
==== Front
Antimicrob Agents Chemother
Antimicrob Agents Chemother
aac
Antimicrobial Agents and Chemotherapy
0066-4804
1098-6596
American Society for Microbiology 1752 N St., N.W., Washington, DC

39133022
aac00272-24
10.1128/aac.00272-24
aac.00272-24
Mechanisms of Resistance
bacteriologyBacteriologyEfficacy of aspergillomarasmine A/meropenem combinations with and without avibactam against bacterial strains producing multiple β-lactamases
https://orcid.org/0000-0002-1654-7903
Rotondo Caitlyn M. 1 2 3
https://orcid.org/0000-0002-9129-7131
Wright Gerard D. 1 2 3 wrightge@mcmaster.ca

1 David Braley Centre for Antibiotic Discovery, McMaster University , Hamilton, Ontario, Canada
2 M.G. DeGroote Institute for Infectious Disease Research, McMaster University , Hamilton, Ontario, Canada
3 Department of Biochemistry and Biomedical Sciences, McMaster University , Hamilton, Ontario, Canada
Editor Poirel Laurent University of Fribourg , Fribourg, Switzerland

Address correspondence to Gerard D. Wright, wrightge@mcmaster.ca
The authors declare no conflict of interest.

9 2024
12 8 2024
12 8 2024
68 9 e00272-2421 2 2024
16 7 2024
Copyright © 2024 Rotondo and Wright.
2024
Rotondo and Wright
https://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

ABSTRACT

The effectiveness of β-lactam antibiotics is increasingly threatened by resistant bacteria that harbor hydrolytic β-lactamase enzymes. Depending on the class of β-lactamase present, β-lactam hydrolysis can occur through one of two general molecular mechanisms. Metallo-β-lactamases (MBLs) require active site Zn2+ ions, whereas serine-β-lactamases (SBLs) deploy a catalytic serine residue. The result in both cases is drug inactivation via the opening of the β-lactam warhead of the antibiotic. MBLs confer resistance to most β-lactams and are non-susceptible to SBL inhibitors, including recently approved diazabicyclooctanes, such as avibactam; consequently, these enzymes represent a growing threat to public health. Aspergillomarasmine A (AMA), a fungal natural product, can rescue the activity of the β-lactam antibiotic meropenem against MBL-expressing bacterial strains. However, the effectiveness of this β-lactam/β-lactamase inhibitor combination against bacteria producing multiple β-lactamases remains unknown. We systematically investigated the efficacy of AMA/meropenem combination therapy with and without avibactam against 10 Escherichia coli and 10 Klebsiella pneumoniae laboratory strains tandemly expressing single MBL and SBL enzymes. Cell-based assays demonstrated that laboratory strains producing NDM-1 and KPC-2 carbapenemases were resistant to the AMA/meropenem combination but became drug-susceptible upon adding avibactam. We also probed these combinations against 30 clinical isolates expressing multiple β-lactamases. E. coli, Enterobacter cloacae, and K. pneumoniae clinical isolates were more susceptible to AMA, avibactam, and meropenem than Pseudomonas aeruginosa and Acinetobacter baumannii isolates. Overall, the results demonstrate that a triple combination of AMA/avibactam/meropenem has potential for empirical treatment of infections caused by multiple β-lactamase-producing bacteria, especially Enterobacterales.

KEYWORDS

antibiotic resistance
aspergillomarasmine A
beta-lactams
carbapenems
cephems
penams
metallo-beta-lactamases
serine-beta-lactamases
Canadian Government | Canadian Institutes of Health Research (CIHR) PJT183745 Wright Gerard D. cover-dateSeptember 2024
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pmcINTRODUCTION

The use of antibiotics selects for resistance mechanisms that readily spread among bacterial pathogens (1, 2). Consequently, many bacterial isolates now demonstrate resistance to multiple antibiotics (3, 4). These multidrug-resistant (MDR) pathogens threaten the effectiveness of antibiotics, such as β-lactams, resulting in one of the biggest threats to public health in the 21st century (5). There is an urgent need to preserve the activity of existing antibiotics to circumvent the threat posed by MDR bacteria.

The predominant resistance mechanism to β-lactams is enzyme-catalyzed inactivation by β-lactamases. These enzymes are divided into four classes based on their sequence identity and mechanism of action. β-Lactamases utilize either an active site serine residue (class A, C, and D serine-β-lactamases) or active site Zn2+ ions (class B metallo-β-lactamases) to inactivate β-lactams by hydrolyzing the β-lactam warhead essential to their activity (1, 6). An increasing number of bacteria contain multiple β-lactamases from different classes (7, 8). Therefore, combination therapy, which uses two or more antibiotics and/or resistance inhibitors to target these β-lactamase-producing bacteria, is increasingly common in recovering the activity of β-lactam antibiotics. A significant advantage of this approach is synergy, where the combined effect of two or more antibiotics and/or inhibitors is greater than the sum of their individual activities (4, 9). Given the rise of MDR strains and the growing diversity of β-lactamases, the discovery of a therapy that targets bacteria producing any combination of all four classes of β-lactamases would be the most advantageous.

Several serine-β-lactamase (SBL) inhibitors have been developed for clinical use (e.g., avibactam, vaborbactam, clavulanic acid) in fixed-dose combinations with various β-lactam antibiotics (10, 11). However, metallo-β-lactamases (MBLs) remain a clinical challenge due to their insensitivity to approved SBL inhibitors and their ability to hydrolyze most β-lactam antibiotics (12, 13). Aspergillomarasmine A (AMA) is a natural product synthesized by Aspergillus versicolor (14). Although AMA exhibits no antimicrobial activity, its inhibitory potency increases when combined with β-lactam antibiotics, especially carbapenems, such as meropenem (15). Indeed, AMA was shown to restore the activity of meropenem against Enterobacteriaceae, Acinetobacter, and Pseudomonas isolates, producing either NDM-1 or VIM-2, two clinically relevant MBLs (14).

Although AMA offers a means to mitigate the presence of MBLs, given the growing number of MDR isolates expressing both MBLs and SBLs, adding an SBL inhibitor provides a strategy to expand the effectiveness of an AMA/meropenem combination therapy. This study used isogenic Escherichia coli and Klebsiella pneumoniae laboratory strains expressing individual MBL and SBL genes together with MDR clinical isolates to evaluate the AMA/meropenem pairing efficacy. The inhibitory potency of different AMA/β-lactam combinations was also explored in the presence of avibactam, which can inhibit several SBL enzymes (16). The resulting data provide a road map to study the effect of combining MBL and SBL inhibitors in treating infections caused by bacteria producing multiple β-lactamases.

RESULTS

Construction of tandem β-lactamase gene expression plasmids

We previously determined that AMA can rescue meropenem activity in MBL-producing bacteria (14, 15). However, the efficiency of an AMA/meropenem combination against bacterial strains producing multiple β-lactamases remains unknown. To investigate this activity, one MBL (NDM-1) and one SBL (KPC-2) were cloned into the low-copy number plasmid pGDP2 in tandem, creating NDM-1/KPC-2 and KPC-2/NDM-1 ordered constructs (17). These plasmids contained a PLac promoter upstream of both genes (Fig. S1). Once transformed into E. coli BW25113, we determined the minimum inhibitory concentration (MIC) for different β-lactam antibiotics to investigate gene expression.

Under individual promoter control, the NDM-1/KPC-2 and KPC-2/NDM-1 constructs had identical meropenem, piperacillin, cefepime, and cefaclor MIC values and similar doripenem and cefotaxime MIC values (Table S1). These results are consistent with the observation that these two β-lactamase enzymes confer resistance to these antibiotics (Table S2). To determine if the genes were being expressed equally in the constructs, we evaluated the MIC for aztreonam, which is known to be susceptible to KPC-2 but not NDM-1 (Table S2). The NDM-1/KPC-2 and KPC-2/NDM-1 constructs demonstrated respective aztreonam MIC values of 128 and 256 µg/mL, indicating similar KPC expression (Table S1).

Inhibition of multiple β-lactamases by the AMA/meropenem combination is dependent on β-lactamase class and carbapenemase activity

We previously demonstrated that the MBL inhibitory potency of AMA varied with the β-lactam partner, and that the optimal antibiotic was a carbapenem, such as meropenem (15). To follow up on this finding, we sought to determine the efficacy of an AMA/meropenem combination against 10 E. coli BW25113 and 10 K. pneumoniae ATCC 33495 strains, producing one MBL and one SBL. These included common β-lactamases from each class, such as KPC-2 and CTX-M-15 (class A), NDM-1 (class B), CMY-2 (class C), and OXA-48 (class D) (18–22). In addition, certain strains were designed to produce OXA-23, which is among the most frequently isolated carbapenem-hydrolyzing class D β-lactamase and a predominant cause of carbapenem resistance in A. baumannii isolates worldwide (23).

Most E. coli and K. pneumoniae strains required an AMA concentration of 8–16 µg/mL to restore the activity of meropenem to its susceptibility breakpoint (Tables 1 and 2). The NDM-1/KPC-2 and KPC-2/NDM-1 constructs demonstrated the highest resistance level, requiring a concentration of AMA of >32 µg/mL to restore susceptibility to meropenem (Tables 1 and 2), consistent with the carbapenemase activity of KPC-2 (Table S2). Furthermore, OXA-23 and OXA-48 showed little to no activity against meropenem and doripenem when produced by E. coli BW25113 (Table S2) and had only modest resistance in K. pneumoniae (Table S3). Singkham-in et al. have reported that carbapenem susceptibility varied between different K. pneumoniae isolates in a strain-specific manner partially related to gene expression levels (24). Overall, the results demonstrated that strains expressing SBL carbapenemases (e.g., KPC-2) are resistant to the AMA/meropenem combination and may also require the presence of an SBL inhibitor (e.g., avibactam) to alleviate β-lactam resistance.

TABLE 1 Concentration of AMA needed to restore the activity of different β-lactam antibiotics to their susceptibility breakpoint concentration against E. coli BW25113 strains producing one MBL and one SBLa,i,j

β-Lactamase b	[AMA] at the susceptibility breakpoint of the antibiotics in different combinations (µg/mL)c,d	
Gene 1	Gene 2	AMA/
MEM	AVI/
MEM	AMA/AVI/
MEM	AMA/AVI/
DOR	AMA/AVI/
PIP	AMA/AVI/
AMP	AMA/AVI/
CTX	AMA/AVI/
FEP	AMA/AVI/
CEC	
NDM-1f	KPC-2e	> 32	16	8	8	16	> 32	32	16	> 32	
NDM-1	CTX-M-15e	16	16	8	8	16	> 32	32	16	> 32	
NDM-1	CMY-2g	16	16	8	8	16	> 32	32	16	> 32	
NDM-1	OXA-23h	16	16	16	16	32	> 32	32	16	> 32	
NDM-1	OXA-48h	16	16	8	16	16	> 32	32	16	> 32	
a Both β-lactamase genes were cloned into pGDP2 with individual promoters.

b Because similar results were obtained regardless of the position of the β-lactamase genes, the data for the constructs with NDM-1 at position two were removed from the table.

c The EUCAST susceptibility breakpoint concentrations for MEM, DOR, PIP, AMP, CTX, FEP, and CEC are 2, 1, 8, 8, 1, 1, and 1 µg/mL.

d AVI was maintained at 4 µg/mL, except during the AVI/MEM combination.

e Class A.

f Class B.

g Class C.

h Class D.

i AMA, aspergillomarasmine A; AVI, avibactam; MEM, meropenem; DOR, doripenem; PIP, piperacillin; AMP, ampicillin; CTX, cefotaxime; FEP, cefepime; CEC, cefaclor.

j All assays were conducted in duplicate.

TABLE 2 Concentration of AMA needed to restore the activity of meropenem to its susceptibility breakpoint concentration against K. pneumoniae ATCC 33495 strains producing one MBL and one SBLa,i,j

β-Lactamase b	[AMA] at 2 µg/mL of MEM in different combinations (µg/mL)c,d	
Gene 1	Gene 2	AMA/
MEM	AVI/
MEM	AMA/AVI/
MEM	
NDM-1f	KPC-2e	> 64	> 64	8	
NDM-1	CTX-M-15e	16	> 64	16	
NDM-1	CMY-2g	8	> 64	8	
NDM-1	OXA-23h	16	> 64	16	
NDM-1	OXA-48h	8	> 64	16	
a Both β-lactamase genes were cloned into pGDP2 with individual promoters.

b Because similar results were obtained regardless of the position of the β-lactamase genes, the data for the constructs with NDM-1 at position two were removed from the table.

c 2 µg/mL is the EUCAST susceptibility breakpoint concentration for MEM.

d AVI was maintained at 4 µg/mL, except during the AVI/MEM combination.

e Class A.

f Class B.

g Class C.

h Class D.

i AMA, aspergillomarasmine A; AVI, avibactam; MEM, meropenem.

j All assays were conducted in duplicate.

β-Lactamase inhibitors showed a greater inhibitory potency when combined with a carbapenem antibiotic

To determine the optimal AMA/avibactam/β-lactam combination, we partnered AMA and avibactam with two carbapenems (meropenem, doripenem), two penams (piperacillin, ampicillin), and three cephems (cefotaxime, cefepime, cefaclor). The inhibitory potency of these combinations was evaluated against 10 laboratory E. coli strains, producing one MBL and one SBL. The results demonstrated that most E. coli strains required 8 µg/mL of AMA and 4 µg/mL of avibactam to restore susceptibility to both meropenem and doripenem (Table 1). For strains producing either NDM-1/KPC-2 or KPC-2/NDM-1, this is an improvement over the AMA/meropenem combination, where resistance to meropenem was observed even at 32 µg/mL of AMA. Furthermore, most strains generally become resensitized to piperacillin and cefepime at 16 µg/mL of AMA and 4 µg/mL of avibactam (Table 1). However, >32 µg/mL of AMA and 4 µg/mL of avibactam were necessary to restore susceptibility to ampicillin and cefaclor (Table 1). The wild-type E. coli BW25113 strain encodes a chromosomal cephalosporinase (AmpC). Therefore, these results are consistent with carbapenems, piperacillin, cefepime, and cefotaxime being poor substrates for AmpC, whereas ampicillin and older cephalosporins are susceptible to hydrolysis by this β-lactamase (25). The results indicated that AMA and avibactam achieved the greatest inhibitory potency when combined with a carbapenem antibiotic such as meropenem.

The efficacy of the AMA/avibactam/meropenem combination depends on the class of β-lactamase and the bacterial order

The meropenem concentration chosen to evaluate the effectiveness of AMA and avibactam was 2 µg/mL, the European Committee on Antimicrobial Susceptibility Testing (EUCAST) susceptibility breakpoint of meropenem (26). These AMA/avibactam/meropenem potentiation assays were then evaluated against 10 K. pneumoniae ATCC 33495 strains producing one MBL and one SBL. Similar to the results against E. coli, all K. pneumoniae strains were resensitized to meropenem at 8–16 µg/mL of AMA and 4 µg/mL of avibactam (Table 2). However, more variability in the effectiveness of the AMA/avibactam/meropenem combination was observed when tested against 30 MDR clinical strains.

Several clinical strains became susceptible to meropenem at 8–16 µg/mL of AMA and 4 µg/mL of avibactam (Table 3). The results were similar whether these strains produced seven different β-lactamases (CTX-M-15, TEM-166, OXA-2, NDM-1, CMY-6, AmpC, and OXA-1), such as E. coli GN610 or just three β-lactamases (ACT-17, VIM-1, and AmpH), such as E. cloacae 397260. If no β-lactamases were being expressed, the AMA/avibactam/meropenem combination was not expected to affect the antibiotic susceptibility of these clinical strains. In addition, the results demonstrated that the addition of avibactam to the AMA/meropenem combination generally increased inhibitory potency against strains producing multiple class D OXA β-lactamases but had minimal effect on strains producing β-lactamases solely from classes A–C. Furthermore, AMA/avibactam/meropenem was less effective against P. aeruginosa isolates, requiring concentrations >64 µg/mL of AMA and 4 µg/mL of avibactam to restore meropenem susceptibility, even if only VIM-2 was being produced (e.g., P. aeruginosa H1010812). In addition, A. baumannii B1NG08a showed a high level of resistance to the AMA/ avibactam/meropenem combination (Table 3; Table S6) due to the production of OXA-23, a carbapenem-hydrolyzing class D β-lactamase, which is non-susceptible to AMA and avibactam (14, 27). Additional findings demonstrated that the efficacy of the AMA/avibactam/ meropenem combination was not necessarily related to the initial degree of avibactam resistance for the strains. For example, E. coli GN688 and P. rettgeri GN570 conferred avibactam MIC values of 16 and 1,024 µg/mL. Yet, both strains were resensitized to meropenem with 16 µg/mL of AMA and 4 µg/mL of avibactam (Table 3; Table S4). Overall, the results indicated that the AMA/avibactam/meropenem combination was effective against Enterobacterales producing β-lactamases from classes A–D but lacked activity against Pseudomonas and Acinetobacter isolates.

TABLE 3 Concentration of AMA needed to restore the activity of meropenem to its susceptibility breakpoint concentration in combination with and without avibactam against clinical strains producing multiple β-lactamasesh,i

Straina	β-Lactamases	[AMA] at 2 µg/mL of MEM (µg/mL)b,c	
AMA/MEM	AVI/MEM	AMA/AVI/MEM	
Acinetobacter baumannii B1NG08a	NDM-1e	OXA-23g	OXA-69g	> 64	> 64	> 64	
Citrobacter freundii
GN978	TEM-1d
NDM-1e
OXA-1g	CTX-M-15d
CMY-6f	CMY-46f	8	> 64	8	
CMY-18f	
Enterobacter cloacae
36749	NDM-1e	TEM-171d	OXA-1g	8	16	8	
AmpHf	ACT-25	
Enterobacter cloacae
47219	ACT-17f	NDM-1e	OXA-1g	8	32	16	
LAP-2d	TEM-1d	AmpHf	
Enterobacter cloacae
86502	ACT-17f	TEM-1d	CTX-M-15d	8	32	16	
OXA-1g	VIM-1e	AmpHf	
Enterobacter cloacae 397260	ACT-17f	VIM-1e	AmpHf	8	32	8	
Enterobacter cloacae GN574	TEM-1d
OXA-1g	NDM-1e	CTX-M-15d	16	> 64	16	
ACT-17f	
Enterobacter cloacae GN579	CTX-M-15
ACT-25f	TEM-1
OXA-1	NDM-1e	16	32	8	
Enterobacter cloacae GN687	NDM-1e
TEM-1d	OXA-9g
ACT-25f	CTX-M-15d	16	> 64	16	
Escherichia coli
130392–1	TEM-1d
CMY-83f	NDM-5e	CMY-42f	32	32	32	
Escherichia coli
376762	NDM-5e
TEM-1d
AmpC1f	CTX-M-15d
AmpHf	OXA-1g
AmpCf	32	8	32	
Escherichia coli
376948	AmpC1f
TEM-1d	AmpHf
AmpCf	NDM-5e	32	8	> 64	
Escherichia coli
387039	AmpHf
AmpCf
OXA-1g	NDM-5e
AmpC1f	OXA-181g
CMY-2f	> 64	64	64	
Escherichia coli
GN610	CTX-M-15d
NDM-1e
OXA-1g	TEM-166d
CMY-6f	OXA-2g
AmpCf	> 64	16	16	
Escherichia coli
GN688	CTX-M-15d
OXA-1g	TEM-1d
AmpCf	NDM-1e	16	8	8	
Klebsiella oxytoca
GN942	NDM-1e
OXA-9g	CTX-M-15d
OXY-2–8d	TEM-1d	8	32	8	
Klebsiella pneumoniae 86500	SHV-11d
NDM-5
AmpHf	CTX-M-15d
OXA-1g	OXA-232g
TEM-1d	> 64	> 64	64	
Klebsiella pneumoniae 110027	CTX-M-14d
OXA-48g
OXA-9g	NDM-1e
SHV-1d	CTX-M-15d
TEM-1d	> 64	> 64	16	
Klebsiella pneumoniae 130392–2	DHA-1f
OXA-232g
OXA-9g	SHV-27d
CTX-M-15d
CMY-42f	NDM-1e
TEM-183d	16	64	16	
Klebsiella pneumoniae 420322	SHV-1d
CTX-M-15d
AmpHf	OXA-181g
OXA-1g	NDM-5e
TEM-1d	> 64	> 64	64	
Klebsiella pneumoniae GN529	TEM-1d
SHV-144d	NDM-1e
OXA-1g	CTX-M-15d
SHV-66d	8	> 64	8	
Klebsiella pneumoniae GN629	SHV-11d
DHA-7f	CTX-M-15d
OXA-1g	NDM-1e	16	> 64	16	
Klebsiella pneumoniae N11-0306	CTX-M-15d
OXA-1g	SHV-11d
DHA-7f	NDM-1e	16	> 64	16	
Klebsiella pneumoniae N11-2218	NDM-1e
SHV-83d	CTX-M-15d
SHV-144d	CMY-6f
SHV-11d	8	> 64	8	
Morganella morganii GN575	CTX-M-15d	NDM-1e	DHA-1f	8	> 64	8	
Providencia rettgeri
GN570	NDM-1e
CMY-6f	VEB-1ad
OXA-1g	TEM-1d	64	> 64	16	
Providencia stuartii
GN576	CMY-2f	NDM-1e		8	> 64	8	
Pseudomonas aeruginosa 411090	NDM-1e
PDC-3f	VEB-9d
OXA-50g	OXA-10g	> 64	> 64	> 64	
Pseudomonas aeruginosa H1010805	VIM-2e			> 64	> 64	> 64	
Pseudomonas aeruginosa H1010812	IMP-7e	PDC-7f	OXA-485g	> 64	> 64	> 64	
a Sequencing data are available in the NCBI BioProject database under accession number PRJNA532924.

b 2 µg/mL is the EUCAST susceptibility breakpoint concentration for MEM.

c AVI was maintained at 4 µg/mL, except during the AVI/MEM combination.

d Class A.

e Class B.

f Class C.

g Class D.

h AMA, aspergillomarasmine A; AVI, avibactam; MEM, meropenem.

i All assays were conducted in duplicate.

DISCUSSION

Although AMA has previously been shown to restore the activity of β-lactam antibiotics against MBL-expressing bacteria (14, 15), many bacterial strains produce multiple β-lactamases from different classes (7, 8). Therefore, tandem β-lactamase pGDP2 expression vectors were constructed to explore the inhibitory potency of the AMA/avibactam/β-lactam antibiotic combinations against 10 E. coli and 10 K. pneumoniae laboratory strains producing one MBL (NDM-1) and one SBL (KPC-2, CTX-M-15, CMY-2, OXA-23, or OXA-48) enzyme.

Generating plasmids with a promoter upstream of both β-lactamase genes resulted in similar antibiograms, regardless of the position of the genes in the constructs (Table S1). These results are consistent with previous studies, which demonstrated that adding a second promoter allowed equal expression of two genes in the same plasmid (28). The effectiveness of AMA and avibactam in combination with β-lactam antibiotics from three subclasses (penam, cephem, and carbapenem) could then be determined against bacterial strains containing these plasmids. Consistent with our previous study, AMA and avibactam demonstrated the most significant inhibitory potency when paired with a carbapenem antibiotic, such as meropenem or doripenem (Table 1), possibly due to β-lactam antibiotics having varying affinities for their targets, the penicillin-binding proteins (PBPs) (15). For example, carbapenems (e.g., meropenem and doripenem) are potent inhibitors of essential PBPs, such as PBP2, which is important for bacterial cell shape and elongation (29, 30). Comparatively, ampicillin’s bactericidal activity results from inhibiting several non-essential PBPs (30).

Consistent with the mode of action of AMA, the AMA/meropenem combination demonstrated no activity against SBL carbapenemases (e.g., KPC-2) in E. coli and K. pneumoniae laboratory strains (14). However, in the presence of AMA and avibactam, the activity of meropenem was restored against all laboratory strains, producing multiple β-lactamases (Tables 1 and 2). These results indicated that combining MBL and SBL inhibitors can restore β-lactam susceptibility against bacterial strains producing multiple β-lactamases. In addition, the inhibitory potency of the AMA/avibactam/meropenem combination did not appear to be pathogen-dependent for two representative Enterobacteriaceae laboratory strains.

The inhibitory potency of AMA, avibactam, and meropenem was also explored against 30 MDR clinical isolates. The AMA/meropenem and AMA/avibactam/meropenem combinations were most effective against Enterobacterales isolates producing SBLs from classes A and C and NDM-1 (Table 3). However, the AMA/meropenem combination was less effective against clinical isolates producing multiple class D OXA β-lactamases or NDM variants (e.g., NDM-5). Still, meropenem susceptibility generally increased upon adding avibactam to the combination. These results are consistent with previous studies demonstrating that AMA is a rapid and potent MBL inhibitor (14),whereas avibactam effectively inhibits class A, C, and some class D enzymes (27).

The AMA/meropenem and AMA/avibactam/meropenem combinations demonstrated little activity against A. baumannii and P. aeruginosa isolates regardless of their β-lactamase content (Table 3). This observed resistance could reflect the notable level of intrinsic and acquired antibiotic resistance mechanisms in P. aeruginosa (31). Furthermore, previous studies have shown that avibactam has little to no activity against carbapenem-hydrolyzing class D β-lactamases, such as OXA-23, OXA-24/40, OXA-48, OXA-58 and OXA-143 (27, 32), which are typically produced by A. baumannii (22, 23). Therefore, different SBL inhibitors may be required to alleviate A. baumannii and P. aeruginosa resistance. For example, durlobactam and taniborbactam have shown potent activity in clinical trials against MDR Acinetobacter spp. and carbapenem-resistant P. aeruginosa, respectively (27).

These results demonstrate that an AMA/avibactam/meropenem combination may have value in infections caused by Enterobacteriaceae producing class A, B, and C β-lactamase enzymes. As MDR isolates increasingly express many β-lactamases, higher-order combinations of antibiotics with β-lactamase inhibitors should be considered for further development to ensure adequate coverage of resistance mechanisms.

MATERIALS AND METHODS

Construction of pGDP2 vectors containing a promoter for each β-lactamase gene

The sequences of the β-lactamase genes were obtained from the Comprehensive Antibiotic Resistance Database (CARD) (33). The pGDP2 plasmids containing a single β-lactamase gene were constructed as described previously (15). These plasmids were used as the foundation for cloning the second β-lactamase gene into the pGDP2 vectors. The construction of the pGDP2:NDM-1/KPC-2 (Fig. S1) began with the polymerase chain reaction (PCR) amplification of blaNDM-1 from the pGDP2:NDM-1 plasmid using 5′–GCC AGC CTA GCC GGG AGA TCT–3′ as a forward primer and 5′–CCG TTG AGC ACC GCC GCC GCA GAA GGC CAT CCT GAC GGA TGG–3′ as the reverse primer. To facilitate gene expression in downstream experiments, the primers were designed to amplify the PLac promoter, the sequence of blaNDM-1, and the rrnB T2 terminator from the template plasmid. In addition, the forward and reverse primers were engineered to amplify approximately 20 nucleotides upstream and downstream, respectively, of the BglII recognition site of the pGDP2 vector. Gibson assembly was then employed to insert the purified blaNDM-1 DNA into a BglII digested pGDP2:KPC-2 plasmid, following the guidelines specified by the manufacturer (34).

Following XbaI digestion, the purified plasmid was transformed into chemically competent E. coli BW25113 or K. pneumoniae ATCC 33,495 cells to verify the second gene’s insertion. Transformation into K. pneumoniae cells was conducted using the freeze–thaw transformation procedure described in reference (35). The sequence of the plasmid was verified by Sanger sequencing. All other pGDP2 vectors containing two β-lactamase genes and two promoters were constructed as described above but using their respective β-lactamase genes.

Cell-based antimicrobial assays

All cell-based assays were conducted in 96-well round base micro test plates (Sarstedt, Nümbrecht, Germany) based on the protocol described in references (15, 36). Most compounds employed in these assays were dissolved in water. However, AMA was diluted in water containing ≤5% (v/v) ammonium hydroxide (NH4OH), avibactam was dissolved in dimethyl sulfoxide (DMSO), and cefaclor was diluted in water containing 1 M hydrochloric acid (HCl). If water was used as a solvent, compounds were filter-sterilized before use.

For all the cell-based assays, a bacterial inoculum was prepared from the bacterial cells of interest using colonies picked from overnight plates whose optical density at 625 nm (OD625) was adjusted to 0.08–0.10. Once the optimal OD625 was reached, a 200-fold dilution of the inoculum in cation-adjusted Mueller Hinton II broth (CAMHB) was conducted before adding it to the micro test plate for a final assay volume of 100 µL.

For minimum inhibitory concentration (MIC) assays, 10 twofold dilutions of the β-lactam antibiotics or β-lactamase inhibitors (3.9–2,000 ng/mL, 0.5–256 µg/mL or 8–4,096 µg/mL) were added along the x-axis of the plate. Two columns were reserved for controls; one served as a growth control as it contained only bacterial inoculum, whereas the other was a sterility control containing only CAMHB. The MIC value was determined as the lowest concentration of β-lactam antibiotic or β-lactam inhibitor showing no bacterial growth.

Two-dimensional checkerboard assays were performed using AMA and meropenem or avibactam and meropenem. Briefly, twofold dilutions of AMA (2–32 µg/mL for E. coli BW25113 strains and 0.5–64 µg/mL for all other strains) or avibactam (1–16 µg/mL for E. coli BW25113 strains and 0.5–64 µg/mL for all other strains) were added along the x-axis. Twofold dilutions of meropenem (1–16 µg/mL for E. coli BW25113 strains and 0.5–64 µg/mL for all other strains) were then added along the y-axis of the plate. Four columns were reserved for controls; one contained twofold dilutions of AMA or avibactam, one possessed twofold dilutions of meropenem to verify MIC values, and the last two contained the bacterial inoculum and CAMHB to serve as growth and sterility controls. The efficacy of the combination was scored based on the minimum concentration of AMA or avibactam required to restore meropenem growth inhibition. According to the European Committee on Antimicrobial Susceptibility Testing (EUCAST), the susceptibility breakpoint concentration for meropenem is 2 µg/mL (26).

Potentiation assays were conducted with AMA, avibactam, and a β-lactam antibiotic. In brief, twofold dilutions of AMA (2–32 µg/mL for E. coli BW25113 strains and 0.5–64 µg/mL for all other strains) were added along the x-axis of a plate, whereas twofold dilutions of avibactam (1–16 µg/mL for E. coli BW25113 strains and 0.5–64 µg/mL for all other strains) were added along the y-axis of the plate. The β-lactam antibiotic was added to both plate axes at its EUCAST susceptibility breakpoint concentration (26). The susceptibility breakpoint concentrations for meropenem, doripenem, piperacillin, ampicillin, cefotaxime, cefepime, and cefaclor were 2, 1, 8, 8, 1, 1, and 1 µg/mL, respectively. Four columns of the plates were reserved for controls; three contained twofold dilutions of either AMA, avibactam, or the β-lactam antibiotic to verify MIC values, whereas one alternatively contained the bacterial inoculum and CAMHB to serve as growth and sterility controls. The efficacy of the combination was scored based on the concentration of AMA required to restore the activity of the different β-lactam antibiotics to their respective EUCAST susceptibility breakpoint concentration at 4 µg/mL of avibactam (26).

After a 16–20 h static incubation at 37°C, bioassay plates containing E. coli BW25113 were shaken for 5 min to resuspend the bacterial cells. However, bioassay plates containing all other strains were resuspended manually using a pipette to minimize the formation of aerosols. The bioassay plates were read spectrophotometrically at a wavelength of 600 nm using a BioTek Synergy H1 plate reader (BioTek, Winooski, VT). All cell-based assays were performed in duplicate.

Genomic DNA extraction

LB medium was inoculated with the appropriate clinical strain. The inoculated medium was incubated at 37°C in a shaking incubator for 16–20 h. Cells were harvested by centrifugation (10,000×g, 3 min, room temperature) using a Fisher Scientific accuSpin Micro 17 microcentrifuge (Thermo Fisher Scientific, Waltham, MA). Cell pellets were resuspended in a combination of 180 µL of genomic digestion buffer [25 mM Tris-HCl, 2.5 mM EDTA, 1% (w/v) SDS, pH 8.0] and 20 µL of proteinase K (final concentration of 1–5 mg/mL). The resuspended cells were incubated at 55°C for 2 h with occasional mixing. The samples were supplemented with 20 µL of RNase A (final concentration of 1 mg/mL) and placed in a 37°C static incubator for 2 h. Following incubation, 200 µL of genomic lysis/binding buffer [30 mM Tris-HCl, 30 mM EDTA, 800 mM guanidine thiocyanate, 5% (v/v) Triton X-100, 5% (v/v) Tween 20, pH 8.0] was added, and the samples were mixed until a homogenous solution was obtained. A volume of 500 µL of phenol:chloroform:isoamyl alcohol (25:24:1) was then used to separate unwanted proteins and cellular debris from the genomic DNA. Following centrifugation (17,000×g, 5 min, room temperature), the genomic DNA, located in the top aqueous layer, was removed and placed in a new microcentrifuge tube. The phenol:chloroform:isoamyl alcohol extraction was repeated until no white precipitate existed between the organic and aqueous phases. The genomic DNA was then supplemented with a 0.1 vol of 3 M sodium acetate (pH 5.2) and one volume of cold 2-propanol. The tube was then gently inverted until a precipitate could be seen. Following centrifugation (12,000×g, 10 min, 4°C), the supernatant was removed, while ensuring that the precipitated DNA remained undisturbed. The pellet was washed with 1 mL of cold 70% (v/v) ethanol. The supernatant was removed after centrifugation (12,000×g, 10 min, 4°C). This ethanol wash was repeated twice. The pellet was then allowed to air dry before being resuspended in 50 µL of Tris-EDTA buffer. The purity of the genomic DNA was analyzed using a NanoDrop Spectrophotometer (Thermo Fisher Scientific) and a 1.0% (w/v) agarose gel. The genomes of the clinical strains were then sequenced by Illumina sequencing. Following sequencing, the genomes of the clinical strains were assembled, and resistance genes were identified by analyzing the genome assemblies using the Resistance Gene Identifier from the CARD, where only perfect and strict hits were retained (33).

ACKNOWLEDGMENTS

We thank Hamilton Health Sciences, Barbara Willey (Mount Sinai Hospital), Dr. Johann Pitout (University of Calgary), Dr. Michael Mulvey (Public Health Agency of Canada), and Dr. Roberto Melano (Public Health Ontario) for the clinical strains. We also thank Allison Guitor for preparing libraries and assembling genomes for the sequenced clinical strains.

This research was funded by a Canadian Institutes of Health Research grant (PJT183745).

DATA AVAILABILITY

Sequencing data are available in the National Institutes of Health (NCBI) BioProject database under accession number PRJNA532924.

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/aac.00272-24.

10.1128/aac.00272-24.SuF1 Supplemental material aac.00272-24-s0001.docx

Figure S1; Tables S1 to S4.

ASM does not own the copyrights to Supplemental Material that may be linked to, or accessed through, an article. The authors have granted ASM a non-exclusive, world-wide license to publish the Supplemental Material files. Please contact the corresponding author directly for reuse.
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