Activity of novel antibiotics against dual metallo-Beta-lactamase producing Enterobacter hormaechei clinical isolates

Abstract

Background

Metallo-β-lactamase (MBL)-producing carbapenemase-resistant Enterobacterales infections are associated with significant mortality. Several β-lactam/β-lactamase inhibitor combinations (BL/BLI) with promising activity against MBLs are in the pipeline.

Objective

To investigate the in vitro activity of upcoming BL/BLI agents against Enterobacter hormaechei clinical isolates co-producing NDM and VIM enzymes.

Methods

Eleven E. hormaechei isolates co-harbouring bla_NDM-7 and bla_VIM-1 were identified from 10 patients admitted to a tertiary hospital in Santiago, Chile, between July 2022 and July 2023. Reference broth microdilution (BMD) panels were developed to determine minimum inhibitory concentrations (MICs) to aztreonam/avibactam, cefepime/taniborbactam, cefepime/zidebactam, cefiderocol and cefiderocol/xeruborbactam. Cefiderocol population analysis profile–area under the curve (PAP–AUC) was performed in strains exhibiting cefiderocol susceptibility to assess for cefiderocol heteroresistance. Long-read sequencing, using the Oxford Nanopore Technologies platform, was conducted on all isolates to characterize the genomic background of bla_NDM-7 and bla_VIM-1.

Results

Among the 11 E. hormaechei isolates assemblies revealed bla_NDM-7 and bla_VIM-1 were located on separate plasmids. All isolates were susceptible to aztreonam/avibactam, cefepime/taniborbactam and cefepime/zidebactam. Cefiderocol susceptibility was variable; the addition of xeruborbactam restored susceptibility.

Conclusions

Our findings indicate that E. hormaechei clinical isolates co-producing NDM and VIM metallo-carbapenemases exhibited susceptibility to all tested novel BL/BLIs, including aztreonam/avibactam, cefepime/taniborbactam and cefepime/zidebactam. The combination of cefiderocol and xeruborbactam restored the activity of cefiderocol.

Introduction

The dissemination of carbapenemase-producing carbapenem-resistant Enterobacterales (CP-CRE) poses a significant global threat. Among CP-CRE, metallo-β-lactamase (MBL) infections are particularly worrisome due to their high mortality and limited availability of effective treatments.¹ An increase in the prevalence of CP-CRE producing multiple carbapenemases has been reported in several regions, including Latin America.² In general, most reports describe the co-occurrence of two serine carbapenemases (e.g. bla_KPC + bla_OXA-48) or combinations of serine and MBL carbapenemases (e.g. bla_KPC + bla_NDM).^2–4 CP-CRE isolates co-producing multiple MBLs remain rare, but their emergence would raise serious concerns as it may further constrain already limited therapeutic options.

A surge in CP-CRE cases has been reported in Chile since 2021, primarily driven by KPC- or NDM-producing strains.⁵ More recently, Enterobacter hormaechei clinical isolates harbouring two distinct MBL genes, bla_NDM and bla_VIM, have been observed. This evolving epidemiology provides a unique opportunity to assess the activity of novel β-lactam/β-lactamase inhibitor (BL/BLI) combinations with anticipated activity against MBLs: either recently approved (e.g. aztreonam/avibactam) or in advanced stages of development (e.g. cefepime/taniborbactam, cefepime/zidebactam, cefiderocol/xeruborbactam). Here, we evaluated the in vitro activity of these novel BL/BLIs against clinical isolates co-producing NDM and VIM, and provide a genomic characterization of these highly resistant E. hormaechei strains.

Methods

Eleven E. hormaechei isolates co-harbouring bla_NDM and bla_VIM were identified from 10 patients admitted to a tertiary hospital in Santiago, Chile, between July 2022 and July 2023 (Table S1, available as Supplementary data at JAC-AMR Online). Species identification was performed using MALDI-TOF MS. Routine antimicrobial susceptibility testing was conducted using the BD Phoenix^™ system (Becton Dickinson, USA). Carbapenemase genes (i.e. bla_KPC, bla_NDM and bla_VIM) were identified by multiplex PCR⁶ and enzyme production was confirmed by immunochromatography (i.e. NG-Test CARBA 5). Reference broth microdilution (BMD) panels (Thermo Fisher Scientific, USA) were developed to determine minimum inhibitory concentrations (MICs) to the following agents: aztreonam/avibactam, cefepime/taniborbactam, cefepime/zidebactam, cefiderocol and cefiderocol/xeruborbactam. β-lactam dilutions ranged from ≤0.125 to ≥256 mg/L. Testing was performed in triplicate to derive modal MICs. Quality control strains for all agents were selected according to guidance from the Clinical and Laboratory Standards Institute (CLSI) M100 document.⁷

Aztreonam/avibactam susceptibility categorization was based on the recently established CLSI criteria of ≤4/4 mg/L. Investigational breakpoints were used for cefepime/taniborbactam (≤16/4 mg/L) and cefepime/zidebactam (≤64/64 mg/L).^8,9 Cefiderocol and cefiderocol/xeruborbactam categorization were based on the CLSI breakpoint for cefiderocol (≤4 mg/L). Cefiderocol population analysis profile–area under the curve (PAP–AUC) was performed in the few strains exhibiting cefiderocol susceptibility to assess for cefiderocol heteroresistance.¹⁰

Long-read sequencing, using Oxford Nanopore Technology platforms, was conducted for all isolates. To determine high-resolution genetic relationships among isolates from the same lineage, core-genome-based SNP analyses were conducted using an all-against-all approach. Detailed methods are provided in the Supplementary material.

Results

Isolate collection and genomic results

Among the 11 E. hormaechei isolates co-harbouring bla_VIM and bla_NDM, three were recovered from clinical specimens; the remaining from rectal surveillance swabs. All isolates carried bla_NDM-7 and bla_VIM-1 alleles (Table 1). Ten isolates belonged to the sequence type (ST) 133 lineage and the remaining (SCL19716) corresponded to a ST45 lineage (Table S1). Core-genome based SNP analysis revealed 8–47 SNP differences among isolates from the ST133 isolates, indicating close genetic relatedness independent of the source of collection (i.e. clinical versus surveillance isolates); Table S2. Assemblies revealed bla_NDM-7 and bla_VIM-1 were located on separate plasmids (Figure S1). Plasmids carrying bla_NDM-7 belonged to the IncX3 incompatibility group, ranged in size from 45.25 to 46.16 kb, and exhibited a high degree of sequence identity (∼99.9%) (Table S3). The bla_NDM-7 gene was positioned upstream of IS3000 and IS5, adjacent to a truncated ISAba125 and a bleomycin resistance gene (ble_MBL), with IS26 and ISKox3e located further downstream (Figure S2). No further antimicrobial resistance determinants were found within these plasmids. By contrast, plasmids containing bla_VIM-1 belonged to the IncHI2A or IncFIB/IncFII incompatibility groups, with sizes ranging from 173.48 to 297.02 kb. bla_VIM-1 was located downstream of an IS4321 insertion sequence and Class I integron (intI1), and upstream of additional resistance genes including aac(6’)-II, dfrA1, aadA1, smr, qacEΔ1 and sul1 (Figure S2). All bla_VIM-1-carrying plasmids harboured bla_CTX-M-9, an extended spectrum β-lactamase, all but two carried bla_TEM-1.

Table 1.

Antimicrobial susceptibility testing results using broth microdilution testing

Isolate	Aztreonam (mg/L)		Aztreonam-avibactama (mg/L)		Cefepime-taniborbactamb (mg/L)		Cefepime-zidebactamc (mg/L)		Cefiderocold (mg/L)		Cefiderocol-xeruborbactame (mg/L)
SCL 14313	2	S	0.25/4	S	0.5/4	S	≤0.25/0.25	S	16	R	≤0.25/4	S
SCL 15653	2	S	0.125/4	S	0.5/4	S	≤0.25/0.25	S	32	R	≤0.25/4	S
SCL 16617	2	S	0.125/4	S	0.5/4	S	≤0.25/0.25	S	16	R	≤0.25/4	S
SCL 16971	8	I	0.25/4	S	1/4	S	≤0.25/0.25	S	16	R	≤0.25/4	S
SCL 17132	4	S	0.125/4	S	0.5/4	S	≤0.25/0.25	S	16	R	≤0.25/4	S
SCL 17230	2	S	0.125/4	S	0.5/4	S	≤0.25/0.25	S	8	I	≤0.25/4	S
SCL 17600	4	S	0.25/4	S	1/4	S	≤0.25/0.25	S	16	R	≤0.25/4	S
SCL 17735	2	S	0.125/4	S	1/4	S	≤0.25/0.25	S	16	R	≤0.25/4	S
SCL 17942	2	S	0.125/4	S	0.5/4	S	≤0.25/0.25	S	16	R	≤0.25/4	S
SCL 17964	4	S	0.125/4	S	0.5/4	S	≤0.25/0.25	S	32	R	≤0.25/4	S
SCL 19716	≥32	R	2/4	S	2/4	S	≤0.25/0.25	S	4	S	≤0.25/4	S

S, susceptible; I, intermediate; R, resistant.

^aBased on the CLSI aztreonam/avibactam breakpoint of ≤8/4 mg/L.

^bBased on a cefepime/taniborbactam investigational breakpoint of ≤16/4 mg/L.

^cBased on a CLSI-endorsed cefepime/zidebactam investigational breakpoint of ≤64/64 mg/L.

^dBased on the CLSI cefiderocol breakpoint of ≤4 mg/L.

^eBased on the CLSI cefiderocol breakpoint of ≤4 mg/L; susceptibility criteria for cefiderocol/xeruborbactam are not available.

Activity of novel β-lactams

All isolates exhibited susceptibility to aztreonam/avibactam, cefepime/taniborbactam and cefepime/zidebactam, with MIC₅₀ and MIC₉₀ values as follows: aztreonam-avibactam (0.125/4 and 0.5/4 mg/L), cefepime/taniborbactam (0.5/4 and 2/4 mg/L) and cefepime/zidebactam (≤0.25/0.25 and ≤0.25/0.25 mg/L); Table 1. By contrast, nine of 11 isolates were resistant to cefiderocol (MICs = 16–32 mg/L), one was intermediate (MIC = 8 mg/L) and only one had an MIC in the susceptible range (MIC = 4 mg/L). The addition of xeruborbactam reduced cefiderocol MICs, rendering all isolates highly susceptible, with MICs ≤0.25/4 mg/L (Table 1).

Cefiderocol activity, heteroresistance and genomic basis of non-susceptibility

To explore the activity of cefiderocol on E. hormaechei harbouring either bla_NDM or bla_VIM, a subset of 10 isolates available in our biorepository were investigated further (Table S4). All isolates displayed cefiderocol susceptibility, with MICs ranging from 0.5–1 mg/L and 1–4 mg/L for those harbouring bla_NDM or bla_VIM, respectively. Genomic analyses did not reveal changes previously associated to cefiderocol resistance, including siderophore-related proteins (e.g. cirA, fhuA, fur, fepA, fecA),^11,12  ftsl (encoding PBP3) or the ampC locus and its regulators (ampD, ampR, ampG) (Table S5). Notably, the cefiderocol-susceptible isolate (SCL 19716, MIC = 4 mg/L) exhibited pronounced heteroresistance by PAP, with median survival rates of 0.04% and 0.035% at 16 and 32 mg/L, respectively (Figure S3). The mean AUC was 156.6: substantially higher than that of the control strain Escherichia coli ATCC 25922 (AUC: 8.23).

Discussion

Our findings indicate that E. hormaechei clinical isolates co-producing NDM and VIM carbapenemases exhibit susceptibility to all novel BL/BLIs, including aztreonam/avibactam, cefepime/taniborbactam, cefepime/zidebactam and cefiderocol/xeruborbactam. These results are encouraging given the limited treatment options with reliable activity against MBLs, especially as the frequency of CP-CRE infections continues to rise. By contrast, cefiderocol alone exhibited poor activity against dual MBL-producing organisms, with most isolates falling into the non-susceptible range. The addition of xeruborbactam, however, successfully restored cefiderocol activity, underscoring the critical role of BLIs in safeguarding the efficacy of β-lactams amid an expanding repertoire of potent β-lactamases.

Although reports of isolates co-producing multiple MBLs are sporadic, the increasing detection of dual CP-CRE highlights an impending epidemiological shift towards a more complex resistance landscape. This threat has been widely documented,¹³ including a recent report of the multispecies emergence of CP-CRE recovered from infections co-harbouring bla_KPC and bla_NDM obtained across a multicentre network in Chile.¹⁴

Several BL/BLIs demonstrating potent in vitro activity against MBL-producers are in the pipeline. The combination of aztreonam/avibactam exploits avibactam’s inhibition of serine β-lactamases—which frequently co-occur with MBLs—with aztreonam’s stability against MBL hydrolysis. In addition, two novel inhibitors taniborbactam and zidebactam, both combined with cefepime, have completed Phase 3 clinical trials.^15,16 Taniborbactam, a cyclic boronate BLI, exhibits broad inhibitory activity against MBLs, with the notable exception of IMP-type carbapenemases.¹⁷ Zidebactam, functioning as a β-lactam enhancer, increases cefepime’s activity through dual PBP2 binding and β-lactamase inhibition.¹⁸ Finally, xeruborbactam, a cyclic boronate BLI with broad-spectrum MBL inhibition, in earlier stages of development provides added protection to cefiderocol.¹⁹

Regrettably, emerging resistance to novel BL/BLI agents has already been observed. The efficacy of aztreonam/avibactam has been compromised by a combination of resistance mechanisms, such as the co-expression of NDM and CMY-42 beta-lactamases in conjunction with amino acid insertions in PBP3: ultimately reducing the binding affinity of aztreonam (i.e. aztreonam-avibactam), cefiderocol and cefepime (i.e. cefepime-taniborbactam).^20–22 Moreover, taniborbactam demonstrates reduced inhibitory activity against specific NDM and VIM variants harbouring single amino acid substitutions (e.g. NDM-9, VIM-83).²³ While MBL variants resistant to zidebactam or xeruborbactam have thus far not been described, potential vulnerabilities are likely to be revealed as their use increases.

Until recently, cefiderocol was the only clinically-available β-lactam with activity against MBL-producing organisms. Cefiderocol resistance has been associated with increased carbapenemase production,^24,25 changes in iron transport receptors,¹¹ amino acid substitutions in Class C β-lactamases²⁶ and structural modifications of PBP3.²⁷ Our genomic analysis did not identify any known resistance-conferring mutations, suggesting decreased cefiderocol activity was primarily driven by MBL production. Encouragingly, the addition of xeruborbactam restored susceptibility in all isolates. Notably, the only cefiderocol-susceptible isolate in our collection exhibited cefiderocol heteroresistance. The clinical relevance of the heteroresistant phenotype remains unclear.

The scope of our study was limited to E. hormaechei isolates co-harbouring bla_NDM-7 and bla_VIM-1. Future investigations should assess the activity of novel BL/BLIs against dual MBL-producers from diverse geographic regions, encompassing additional bacterial species and alleles. Nevertheless, our findings provide insights into the promising efficacy of upcoming BL/BLIs in combating CP-CRE co-producing multiple MBLs. Continued surveillance and mechanistic studies will be essential to track resistance evolution and optimize therapeutic strategies in this increasingly complex resistance landscape.

Funding

This study was supported by the Agencia Nacional de Investigación y Desarrollo (ANID, Chile) through Fondo Nacional de Desarrollo Científico y Tecnológico regular #1250588 to J.M.M. and Agencia Nacional de Investigación y Desarrollo de Chile Beca Doctorado Nacional #21231099 to C.S. The funders had no role in study design, data collection nor interpretation.

Transparency declarations

J.M.M has received unrestricted research grants from Pfizer and MSD. The remaining authors do not have any disclosures.

Supplementary data

Figures S1 to S3 and Tables S1 to S5 are available as Supplementary data at JAC-AMR Online.