direct antibacterial activity and acts by increasing cell permeability, and when combined with an antibacterial agent, SPR741 facilitates the entry of the active compounds.¹¹ In this study, the antimicrobial activity of several clinically available β-lactam agents was tested alone and combined with SPR741 in vitro against a challenge set of 423 E. coli and K. pneumoniae producing a variety of current and relevant β-lactamase enzymes.

Materials and Methods

Clinical isolates

A challenge set of 423 clinical isolates (202 E. coli and 221 K. pneumoniae) was selected by the presence of β-lactamases that included plasmid AmpCs (pAmpCs), extended-spectrum β-lactamases (ESBLs), KPCs, metallo-β-lactamases (MBLs), and OXA-48-like enzymes (Supplementary Table S1). All K. pneumoniae isolates were recovered during 2016, whereas most E. coli (80%) were recovered during 2013 and 2016, and isolates from 2002 to 2012 were added to include a greater number of isolates with less common resistance genotypes. This set of isolates also originated from the SENTRY Antimicrobial Surveillance Program collection of microorganisms. These isolates were received from medical centers worldwide, including North America (n = 218), Europe (n = 111), Asia-Pacific (n = 57), and Latin America (n = 37).

Bacterial identification was performed by the participating microbiology laboratory and confirmed by the central monitoring laboratory (JMI Laboratories, North Liberty, IA). Isolates were screened for β-lactamase-encoding genes by polymerase chain reaction/microarray, followed by sequencing or by genome sequence and in silico analysis.^2–4 A line list containing all isolates included in this study and respective β-lactamase-encoding genes is presented as a Supplementary Table S1.

Antimicrobial susceptibility testing

Isolates were tested for susceptibility by broth microdilution methods, according to the recommendations of CLSI.¹² Minimum inhibitory concentration (MIC) results were obtained using reference frozen-form panels manufactured by JMI Laboratories. Based on previous in vitro checkerboard studies, SPR741 exhibits a dose/response to its potentiation effect beyond 8 mg/L.⁹ Also, plasma toxicokinetic analysis from good laboratory practice repeat dose 14-day nonhuman primate toxiocology studies show that plasma C_max for SPR741 at the no observed adverse effect level dose was >44 mg/L, and the concentration of SPR741 in plasma exceeded 8 mg/L for >50% of the dosing interval. Additioanally, a Phase 1 single ascending dose/multiple ascending dose assessment of SPR741 in healthy adult volunteers showed C_max of >35 mg/L and the concentration of SPR741 exceeded 8 mg/L for >50% of the dosing interval at safe and well-tolerated doses; therefore, SPR741 was tested at a fixed concentration of 8 mg/L in combination with several β-lactam agents.^13,14 In addition, these β-lactam agents were tested alone against this collection, as was SPR741. SPR741 showed MIC results of >8 mg/L against all but two isolates (MIC, 8 mg/L).

MIC values were validated by concurrently testing quality control (QC) strains, including E. coli ATCC 25922 and 35218, and NCTC 10418; Pseudomonas aeruginosa ATCC 27853; and Staphylococcus aureus ATCC 29213, following the CLSI M100 guidelines; and acceptable MIC ranges were those published in the CLSI M100.¹⁵ Target MIC QC values expected for temocillin and mecillinam were those published by the British Society for Antimicrobial Chemotherapy (BSAC).¹⁶

MIC results obtained against clinical isolates were interpreted using the CLSI M100 and EUCAST documents, as available.^15,17 MIC results obtained for temocillin were interpreted according to the BSAC systemic (≤8 mg/L for susceptible) and urinary tract infection (UTI) (≤32 mg/L for susceptible) breakpoints.¹⁸

Data analysis

The possible effect of SPR741 on the in vitro activity of antimicrobial agents was evaluated based on (1) the decrease of MIC results, MIC₅₀, and/or MIC₉₀ values in the presence of SPR741 compared with values obtained for the active agent tested alone; and (2) the increase in susceptibility rates obtained for the combinations compared with rates obtained for the active agent tested alone when applying currently available breakpoints.

Results

Antimicrobial activity of β-lactam–SPR741 combinations

When combined with SPR741 at a fixed concentration of 8 mg/L against pAmpC-producing isolates, several β-lactams had MIC₅₀ and MIC₉₀ results lower than the respective codrugs tested alone (Table 1). Piperacillin–tazobactam–SPR741 (MIC_50/90, ≤0.12/1 mg/L) showed MIC₅₀ and MIC₉₀ results ≥64- and 128-fold lower than piperacillin–tazobactam (MIC_50/90, 8/128 mg/L). Similarly, aztreonam–SPR741 (MIC_50/90, 0.5/2 mg/L) and ceftazidime–SPR741 (MIC_50/90, 1/4 mg/L) had MIC values 16- to 32-fold lower than the respective drugs tested alone against pAmpC-producing isolates (MIC_50/90, 8/64 mg/L and MIC_50/90, 32/128 mg/L, respectively). These lower MIC values obtained for the SPR741 combinations resulted in increased susceptibility rates (93.8%) for aztreonam, ceftazidime, and piperacillin–tazobactam when applying the current CLSI breakpoints available for the β-lactam tested alone (Table 1). Other drugs, such as mecillinam, temocillin, cefotaxime, and ceftaroline tested with SPR741 showed MIC values 4- to 16-fold lower than the respective codrug tested alone against pAmpC-producing isolates. While these lower MIC results obtained for mecillinam and temocillin (systemic breakpoint) resulted in absolute susceptibility (100.0%), susceptibility results for the cefoxitin, ceftaroline, and cefotaxime combinations remained at 6.2–50.0% (Table 1).

Table 1.

Antimicrobial Activity of β-Lactam and β-Lactam–SPR741 Combinations (Fixed 8 mg/L) Against AmpC-Producing Escherichia coli (10) and Klebsiella pneumoniae (6) Isolates

Antimicrobial agent	MIC₅₀	MIC₉₀	Range	CLSI^a			EUCAST^a
Antimicrobial agent	MIC₅₀	MIC₉₀	Range	% S	% I	% R	% S	% I	% R
AmpC (16)
Aztreonam	8	64	≤1 to 64	37.5	43.8	18.8	6.2	31.2	62.5
Aztreonam–SPR741	0.5	2	≤0.25 to 8	93.8	6.2	0.0	75.0	18.8	6.2
Mecillinam	1	8	≤0.25 to 16				93.8		6.2^b
Mecillinam–SPR741	0.25	1	≤0.06 to 4				100.0		0.0^b
Temocillin	4	16	1 to 16				87.5		12.5^c
Temocillin	4	16	1 to 16				100.0		0.0^b
Temocillin–SPR741	0.5	1	≤0.25 to 8				100.0		0.0^c
Temocillin–SPR741	0.5	1	≤0.25 to 8				100.0		0.0^b
Cefoxitin	128	>256	32 to >256	0.0	0.0	100.0
Cefoxitin–SPR741	16	>32	≤0.25 to >32	12.5	37.5	50.0
Ceftazidime	32	128	16 to >256	0.0	0.0	100.0	0.0	0.0	100.0
Ceftazidime–SPR741	1	4	≤0.12 to 32	93.8	0.0	6.2	62.5	31.2	6.2
Cefotaxime	8	64	2 to 64	0.0	6.2	93.8	0.0	6.2	93.8
Cefotaxime–SPR741	1	8	≤0.25 to 32	50.0	18.8	31.2	50.0	18.8	31.2
Cefepime	≤1	≤1	≤1 to 2	100.0	0.0	0.0^c	93.8	6.2	0.0
Cefepime–SPR741	≤0.25	0.5	≤0.25 to 0.5	100.0	0.0	0.0^c	100.0	0.0	0.0
Ceftaroline	16	128	4 to >256	0.0	0.0	100.0	0.0		100.0
Ceftaroline–SPR741	2	32	≤0.25 to >32	6.2	31.2	62.5	6.2		93.8
Meropenem	0.03	0.12	≤0.015 to 0.12	100.0	0.0	0.0	100.0	0.0	0.0
Meropenem–SPR741	0.03	0.12	≤0.015 to 0.12	100.0	0.0	0.0	100.0	0.0	0.0
Piperacillin–tazobactam	8	128	4 to 256	81.2	6.2	12.5	75.0	6.2	18.8
Piperacillin–tazobactam–SPR741	≤0.12	1	≤0.12 to 128	93.8	0.0	6.2	93.8	0.0	6.2

Criteria as published by CLSI¹⁵ and EUCAST.¹⁷ MIC interpretations for the β-lactam–SPR741 combinations utilized the breakpoints available for the respective β-lactam from CLSI or EUCAST, for comparison purposes.

Mecillinam (≤8 mg/L for susceptible) and temocillin (≤32 mg/L for susceptible) MIC results were interpreted based on the EUCAST¹⁷ and BSAC¹⁸ criteria, respectively, for uncomplicated urinary tract infection.

MIC results obtained for temocillin were interpreted according to the BSAC¹⁸ systemic criterion (≤8 mg/L for susceptible).

BSAC, British Society for Antimicrobial Chemotherapy; CLSI, Clinical and Laboratory Standards Institute; EUCAST, European Committee on Antimicrobial Susceptibility Testing; MIC, minimum inhibitory concentration.

The highest increase in activity against ESBL-producing E. coli was observed for ceftazidime–SPR741 (MIC_50/90, 0.25/2 mg/L) and piperacillin–tazobactam–SPR741 (MIC_50/90, ≤0.12/0.5 mg/L), where the MIC₉₀ values decreased 64-fold compared with ceftazidime (MIC_50/90, 16/128 mg/L) and piperacillin–tazobactam (MIC_50/90, 4/32 mg/L) (Table 2). Aztreonam, mecillinam, and temocillin also had their in vitro activity potentiated in the presence of SPR741 against ESBL-producing E. coli, with MIC values 8- to 16-fold lower than the respective codrug. Although the susceptibility rates for aztreonam-SPR741 remained limited (39.2–77.3%) against ESBL-producing E. coli, mecillinam and temocillin inhibited 97.9–100.0% of isolates at ≤8 mg/L (UTI and systemic breakpoints, respectively). When cefoxitin, cefotaxime, cefepime, and ceftaroline were tested against ESBL-producing E. coli in the presence of SPR741, decreases in the MIC values were not very pronounced; however, the susceptibility rate for cefoxitin–SPR741 increased to 92.8% (Table 2).

Table 2.

Antimicrobial Activity of β-Lactam and β-Lactam–SPR741 Combinations (Fixed 8 mg/L) Against β-Lactamase-Producing Escherichia coli Isolates

Antimicrobial agent	MIC₅₀	MIC₉₀	Range	CLSI^a			EUCAST^a
Antimicrobial agent	MIC₅₀	MIC₉₀	Range	% S	% I	% R	% S	% I	% R
ESBL-producing E. coli (97)
Aztreonam	32	256	≤1 to >256	11.3	9.3	79.4	1.0	10.3	88.7
Aztreonam–SPR741	2	16	≤0.25 to >32	77.3	10.3	12.4	39.2	38.1	22.7
Mecillinam	2	32	≤0.25 to >256				81.4		18.6^b
Mecillinam–SPR741	0.25	2	≤0.06 to >32				97.9		2.1^b
Temocillin	4	16	1 to 64				88.7		11.3^c
Temocillin	4	16	1 to 64				99.0		1.0^b
Temocillin–SPR741	0.5	1	≤0.25 to 4				100.0		0.0^c
Temocillin–SPR741	0.5	1	≤0.25 to 4				100.0		0.0^b
Cefoxitin	8	32	2 to >256	66.0	14.4	19.6
Cefoxitin–SPR741	4	8	≤0.25 to >32	92.8	4.1	3.1
Ceftazidime	16	128	≤2 to >256	28.9	9.3	61.9	0.0	28.9	71.1
Ceftazidime–SPR741	0.25	2	≤0.12 to 128	95.9	1.0	3.1	80.4	15.5	4.1
Cefotaxime	>256	>256	2 to >256	0.0	2.1	97.9	0.0	2.1	97.9
Cefotaxime–SPR741	>32	>32	≤0.25 to >32	6.2	2.1	91.8	6.2	2.1	91.8
Cefepime	16	>256	≤1 to >256	14.4	24.7	60.8 ^c	10.3	18.6	71.1
Cefepime–SPR741	4	>32	≤0.25 to >32	47.4	23.7	28.9 ^c	29.9	33.0	37.1
Ceftaroline	256	>256	≤2 to >256	0.0	0.0	100.0	0.0		100.0
Ceftaroline–SPR741	32	>32	≤0.25 to >32	10.3	1.0	88.7	10.3		89.7
Meropenem	0.03	0.06	≤0.015 to 1	100.0	0.0	0.0	100.0	0.0	0.0
Meropenem–SPR741	≤0.015	0.03	≤0.015 to 0.06	100.0	0.0	0.0	100.0	0.0	0.0
Piperacillin–tazobactam	4	32	≤1 to >256	86.6	9.3	4.1	74.2	12.4	13.4
Piperacillin–tazobactam–SPR741	≤0.12	0.5	≤0.12 to 8	100.0	0.0	0.0	100.0	0.0	0.0
KPC-producing E. coli (46)
Aztreonam	256	>256	4 to >256	2.2	0.0	97.8	0.0	2.2	97.8
Aztreonam–SPR741	16	>32	≤0.25 to >32	28.3	10.9	60.9	8.7	19.6	71.7
Mecillinam	>256	>256	8 to >256				2.2		97.8^b
Mecillinam–SPR741	>32	>32	≤0.06 to >32				26.1		73.9^b
Temocillin	8	16	2 to 128				65.2		34.8^c
Temocillin	8	16	2 to 128				95.7		4.3^b
Temocillin–SPR741	0.5	2	≤0.25 to 32				97.8		2.2^c
Temocillin–SPR741	0.5	2	≤0.25 to 32				100.0		0.0^b
Cefoxitin	32	128	4 to >256	19.6	23.9	56.5
Cefoxitin–SPR741	8	32	≤0.25 to >32	78.3	6.5	15.2
Ceftazidime	64	128	4 to >256	2.2	2.2	95.7	0.0	2.2	97.8
Ceftazidime–SPR741	1	8	≤0.12 to 128	87.0	6.5	6.5	60.9	26.1	13.0
Cefotaxime	128	>256	4 to >256	0.0	0.0	100.0	0.0	0.0	100.0
Cefotaxime–SPR741	32	>32	≤0.25 to >32	6.5	10.9	82.6	6.5	10.9	82.6
Cefepime	16	256	≤1 to >256	4.3	21.7	73.9 ^c	4.3	8.7	87.0
Cefepime–SPR741	4	>32	≤0.25 to >32	28.3	41.3	30.4^c	15.2	39.1	45.7
Ceftaroline	>256	>256	16 to >256	0.0	0.0	100.0	0.0		100.0
Ceftaroline–SPR741	>32	>32	≤0.25 to >32	2.2	2.2	95.7	2.2		97.8
Meropenem	4	8	0.03 to >32	10.9	17.4	71.7	28.3	63.0	8.7
Meropenem–SPR741	1	4	≤0.015 to 8	52.2	21.7	26.1	73.9	26.1	0.0
Piperacillin–tazobactam	256	>256	32 to >256	0.0	10.9	89.1	0.0	0.0	100.0
Piperacillin–tazobactam–SPR741	4	32	≤0.12 to >256	84.8	8.7	6.5	71.7	13.0	15.2
MBL-producing E. coli (32)
Aztreonam	256	>256	≤1 to >256	6.2	3.1	90.6	6.2	0.0	93.8
Aztreonam–SPR741	8	32	≤0.25 to >32	46.9	12.5	40.6	28.1	18.8	53.1
Mecillinam	128	>256	0.5 to >256				9.4		90.6^b
Mecillinam–SPR741	1	4	≤0.06 to >32				96.9		3.1^b
Temocillin	128	>128	2 to >128				6.2		93.8^c
Temocillin	128	>128	2 to >128				18.8		81.2^b
Temocillin–SPR741	4	>32	≤0.25 to >32				68.8		31.2^c
Temocillin–SPR741	4	>32	≤0.25 to >32				84.4		15.6^b
Cefoxitin	>256	>256	8 to >256	3.1	0.0	96.9
Cefoxitin–SPR741	>32	>32	≤0.25 to >32	9.4	3.1	87.5
Ceftazidime	>256	>256	≤2 to >256	3.1	0.0	96.9	0.0	0.0	100.0
Ceftazidime–SPR741	256	>256	≤0.12 to >256	12.5	6.2	81.2	9.4	3.1	87.5
Cefotaxime	>256	>256	64 to >256	0.0	0.0	100.0	0.0	0.0	100.0
Cefotaxime–SPR741	>32	>32	≤0.25 to >32	6.2	0.0	93.8	6.2	0.0	93.8
Cefepime	>256	>256	2 to >256	3.1	0.0	96.9^c	0.0	3.1	96.9
Cefepime–SPR741	>32	>32	≤0.25 to >32	9.4	15.6	75.0^c	9.4	3.1	87.5
Ceftaroline	>256	>256	64 to >256	0.0	0.0	100.0	0.0		100.0
Ceftaroline–SPR741	>32	>32	≤0.25 to >32	6.2	0.0	93.8	6.2		93.8
Meropenem	>32	>32	≤0.015 to >32	6.2	3.1	90.6	9.4	9.4	81.2
Meropenem–SPR741	16	>32	≤0.015 to >32	18.8	3.1	78.1	21.9	21.9	56.2
Piperacillin–tazobactam	>256	>256	≤1 to >256	9.4	3.1	87.5	6.2	3.1	90.6
Piperacillin–tazobactam–SPR741	64	>256	≤0.12 to >256	31.2	18.8	50.0	25.0	6.2	68.8
OXA-48-like-producing E. coli (17)
Aztreonam	64	>256	≤1 to >256	29.4	0.0	70.6	29.4	0.0	70.6
Aztreonam–SPR741	0.5	8	≤0.25 to 32	88.2	5.9	5.9	52.9	35.3	11.8
Mecillinam	4	64	2 to >256				76.5		23.5^b
Mecillinam–SPR741	0.5	1	≤0.06 to 1				100.0		0.0^b
Temocillin	>128	>128	128 to >128				0.0		100.0 ^c
Temocillin	>128	>128	128 to >128				0.0		100.0 ^b
Temocillin–SPR741	16	>32	2 to >32				35.3		64.7^c
Temocillin–SPR741	16	>32	2 to >32				58.8		41.2^b
Cefoxitin	16	>256	2 to >256	35.3	23.5	41.2
Cefoxitin–SPR741	4	32	2 to >32	88.2	0.0	11.8
Ceftazidime	16	256	≤2 to 256	47.1	0.0	52.9
Ceftazidime–SPR741	0.25	2	≤0.12 to 4	100.0	0.0	0.0	88.2	11.8	0.0
Cefotaxime	256	>256	≤1 to >256	11.8	17.6	70.6	11.8	17.6	70.6
Cefotaxime–SPR741	>32	>32	≤0.25 to >32	35.3	0.0	64.7	35.3	0.0	64.7
Cefepime	8	>256	≤1 to >256	35.3	17.6	47.1^c	29.4	17.6	52.9
Cefepime–SPR741	1	>32	≤0.25 to >32	76.5	5.9	17.6^c	52.9	29.4	17.6
Ceftaroline	128	>256	≤2 to >256	0.0	0.0	100.0	0.0		100.0
Ceftaroline–SPR741	32	>32	≤0.25 to >32	17.6	11.8	70.6	17.6		82.4
Meropenem	0.5	32	0.12 to >32	70.6	0.0	29.4	70.6	5.9	23.5
Meropenem–SPR741	0.5	2	0.12 to 4	76.5	17.6	5.9	94.1	5.9	0.0
Piperacillin–tazobactam	128	>256	64 to >256	0.0	23.5	76.5	0.0	0.0	100.0
Piperacillin–tazobactam–SPR741	2	4	≤0.12to 16	100.0	0.0	0.0	94.1	5.9	0.0

Mecillinam (≤8 mg/L for susceptible) and temocillin (≤32 mg/L for susceptible) MIC results were interpreted based on the EUCAST¹⁷ and BSAC¹⁸ criteria, respectively, for uncomplicated UTI.

MIC results obtained for temocillin were interpreted according to the BSAC¹⁸ systemic criterion (≤8 mg/L for susceptible).

ESBL, extended-sprectrum β-lactamase; KPC, K. pneumoniae carbapenemase; MBL, metallo-β-lactamase.

SPR741 decreased the ceftazidime (MIC_50/90, 1/8 mg/L; 60.9–87.0% susceptible) and piperacillin–tazobactam (MIC_50/90, 4/32 mg/L; 71.7–84.8% susceptible) MIC values 16- to 64-fold when compared with the results obtained for these drugs tested alone against KPC-producing E. coli (Table 2). The cefoxitin–SPR741 (MIC_50/90, 8/32 mg/L) MIC₅₀ and MIC₉₀ results were fourfold lower than cefoxitin (MIC_50/90, 32/128 mg/L) against KPC-producing E. coli. The potentiation observed for ceftazidime, piperacillin–tazobactam, and cefoxitin in the presence of SPR741 resulted in susceptibility rates 58.7–84.8% higher than those obtained for these drugs tested alone; however, these rates were still suboptimal (60.9–87.0%). Temocillin–SPR741 had MIC₅₀ and MIC₉₀ results of 0.5 and 2 mg/L, respectively, which were 8- to 16-fold lower than temocillin (MIC_50/90, 8/16 mg/L). The increased temocillin potency provided by SPR741 also increased the temocillin susceptibility rate (from 65.2%) to 97.8% when applying the systemic breakpoint (Table 2).

Overall, SPR741 did not provide increased potency to β-lactam agents against MBL-producing E. coli (MIC₉₀, >32 mg/L), except for aztreonam and mecillinam (Table 2). Although the aztreonam MIC values decreased 16- to 32-fold in the presence of SPR741 compared with aztreonam tested alone, susceptibility rates obtained by current breakpoints remained low (28.1–46.9%). However, the mecillinam MIC₅₀ and MIC₉₀ results dropped to 1 and 4 mg/L, respectively, (from MIC_50/90, 128/>256 mg/L when tested alone) against MBL-producing E. coli. These MIC values for mecillinam–SPR741 resulted in a susceptibility result of 96.9% versus 9.4% for mecillinam against MBL-producing E. coli (Table 2).

Aztreonam–SPR741 (MIC_50/90, 0.5/8 mg/L), mecillinam–SPR741 (MIC_50/90, 0.5/1 mg/L), ceftazidime–SPR741 (MIC_50/90, 0.25/2 mg/L), and piperacillin–tazobactam–SPR741 (MIC_50/90, 2/4 mg/L) showed MIC₉₀ results ≥64-fold lower than the respective codrugs tested alone against OXA-48-like-producing E. coli (Table 2). These same combinations inhibited 88.2–100.0% of these isolates at their respective CLSI or EUCAST breakpoints, except for aztreonam–SPR741 (52.9–88.2%).

Piperacillin–tazobactam–SPR741 (MIC_50/90, 0.5/1 mg/L) and temocillin–SPR741 (MIC_50/90, 0.5/1 mg/L), followed by mecillinam–SPR741 (MIC_50/90, 0.5/4 mg/L) showed the lowest MIC₉₀ values against ESBL-producing K. pneumoniae, with susceptibility rates of 96.0–99.0% (Table 3). In contrast, a decrease in MIC results for β-lactam agents (MIC₉₀, >32 mg/L) in the presence of SPR741 was not generally observed against KPC-, MBL-, or OXA-48-like-producing K. pneumoniae (Table 3). However, temocillin–SPR741 (MIC_50/90, 1/32 mg/L) showed results against KPC-producing K. pneumoniae that were 2- to 32-fold lower than temocillin. Therefore, when applying the systemic breakpoint, SPR741 increased the temocillin susceptibility rate to 78.4% from 14.9% when tested alone, while the rate increased to 94.6% when the UTI breakpoint was used. When tested against OXA-48-like-producing K. pneumoniae, SPR741 increased the susceptibility rates for mecillinam and ceftazidime 66.6–73.4% (Table 3).

Table 3.

Antimicrobial Activity of β-Lactam and β-Lactam–SPR741 Combinations (Fixed 8 mg/L) Against β-Lactamase-Producing Klebsiella pneumoniae Isolates

Antimicrobial agent	MIC₅₀	MIC₉₀	Range	CLSI^a			EUCAST^a
Antimicrobial agent	MIC₅₀	MIC₉₀	Range	% S	% I	% R	% S	% I	% R
ESBL-producing K. pneumoniae (101)
Aztreonam	64	>256	≤1 to >256	10.9	5.9	83.2	1.0	9.9	89.1
Aztreonam–SPR741	4	32	≤0.25 to >32	55.4	15.8	28.7	26.7	28.7	44.6
Mecillinam	16	>256	0.5 to >256				49.5		50.5^b
Mecillinam–SPR741	0.5	4	≤0.06 to >32				96.0		4.0^b
Temocillin	4	16	≤0.5 to 64				88.1		11.9^c
Temocillin	4	16	≤0.5 to 64				99.0		1.0^b
Temocillin–SPR741	0.5	1	≤0.25 to 16				99.0		1.0^c
Temocillin–SPR741	0.5	1	≤0.25 to 16				100.0		0.0^b
Cefoxitin	4	32	2 to >256	65.3	11.9	22.8
Cefoxitin–SPR741	4	16	0.5 to >32	79.2	11.9	8.9
Ceftazidime	32	256	≤2 to >256	15.8	5.0	79.2	0.0	7.6	92.4
Ceftazidime–SPR741	1	8	≤0.12 to 256	85.1	6.9	7.9	55.4	29.7	14.9
Cefotaxime	256	>256	≤1 to >256	1.0	4.0	95.0	1.0	4.0	95.0
Cefotaxime–SPR741	>32	>32	≤0.25 to >32	4.0	4.0	92.1	4.0	4.0	92.1
Cefepime	16	>256	≤1 to >256	15.8	25.7	58.4^c	7.9	18.8	73.3
Cefepime–SPR741	8	>32	≤0.25 to >32	34.7	20.8	44.6^c	25.7	18.8	55.4
Ceftaroline	256	>256	≤2 to >256	0.0	0.0	100.0	0.0		100.0
Ceftaroline–SPR741	8	>32	≤0.25 to >32	4.0	23.8	72.3	4.0		96.0
Meropenem	0.03	0.12	≤0.015 to 16	95.0	2.0	3.0	97.0	2.0	1.0
Meropenem–SPR741	0.03	0.12	≤0.015 to 1	100.0	0.0	0.0	100.0	0.0	0.0
Piperacillin–tazobactam	8	>256	≤1 to >256	65.3	15.8	18.8	51.5	13.9	34.7
Piperacillin–tazobactam–SPR741	0.5	1	≤0.12 to >256	98.0	0.0	2.0	97.0	1.0	2.0
KPC-producing K. pneumoniae (74)
Aztreonam	>256	>256	128 to >256	0.0	0.0	100.0	0.0	0.0	100.0
Aztreonam–SPR741	>32	>32	8 to >32	0.0	2.7	97.3	0.0	0.0	100.0
Mecillinam	>256	>256	256 to >256				0.0		100.0^b
Mecillinam–SPR741	16	>32	0.5 to >32				43.2		56.8^b
Temocillin	32	64	2 to >128				14.9		85.1^c
Temocillin	32	64	2 to >128				83.8		16.2^b
Temocillin–SPR741	1	32	0.5 to >32				78.4		21.6^c
Temocillin–SPR741	1	32	0.5 to >32				94.6		5.4^b
Cefoxitin	64	>256	8 to >256	1.4	9.5	89.2
Cefoxitin–SPR741	16	>32	≤0.25 to >32	48.6	20.3	31.1
Ceftazidime	>256	>256	32 to >256	0.0	0.0	100.0	0.0	0.0	100.0
Ceftazidime–SPR741	8	>256	0.5 to >256	36.5	24.3	39.2	9.5	27.0	63.5
Cefotaxime	>256	>256	8 to >256	0.0	0.0	100.0	0.0	0.0	100.0
Cefotaxime–SPR741	>32	>32	4 to >32	0.0	0.0	100.0	0.0	0.0	100.0
Cefepime	128	>256	4 to >256	0.0	2.7	97.3^c	0.0	2.7	97.3
Cefepime–SPR741	>32	>32	1 to >32	1.4	18.9	79.7^c	1.4	9.5	89.2
Ceftaroline	>256	>256	64 to >256	0.0	0.0	100.0	0.0		100.0
Ceftaroline–SPR741	8	>32	≤0.25 to >32	5.4	18.9	75.7	5.4		94.6
Meropenem	32	>32	4 to >32	0.0	0.0	100.0	0.0	13.5	86.5
Meropenem–SPR741	8	>32	0.5 to >32	6.8	4.1	89.2	10.8	45.9	43.2
Piperacillin–tazobactam	>256	>256	128 to >256	0.0	0.0	100.0	0.0	0.0	100.0
Piperacillin–tazobactam–SPR741	32	>256	0.25 to >256	43.2	14.9	41.9	35.1	8.1	56.8
MBL-producing K. pneumoniae (25)
Aztreonam	>256	>256	≤1 to >256	4.0	0.0	96.0	4.0	0.0	96.0
Aztreonam–SPR741	16	>32	≤0.25 to >32	32.0	16.0	52.0	8.0	24.0	68.0
Mecillinam	256	>256	16 to >256				0.0		100.0^b
Mecillinam–SPR741	2	32	0.5 to >32				80.0		20.0^b
Temocillin	128	>128	16 to >128				0.0		100.0^c
Temocillin	128	>128	16 to >128				20.0		80.0^b
Temocillin–SPR741	4	>32	1 to >32				76.0		24.0^c
Temocillin–SPR741	4	>32	1 to >32				84.0		16.0^b
Cefoxitin	>256	>256	64 to >256	0.0	0.0	100.0
Cefoxitin–SPR741	>32	>32	1 to >32	12.0	8.0	80.0
Ceftazidime	>256	>256	128 > 256	0.0	0.0	100.0	0.0	0.0	100.0
Ceftazidime–SPR741	>256	>256	16 to >256	0.0	0.0	100.0	0.0	0.0	100.0
Cefotaxime	>256	>256	64 to >256	0.0	0.0	100.0	0.0	0.0	100.0
Cefotaxime–SPR741	>32	>32	>32 to >32	0.0	0.0	100.0	0.0	0.0	100.0
Cefepime	>256	>256	8 to >256	0.0	4.0	96.0^c	0.0	0.0	100.0
Cefepime–SPR741	>32	>32	16 to >32	0.0	0.0	100.0^c	0.0	0.0	100.0
Ceftaroline	>256	>256	64 to >256	0.0	0.0	100.0	0.0		100.0
Ceftaroline–SPR741	32	>32	1 to >32	0.0	8.0	92.0	0.0		100.0
Meropenem	>32	>32	0.5 to >32	4.0	4.0	92.0	8.0	0.0	92.0
Meropenem–SPR741	>32	>32	0.5 to >32	4.0	4.0	92.0	8.0	0.0	92.0
Piperacillin–tazobactam	>256	>256	32 to >256	0.0	4.0	96.0	0.0	0.0	100.0
Piperacillin–tazobactam–SPR741	128	>256	1 to >256	4.0	20.0	76.0	4.0	0.0	96.0
OXA-48-like-producing K. pneumoniae (15)
Aztreonam	>256	>256	16 to >256	0.0	0.0	100.0	0.0	0.0	100.0
Aztreonam–SPR741	16	>32	1 to >32	40.0	0.0	60.0	13.3	26.7	60.0
Mecillinam	256	>256	8 to >256				13.3		86.7^b
Mecillinam–SPR741	1	16	0.5 to 16				86.7		13.3^b
Temocillin	>128	>128	128 to >128				0.0		100.0^c
Temocillin	>128	>128	128 to >128				0.0		100.0^b
Temocillin–SPR741	>32	>32	4 to >32				33.3		66.7^c
Temocillin–SPR741	>32	>32	4 to >32				46.7		53.3^b
Cefoxitin	64	256	4 to 256	13.3	6.7	80.0
Cefoxitin–SPR741	16	>32	4 to >32	46.7	26.7	26.7
Ceftazidime	128	256	4 to 256	6.7	6.7	86.7	0.0	6.7	93.3
Ceftazidime–SPR741	2	128	≤0.12 to 128	73.3	0.0	26.7	33.3	40.0	26.7
Cefotaxime	>256	>256	128 to >256	0.0	0.0	100.0	0.0	0.0	100.0
Cefotaxime–SPR741	>32	>32	>32 to >32	0.0	0.0	100.0	0.0	0.0	100.0
Cefepime	>256	>256	16 to >256	0.0	0.0	100.0^c	0.0	0.0	100.0
Cefepime–SPR741	>32	>32	8 to >32	0.0	26.7	73.3^c	0.0	0.0	100.0
Ceftaroline	>256	>256	128 to >256	0.0	0.0	100.0	0.0		100.0
Ceftaroline–SPR741	>32	>32	1 to >32	0.0	6.7	93.3	0.0		100.0
Meropenem	16	32	1 to >32	6.7	13.3	80.0	20.0	26.7	53.3
Meropenem–SPR741	4	32	0.5 to >32	26.7	13.3	60.0	40.0	40.0	20.0
Piperacillin–tazobactam	>256	>256	64 to >256	0.0	6.7	93.3	0.0	0.0	100.0
Piperacillin–tazobactam–SPR741	8	>256	0.5 to >256	66.7	13.3	20.0	53.3	13.3	33.3

MIC results obtained for temocillin were interpreted according to the BSAC¹⁸ systemic criterion (≤8 mg/L for susceptible).

Discussion

Previous studies have reported on the synergistic and/or increased activity of several antimicrobial agents in combination with SPR741.^9,10 The study presented here expands on the previous results by including a greater number of isolates with varying resistance phenotypes/genotypes and antimicrobial agents tested in combination with SPR741. MIC values and susceptibility rates obtained by the combinations were compared with the codrugs tested alone. The in vitro data suggest that SPR741 decreases the MIC results for several agents tested against E. coli and K. pneumoniae carrying relevant β-lactamase genes. SPR741 possesses the ability to disrupt the outer membrane and permeabilize the Gram-negative cell. One can hypothesize that the disruption of the outer membrane may cause the periplasmic space components, including β-lactamases, to be released extracellularly, potentially decreasing the concentration of β-lactamases, minimizing β-lactam hydrolysis and increasing codrug concentration. In addition, a more permeable cell may cause a given codrug to reach its target faster^9–11; however, the activity of β-lactam–SPR741 combinations were not uniform and appeared to be dependent on the β-lactam agent, β-lactamase produced, and bacterial species.

When applying current CLSI breakpoints, the aztreonam and ceftazidime susceptibility rates increased significantly (to 93.8% from 0.0–37.5%) against AmpC-producing isolates. Other agents, such as mecillinam, temocillin, and piperacillin–tazobactam also showed high susceptibility rates (93.8–100.0%) when tested with SPR741. However, these agents had initially high susceptibility rates when tested alone (81.2–100.0%), nevertheless the impact on MIC results was significant (4- to 128-fold). In addition, the MIC values for aztreonam, cefoxitin, and ceftazidime decreased in the presence of SPR741 against ESBL-producing E. coli and these drugs are generally less efficiently hydrolyzed (read k_cat/K_M) by CTX-M enzymes,^19–24 which encompassed the vast majority of ESBL enzymes included in this study. Therefore, it may be possible that the lower enzymatic concentration in the periplasmic space, a less efficient β-lactam hydrolysis, and consequently higher β-lactam concentration act synergistically and explain the increased potency observed.

SPR741 also increased the potency of mecillinam and temocillin 8- to 16-fold against ESBL-producing E. coli, although the increase in susceptibility was less pronounced due to the high in vitro susceptibility observed for these compounds alone.^25–28 The α-methoxy group in the temocillin molecule blocks the entry of a water molecule into the β-lactamase active site cavity, preventing activation of the serine and the chemical events leading to hydrolysis,^28,29 while mecillinam seems to be more stable against a broad range of β-lactamases, including many ESBL enzymes.^29,30 Therefore, temocillin and mecillinam decreased hydrolysis by ESBL enzymes, coupled with increased cell permeability could, at least in part, also explain the potentiation observed with SPR741 against ESBL-producing E. coli. However, these features would not explain the increased potency of piperacillin–tazobactam–SPR741 (64-fold based on MIC₉₀ values), since piperacillin is well hydrolyzed by CTX-M and tazobactam is a good inhibitor of CTX-M.²³ Thus, we speculate that the increased membrane permeability provided by SPR741 could increase the entry of both piperacillin and tazobactam into the bacterial cell, therefore resulting in increased drug concentration and overall activity.^9,11

Several agents, including temocillin, cefoxitin, ceftazidime, and piperacillin–tazobactam showed potentiation in the presence of SPR741 against KPC-producing E. coli, although the lowered MIC results did not result in acceptable (>90% susceptible) in vitro activity, except for temocillin. This agent demonstrated good in vitro activity against KPC producers in previous studies.^29,31,32 The previous temocillin in vitro data against KPC-producing E. coli was confirmed in this study (65.2% and 95.7% susceptibility for systemic and UTI breakpoints, respectively), which increased to 97.8–100.0% when tested with SPR741, regardless of breakpoint. These results indicate that such combination could potentially treat infections caused by KPC-producing E. coli based on the current BSAC systemic breakpoint (≤8 mg/L).¹⁸ In contrast, temocillin was less active against MBL and OXA-48-like E. coli producers, irrespective of SPR741, which may be because this agent is a good substrate for these enzymes.²⁸

Similar to the β-lactam–SPR741 activity profiles observed against ESBL-producing E. coli, the activity of combinations tested against KPC-producing E. coli was not uniform and activity may be associated with the β-lactam hydrolysis rates by the β-lactamase enzymes. E. coli isolates included in this study produced either KPC-2 or KPC-3 (Supplementary Table S1). These enzymes hydrolyze temocillin, ceftazidime, and cefoxitin less efficiently than aztreonam, cefotaxime, or meropenem, and based on MIC₅₀ and MIC₉₀ values, a greater potentiation was observed for the former three agents when paired with SPR741.^{28,29,33–36} However, piperacillin was shown to be well hydrolyzed by KPC-2, whereas tazobactam was unable to provide any protection.³⁷ Still, SPR741 was able to decrease the piperacillin–tazobactam MIC values up to 64-fold compared with piperacillin–tazobactam tested alone against KPC-producing E. coli or to provide a 71.7–84.8% increase in the susceptibility rate. These results favor again the hypothesis that the codrug is reaching its target faster and/or in higher concentration.

A previous study reported that mecillinam was active against NDM and IMP, but not VIM producers.³⁸ The results presented in this study showed that all (57) but 3 MBL-producing isolates (IMP-26, VIM-1, and NDM-1) had mecillinam MIC values above the current UTI breakpoint (≤8 mg/L). In contrast, all strains except a VIM-1-producing E. coli isolate had the mecillinam MICs lowered significantly in the presence of SPR741, resulting in a susceptibility rate of 96.9% (from 9.4% when tested alone). In addition, one could expect the aztreonam–SPR741 combination to be potentially active against MBL-producing E. coli. Although SPR741 provided some degree of potentiation (up to 32-fold), this effect was not enough to increase susceptibility rates above 90% using currently available breakpoints. This may be due to the presence of β-lactamases other than MBL among these isolates (Supplementary Table S1).

SPR741 decreased significantly the MIC₉₀ values (≥64 mg/L) of mecillinam (100% susceptible), ceftazidime (88.2–100% susceptible), and piperacillin–tazobactam (94.1–100.0% susceptible) when tested against OXA-48-like-producing E. coli. Mecillinam and ceftazidime are poor substrates for OXA-48 enzymes, which could at least partially help to maximize the antibacterial activity, once these drugs reach their targets in greater amounts due to the increased cell permeability.^39–41 In contrast, piperacillin is efficiently hydrolyzed by OXA-48, while tazobactam does not possess inhibitory activity toward this enzyme,⁴¹ yet based on MIC results, SPR741 provided a robust potentiation for piperacillin–tazobactam, and susceptibility rates increased to 94.1–100% from 0.0% when tested alone. The reasons for this significant effect on antimicrobial activity remain unclear.

Similar combinations that are active against ESBL-producing E. coli were also active against ESBL-producing K. pneumoniae, including mecillinam, temocillin, ceftazidime, and piperacillin–tazobactam. The reasons for SPR741 to expand the antimicrobial coverage of these agents may be the same reasons as hypothesized earlier, or at least a combination of increased drug cell influx and lower hydrolysis rates. However, the β-lactam–SPR741 combinations tested against ESBL-producing E. coli tended to be more potent than against ESBL-producing K. pneumoniae. These results were generally observed and reported in a previous study.⁹ Considering that both species tested in this study carried similar β-lactamase-encoding genes, these results may suggest that E. coli are more prone than K. pneumoniae to be permeabilized by SPR741.

The combinations tested did not show appreciable activity against MBL- and KPC-producing K. pneumoniae, except for temocillin. SPR741 lowered the temocillin MIC results up to 32-fold against KPC-producing K. pneumoniae, which resulted in susceptibility rates of 78.4% and 94.6% based on current respective systemic and UTI breakpoints. This increased susceptibility indicates that temocillin–SPR741 could be efficacious for treating UTI caused by such pathogens, although some have suggested that temocillin alone could be efficacious for treating UTI caused by KPC producers due to the high concentrations achieved in urine (500 mg/L after 500 mg dose); however, there has not yet been clinical evidence generated to support this statement.^28,29,32 In the present study, temocillin-SPR741 could potentially be even useful for some systemic infections, although it could likely benefit from dosing optimization.^32,42 In fact, a more recent study demonstrated that 6 g of temocillin given in three daily administrations or continuous infusion reached, on average, the necessary free drug concentrations to treat infections caused by Enterobacteriaceae with a MIC of 16 mg/L.⁴² Temocillin inhibited 47.3% of KPC-producing K. pneumoniae at ≤16 mg/L, whereas 85.1% of these isolates were inhibited at ≤16 mg/L with the addition of SPR741 (data not shown).

In summary, this study reports on the activity of several β-lactam agents tested alone and in combination with a new polymyxin derivative, SPR741, against a contemporary challenge set of E. coli and K. pneumoniae. The results presented in this study indicate that some antimicrobial agents were significantly more active in vitro in the presence of SPR741 and could become potential options for treating infections caused by ESBL, KPC, MBL, or OXA-48-like producers. Whether infections caused by a high bacterial inoculum would be refractory to such combination strategy remains to be established. However, it has been documented that inoculum effect is absent or modest for certain β-lactam–β-lactamase combinations.^32,43 It is important to mention that decrease in the codrug MIC values in the presence of SPR741 were not observed in this study against isolates that had elevated MIC results for polymyxins (i.e., >2 mg/L) (data not shown). Also, SPR741 is not expected to decrease the MIC results of any given agents against isolates harboring mutations associated with polymyxin resistance or isolates that are intrinsically resistant to polymyxins (i.e., indole-positive Proteae).⁹ Moreover, the lack of a more uniform activity of β-lactam agents in the presence of SPR741, even against isolates with low polymyxin MIC results (i.e., ≤2 mg/L) suggest that other resistance mechanisms, such as overexpression of the AcrAB-TolC operon could be present in certain isolates.⁹ Additional investigations are needed to better understand the synergistic effects of β-lactam–SPR741 combinations and also the lack thereof. Nevertheless, the promising safety profile of SPR741 compared with that of polymyxin B or colistin along with increased in vitro activity when paired with some agents suggest that these combination strategies deserve further exploration.^13,14

Footnotes

Acknowledgments

The authors express appreciation to the following individuals for technical support and/or article assistance: L. Deshpande, R. Donatelli, Y. Edah, L. Flanigan, H. Huynh, J. Oberholser, B. Roth, and B. Schaefer.

Disclosure Statement

There are no speakers' bureaus or stock options to declare. A.R., N.C., and T.L. are employees of and may hold stock in Spero Therapeutics. All other authors have no competing financial interests exist.

JMI Laboratories contracted to perform services in 2018 for Achaogen, Inc., Albany College of Pharmacy and Health Sciences, Allecra Therapeutics, Allergan, AmpliPhi Biosciences Corp., Amplyx, Antabio, American Proficiency Institute, Arietis Corp., Arixa Pharmaceuticals, Inc., Astellas Pharma, Inc., Athelas, Basilea Pharmaceutica Ltd., Bayer AG, Becton, Dickinson and Company, bioMerieux SA, Boston Pharmaceuticals, Bugworks Research, Inc., CEM-102 Pharmaceuticals, Cepheid, Cidara Therapeutics, Inc., CorMedix, Inc., DePuy Synthes, Destiny Pharma, Discuva Ltd., Dr. Falk Pharma GmbH, Emery Pharma, Entasis Therapeutics, Eurofarma Laboratorios SA, U.S. Food and Drug Administration, Fox Chase Chemical Diversity Center, Inc., Gateway Pharmaceutical, LLC, GenePOC, Inc., Geom Therapeutics, Inc., GlaxoSmithKline plc, Harvard University, Helperby, HiMedia Laboratories, F. Hoffmann-La Roche Ltd., ICON plc, Idorsia Pharmaceuticals Ltd., Iterum Therapeutics plc, Laboratory Specialists, Inc., Melinta Therapeutics, Inc., Merck & Co., Inc., Microchem Laboratory, Micromyx, MicuRx Pharmaceuticals, Inc., Mutabilis Co., Nabriva Therapeutics plc, NAEJA-RGM, Novartis AG, Oxoid Ltd., Paratek Pharmaceuticals, Inc., Pfizer, Inc., Polyphor Ltd., Pharmaceutical Product Development, LLC, Prokaryotics Inc., Qpex Biopharma, Inc., Ra Pharmaceuticals, Inc., Roivant Sciences, Ltd., Safeguard Biosystems, Scynexis, Inc., SeLux Diagnostics, Inc., Shionogi and Co., Ltd., SinSa Labs, Spero Therapeutics, Summit Pharmaceuticals International Corp., Synlogic, T2 Biosystems, Inc., Taisho Pharmaceutical Co., Ltd., TenNor Therapeutics Ltd., Tetraphase Pharmaceuticals, The Medicines Company, Theravance Biopharma, University of Colorado, University of Southern California-San Diego, University of North Texas Health Science Center, VenatoRx Pharmaceuticals, Inc., Vyome Therapeutics, Inc., Wockhardt, Yukon Pharmaceuticals, Inc., Zai Laboratory, and Zavante Therapeutics, Inc.

Funding Information

The development of SPR741 is partially supported by CARB-X. This study was performed by JMI Laboratories and supported by Spero Therapeutics. JMI Laboratories received compensation for services related to performing the in vitro study and preparing this manuscript.

Supplementary Material

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Supplementary Material

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