Emergence of silent NDM-1 carbapenemase gene in carbapenem-susceptible Klebsiella pneumoniae: Clinical implications and epidemiological insights.

The global dissemination of carbapenemase genes, particularly blaNDM-1, poses a significant threat to public health. While research has mainly focused on strains with phenotypic resistance, the impact of silent resistance genes has been largely overlooked. This study documents the first instance of silent blaNDM-1 in a cluster of clonally related carbapenem-susceptible K. pneumoniae strains from a single patient. Despite initial effectiveness of carbapenem therapy, the patient experienced four recurrent lung infections over five months, indicating persistent K. pneumoniae infection. Genomic sequencing revealed all strains harbored blaNDM-1 on the epidemic IncX3 plasmid. A deletion within the upstream promoter region (PISAba125) of blaNDM-1 hindered its expression, resulting in phenotypic susceptibility to carbapenems. However, in vitro bactericidal assays and a mouse infection model showed that K. pneumoniae strains with silent blaNDM-1 exhibited significant tolerance to carbapenem-mediated killing. These findings demonstrate that silent blaNDM-1 can mediate both phenotypic susceptibility and antibiotic tolerance. In silico analysis of 1986 blaNDM sequences showed that 1956 (98.5%) retained the original promoter PISAba125. Given that previous genomic sequencing typically targets carbapenem-resistant strains, accurately assessing the prevalence of silent blaNDM remains challenging. This study highlights the hidden threat of silent resistance genes to clinical antimicrobial therapy and calls for enhanced clinical awareness and laboratory detection.

Studies on enmetazobactam clarify mechanisms of widely used ß-lactamase inhibitors.

ß-Lactams are the most important class of antibacterials, but their use is increasingly compromised by resistance, most importantly via serine ß-lactamase (SBL)-catalyzed hydrolysis. The scope of ß-lactam antibacterial activity can be substantially extended by coadministration with a penicillin-derived SBL inhibitor (SBLi), i.e., the penam sulfones tazobactam and sulbactam, which are mechanism-based inhibitors working by acylation of the nucleophilic serine. The new SBLi enmetazobactam, an N-methylated tazobactam derivative, has recently completed clinical trials. Biophysical studies on the mechanism of SBL inhibition by enmetazobactam reveal that it inhibits representatives of all SBL classes without undergoing substantial scaffold fragmentation, a finding that contrasts with previous reports on SBL inhibition by tazobactam and sulbactam. We therefore reinvestigated the mechanisms of tazobactam and sulbactam using mass spectrometry under denaturing and nondenaturing conditions, X-ray crystallography, and NMR spectroscopy. The results imply that the reported extensive fragmentation of penam sulfone­derived acyl­enzyme complexes does not substantially contribute to SBL inhibition. In addition to observation of previously identified inhibitor-induced SBL modifications, the results reveal that prolonged reaction of penam sulfones with SBLs can induce dehydration of the nucleophilic serine to give a dehydroalanine residue that undergoes reaction to give a previously unobserved lysinoalanine cross-link. The results clarify the mechanisms of action of widely clinically used SBLi, reveal limitations on the interpretation of mass spectrometry studies concerning mechanisms of SBLi, and will inform the development of new SBLi working by reaction to form hydrolytically stable acyl­enzyme complexes.

Extended-Spectrum Beta-Lactamase Shigella sonnei Cluster Among Men Who Have Sex with Men in Chicago, Illinois-July-October 2022.

Drug-resistant shigellosis is increasing, particularly among men who have sex with men (MSM). During July-October 2022, an extended-spectrum beta-lactamase producing Shigella sonnei cluster of 9 patients was identified in Chicago, of whom 8 were MSM and 6 were festival attendees. The cluster also included 4 domestic travelers to Chicago. Sexual health care for MSM should include shigellosis diagnosis and prevention.

What Contributes to the MIC? Beyond ß-Lactamase Gene Detection in Klebsiella pneumoniae.

BACKGROUND: K. pneumoniae is capable of resistance to ß-lactam antibiotics through expression of ß-lactamases (both chromosomal and plasmid-encoded) and downregulation of outer membrane porins. However, the extent to which these mechanisms interplay in a resistant phenotype is not well understood. The purpose of this study was to determine the extent to which ß-lactamases and outer membrane porins affected ß-lactam resistance. METHODS: MICs to ß-lactams and inhibitor combinations were determined by agar dilution or E-test. Outer membrane porin production was evaluated by western blot of outer membrane fractions. ß-lactamase carriage was determined by whole genome sequencing and expression evaluated by RT-qPCR. RESULTS: Plasmid-encoded ß--lactamases were important for cefotaxime and ceftazidime resistance. Elevated expression of chromosomal SHV was important for ceftolozane/tazobactam resistance. Loss of outer membrane porins was predictive of meropenem resistance. ESßLs and pAmpCs in addition to porin loss were sufficient to confer resistance to the third generation cephalosporins, pipercillin/tazobactam, ceftolozane/tazobactam, and meropenem. pAmpCs (CMY-2 and DHA) alone conferred resistance to pipercillin/tazobactam. DISCUSSION: Detection of a resistance gene by whole genome sequencing was not sufficient to predict resistance to all antibiotics tested. some ß-lactam resistance was dependent on the expression of both plasmid-encoded and chromosomal ß-lactamases and loss of porins.

Comparison of Daily versus Admission and Discharge Surveillance Cultures for Multidrug-Resistant Organism Detection in an Intensive Care Unit.

BACKGROUND: Admission and discharge screening of patients for asymptomatic gut colonization with multidrug-resistant organisms (MDROs) is a traditional approach to active surveillance, but its sensitivity for detecting colonization is uncertain. METHODS: Daily rectal or fecal swab samples and clinical data were collected over 12 months from patients in one 25-bed intensive care unit (ICU) in Chicago, IL USA and tested for the following multidrug-resistant organisms (MDROs): vancomycin-resistant enterococci (VRE); third-generation cephalosporin-resistant Enterobacterales, including extended-spectrum ß-lactamase-producing Enterobacterales (ESBL); and carbapenem-resistant Enterobacterales (CRE). MDRO detection by (1) admission/discharge surveillance cultures or (2) clinical cultures were compared to daily surveillance cultures. Samples underwent 16S rRNA gene sequencing to measure the relative abundance of operational taxonomic units (OTUs) corresponding to each MDRO. RESULTS: Compared with daily surveillance cultures, admission/discharge cultures detected 91% of prevalent MDRO colonization and 63% of incident MDRO colonization among medical ICU patients. Only a minority (7%) of MDRO carriers were identified by clinical cultures. Higher relative abundance of MDRO-associated OTUs and specific antibiotic exposures were independently associated with higher probability of MDRO detection by culture. CONCLUSION: Admission and discharge surveillance cultures underestimated MDRO acquisitions in an ICU. These limitations should be considered when designing sampling strategies for epidemiologic studies that use culture-based surveillance.

Mutagenesis and structural analysis reveal the CTX-M ß-lactamase active site is optimized for cephalosporin catalysis and drug resistance.

CTX-M ß-lactamases are a widespread source of resistance to ß-lactam antibiotics in Gram-negative bacteria. These enzymes readily hydrolyze penicillins and cephalosporins, including oxyimino-cephalosporins such as cefotaxime. To investigate the preference of CTX-M enzymes for cephalosporins, we examined eleven active-site residues in the CTX-M-14 ß-lactamase model system by alanine mutagenesis to assess the contribution of the residues to catalysis and specificity for the hydrolysis of the penicillin, ampicillin, and the cephalosporins cephalothin and cefotaxime. Key active site residues for class A ß-lactamases, including Lys73, Ser130, Asn132, Lys234, Thr216, and Thr235, contribute significantly to substrate binding and catalysis of penicillin and cephalosporin substrates in that alanine substitutions decrease both kcat and kcat/KM. A second group of residues, including Asn104, Tyr105, Asn106, Thr215, and Thr216, contribute only to substrate binding, with the substitutions decreasing only kcat/KM. Importantly, calculating the average effect of a substitution across the 11 active-site residues shows that the most significant impact is on cefotaxime hydrolysis while ampicillin hydrolysis is least affected, suggesting the active site is highly optimized for cefotaxime catalysis. Furthermore, we determined X-ray crystal structures for the apo-enzymes of the mutants N106A, S130A, N132A, N170A, T215A, and T235A. Surprisingly, in the structures of some mutants, particularly N106A and T235A, the changes in structure propagate from the site of substitution to other regions of the active site, suggesting that the impact of substitutions is due to more widespread changes in structure and illustrating the interconnected nature of the active site.

Interactions of hydrolyzed ß-lactams with the L1 metallo-ß-lactamase: Crystallography supports stereoselective binding of cephem/carbapenem products.

L1 is a dizinc subclass B3 metallo-ß-lactamase (MBL) that hydrolyzes most ß-lactam antibiotics and is a key resistance determinant in the Gram-negative pathogen Stenotrophomonas maltophilia, an important cause of nosocomial infections in immunocompromised patients. L1 is not usefully inhibited by MBL inhibitors in clinical trials, underlying the need for further studies on L1 structure and mechanism. We describe kinetic studies and crystal structures of L1 in complex with hydrolyzed ß-lactams from the penam (mecillinam), cephem (cefoxitin/cefmetazole), and carbapenem (tebipenem, doripenem, and panipenem) classes. Despite differences in their structures, all the ß-lactam-derived products hydrogen bond to Tyr33, Ser221, and Ser225 and are stabilized by interactions with a conserved hydrophobic pocket. The carbapenem products were modeled as Δ1-imines, with (2S)-stereochemistry. Their binding mode is determined by the presence of a 1ß-methyl substituent: the Zn-bridging hydroxide either interacts with the C-6 hydroxyethyl group (1ß-hydrogen-containing carbapenems) or is displaced by the C-6 carboxylate (1ß-methyl-containing carbapenems). Unexpectedly, the mecillinam product is a rearranged N-formyl amide rather than penicilloic acid, with the N-formyl oxygen interacting with the Zn-bridging hydroxide. NMR studies imply mecillinam rearrangement can occur nonenzymatically in solution. Cephem-derived imine products are bound with (3R)-stereochemistry and retain their 3' leaving groups, likely representing stable endpoints, rather than intermediates, in MBL-catalyzed hydrolysis. Our structures show preferential complex formation by carbapenem- and cephem-derived species protonated on the equivalent (ß) faces and so identify interactions that stabilize diverse hydrolyzed antibiotics. These results may be exploited in developing antibiotics, and ß-lactamase inhibitors, that form long-lasting complexes with dizinc MBLs.

Mutation Rate of AmpC ß-Lactamase-Producing Enterobacterales and Treatment in Clinical Practice: A Word of Caution.

In a retrospective multicenter study of 575 patients with bloodstream infections or pneumonia due to wild-type AmpC ß-lactamase-producing Enterobacterales, species with low in vitro mutation rates for AmpC derepression were associated with fewer treatment failures due to AmpC overproduction (adjusted hazard ratio, 0.5 [95% CI, .2-.9]). However, compared to cefepime/carbapenems, using third-generation cephalosporins as definitive therapy remained associated with this adverse outcome (15% vs 1%).

Sequential Treatment Failure With Aztreonam-Ceftazidime-Avibactam Followed by Cefiderocol Due to Preexisting and Acquired Mechanisms in a New Delhi Metallo-ß-lactamase-Producing Escherichia coli Causing Fatal Bloodstream Infection.

We report a fatal case of New Delhi metallo-ß-lactamase (NDM)-producing Escherichia coli in a bacteremic patient with sequential failure of aztreonam plus ceftazidime-avibactam followed by cefiderocol. Acquired resistance was documented phenotypically and mediated through preexisting and acquired mutations. This case highlights the need to rethink optimal treatment for NDM-producing organisms.

The growing threat of NDM-producing E. coli with penicillin-binding protein 3 mutations in the United States - is there a potential role for durlobactam?

We report identification of 5 patients with infections caused by NDM-5-producing E. coli harboring PBP3 mutations that showed reduced susceptibility to aztreonam-avibactam and cefiderocol. Durlobactam, a novel diazabicyclooctane ß-lactamase inhibitor, demonstrated minimum inhibitory concentrations ranging from 0.5 to 2 µg/mL supporting future investigations into a potential role in clinical management.

Potential involvement of beta-lactamase homologous proteins in resistance to beta-lactam antibiotics in gram-negative bacteria of the ESKAPEE group.

BACKGROUND: Enzymatic degradation mediated by beta-lactamases constitutes one of the primary mechanisms of resistance to beta-lactam antibiotics in gram-negative bacteria. This enzyme family comprises four molecular classes, categorized into serine beta-lactamases (Classes A, C, and D) and zinc-dependent metallo-beta-lactamases (Class B). Gram-negative bacteria producing beta-lactamase are of significant concern, particularly due to their prevalence in nosocomial infections. A comprehensive understanding of the evolution and dissemination of this enzyme family is essential for effective control of these pathogens. In this study, we conducted the prospecting, phylogenetic analysis, and in silico analysis of beta-lactamases and homologous proteins identified in 1827 bacterial genomes with phenotypic data on beta-lactam resistance. These genomes were distributed among Klebsiella pneumoniae (45%), Acinetobacter baumannii (31%), Pseudomonas aeruginosa (14%), Escherichia coli (6%), and Enterobacter spp. (4%). Using an HMM profile and searching for conserved domains, we mined 2514, 8733, 5424, and 2957 proteins for molecular classes A, B, C, and D, respectively. This set of proteins encompasses canonical subfamilies of beta-lactamases as well as hypothetical proteins and other functional groups. Canonical beta-lactamases were found to be phylogenetically distant from hypothetical proteins, which, in turn, are closer to other representatives of the penicillin-binding-protein (PBP-like) and metallo-beta-lactamase (MBL) families. The catalytic amino acid residues characteristic of beta-lactamases were identified from the sequence alignment and revealed that motifs are less conserved in homologous groups than in beta-lactamases. After comparing the frequency of protein groups in genomes of resistant strains with those of sensitive ones applying Fisher's exact test and relative risk, it was observed that some groups of homologous proteins to classes B and C are more common in the genomes of resistant strains, particularly to carbapenems. We identified the beta-lactamase-like domain widely distributed in gram-negative species of the ESKAPEE group, which highlights its importance in the context of beta-lactam resistance. Some hypothetical homologous proteins have been shown to potentially possess promiscuous activity against beta-lactam antibiotics, however, they do not appear to expressly determine the resistance phenotype. The selective pressure due to the widespread use of antibiotics may favor the optimization of these functions for specialized resistance enzymes.

Pseudomonas guariconensis Necrotizing Fasciitis, United Kingdom.

We describe a case of necrotizing fasciitis in the United Kingdom in which Pseudomonas guariconensis was isolated from multiple blood culture and tissue samples. The organism carried a Verona integron-encoded metallo-ß-lactamase gene and evidence of decreased susceptibility to ß-lactam antimicrobial agents. Clinicians should use caution when treating infection caused by this rare pathogen.

Evolutionary "Crowdsourcing": Alignment of Fitness Landscapes Allows for Cross-species Adaptation of a Horizontally Transferred Gene.

Genes that undergo horizontal gene transfer (HGT) evolve in different genomic backgrounds. Despite the ubiquity of cross-species HGT, the effects of switching hosts on gene evolution remains understudied. Here, we present a framework to examine the evolutionary consequences of host-switching and apply this framework to an antibiotic resistance gene commonly found on conjugative plasmids. Specifically, we determined the adaptive landscape of this gene for a small set of mutationally connected genotypes in 3 enteric species. We uncovered that the landscape topographies were largely aligned with minimal host-dependent mutational effects. By simulating gene evolution over the experimentally gauged landscapes, we found that the adaptive evolution of the mobile gene in one species translated to adaptation in another. By simulating gene evolution over artificial landscapes, we found that sufficient alignment between landscapes ensures such "adaptive equivalency" across species. Thus, given adequate landscape alignment within a bacterial community, vehicles of HGT such as plasmids may enable a distributed form of genetic evolution across community members, where species can "crowdsource" adaptation.

The mechanism of ceftazidime and cefiderocol hydrolysis by D179Y variants of KPC carbapenemases is similar and involves the formation of a long-lived covalent intermediate.

Klebsiella pneumoniae carbapenemase (KPC) variants have been described that confer resistance to both ceftazidime-avibactam and cefiderocol. Of these, KPC-33 and KPC-31 are D179Y-containing variants derived from KPC-2 and KPC-3, respectively. To better understand this atypical phenotype, the catalytic mechanism of ceftazidime and cefiderocol hydrolysis by KPC-33 and KPC-31 as well as the ancestral KPC-2 and KPC-3 enzymes was studied. Steady-state kinetics showed that the D179Y substitution in either KPC-2 or KPC-3 is associated with a large decrease in both kcat and KM such that kcat/KM values were largely unchanged for both ceftazidime and cefiderocol substrates. A decrease in both kcat and KM is consistent with a decreased and rate-limiting deacylation step. We explored this hypothesis by performing pre-steady-state kinetics and showed that the acylation step is rate-limiting for KPC-2 and KPC-3 for both ceftazidime and cefiderocol hydrolysis. In contrast, we observed a burst of acyl-enzyme formation followed by a slow steady-state rate for the D179Y variants of KPC-2 and KPC-3 with either ceftazidime or cefiderocol, indicating that deacylation of the covalent intermediate is the rate-limiting step for catalysis. Finally, we show that the low KM value for ceftazidime or cefiderocol hydrolysis of the D179Y variants is not an indication of tight binding affinity for the substrates but rather is a reflection of the deacylation reaction becoming rate-limiting. Thus, the hydrolysis mechanism of ceftazidime and cefiderocol by the D179Y variants is very similar and involves the formation of a long-lived covalent intermediate that is associated with resistance to the drugs.

The interaction of the azetidine thiazole side chain with the active site loop (ASL) 3 drives the evolution of IMP metallo-ß-lactamase against tebipenem.

Imipenemase (IMP) metallo-ß-lactamases (MBLs) hydrolyze almost all available ß-lactams including carbapenems and are not inhibited by any commercially available ß-lactamase inhibitor. Tebipenem (TP) pivoxil is the first orally available carbapenem and possesses a unique bicyclic azetidine thiazole moiety located at the R2 position. TP has potent in vitro activity against Enterobacterales producing extended-spectrum and/or AmpC ß-lactamases. Thus far, the activity of TP against IMP-producing strains is understudied. To address this knowledge gap, we explored the structure activity relationships of IMP MBLs by investigating whether IMP-6, IMP-10, IMP-25, and IMP-78 [MBLs with expanded hydrolytic activity against meropenem (MEM)] would demonstrate enhanced activity against TP. Most of the Escherichia coli DH10B strains expressing IMP-1 variants displayed a ≥twofold MIC difference between TP and MEM, while those expressing VIM or NDM variants demonstrated comparable MICs. Catalytic efficiency (kcat/KM) values for the TP hydrolysis by IMP-1, IMP-6, IMP-10, IMP-25, and IMP-78 were significantly lower than those obtained for MEM. Molecular dynamic simulations reveal that V67F and S262G substitutions (found in IMP-78) reposition active site loop 3, ASL-3, to better accommodate the bicyclic azetidine thiazole side chain, allowing microbiological/catalytic activity to approach that of comparison MBLs used in this study. These findings suggest that modifying the R2 side chain of carbapenems can significantly impact hydrolytic stability. Furthermore, changes in conformational dynamics due to single amino acid substitutions should be used to inform drug design of novel carbapenems.

Structural role of K224 in taniborbactam inhibition of NDM-1.

Taniborbactam (TAN; VNRX-5133) is a novel bicyclic boronic acid ß-lactamase inhibitor (BLI) being developed in combination with cefepime (FEP). TAN inhibits both serine and some metallo-ß-lactamases. Previously, the substitution R228L in VIM-24 was shown to increase activity against oxyimino-cephalosporins like FEP and ceftazidime (CAZ). We hypothesized that substitutions at K224, the homologous position in NDM-1, could impact FEP/TAN resistance. To evaluate this, a library of codon-optimized NDM K224X clones for minimum inhibitory concentration (MIC) measurements was constructed; steady-state kinetics and molecular docking simulations were next performed. Surprisingly, our investigation revealed that the addition of TAN restored FEP susceptibility only for NDM-1, as the MICs for the other 19 K224X variants remained comparable to those of FEP alone. Moreover, compared to NDM-1, all K224X variants displayed significantly lower MICs for imipenem, tebipenem, and cefiderocol (32-, 133-, and 33-fold lower, respectively). In contrast, susceptibility to CAZ was mostly unaffected. Kinetic assays with the K224I variant, the only variant with hydrolytic activity to FEP comparable to NDM-1, confirmed that the inhibitory capacity of TAN was modestly compromised (IC50 0.01 µM vs 0.14 µM for NDM-1). Lastly, structural modeling and docking simulations of TAN in NDM-1 and in the K224I variant revealed that the hydrogen bond between TAN's carboxylate with K224 is essential for the productive binding of TAN to the NDM-1 active site. In addition to the report of NDM-9 (E149K) as FEP/TAN resistant, this study demonstrates the fundamental role of single amino acid substitutions in the inhibition of NDM-1 by TAN.

Relative inhibitory activities of the broad-spectrum ß-lactamase inhibitor taniborbactam against metallo-ß-lactamases.

Taniborbactam (TAN) is a novel broad-spectrum ß-lactamase inhibitor with significant activity against subclass B1 metallo-ß-lactamases (MBLs). Here, we showed that TAN exhibited an overall excellent activity against B1 MBLs including most NDM- and VIM-like as well as SPM-1, GIM-1, and DIM-1 enzymes, but not against SIM-1. Noteworthy, VIM-1-like enzymes (particularly VIM-83) were less inhibited by TAN than VIM-2-like. Like NDM-9, NDM-30 (also differing from NDM-1 by a single amino acid substitution) was resistant to TAN.

Inter-phylum circulation of a beta-lactamase-encoding gene: a rare but observable event.

Beta-lactamase-mediated degradation of beta-lactams is the most common mechanism of beta-lactam resistance in Gram-negative bacteria. Beta-lactamase-encoding genes can be transferred between closely related bacteria, but spontaneous inter-phylum transfers (between distantly related bacteria) have never been reported. Here, we describe an extended-spectrum beta-lactamase (ESBL)-encoding gene (blaMUN-1) shared between the Pseudomonadota and Bacteroidota phyla. An Escherichia coli strain was isolated from a patient in Münster (Germany). Its genome was sequenced. The ESBL-encoding gene (named blaMUN-1) was cloned, and the corresponding enzyme was characterized. The distribution of the gene among bacteria was investigated using the RefSeq Genomes database. The frequency and relative abundance of its closest homolog in the global microbial gene catalog (GMGC) were analyzed. The E. coli strain exhibited two distinct morphotypes. Each morphotype possessed two chromosomal copies of the blaMUN-1 gene, with one morphotype having two additional copies located on a phage-plasmid p0111. Each copy was located within a 7.6-kb genomic island associated with mobility. blaMUN-1 encoded for an extended-spectrum Ambler subclass A2 beta-lactamase with 43.0% amino acid identity to TLA-1. blaMUN-1 was found in species among the Bacteroidales order and in Sutterella wadsworthensis (Pseudomonadota). Its closest homolog in GMGC was detected frequently in human fecal samples. This is, to our knowledge, the first reported instance of inter-phylum transfer of an ESBL-encoding gene, between the Bacteroidota and Pseudomonadota phyla. Although the gene was frequently detected in the human gut, inter-phylum transfer was rare, indicating that inter-phylum barriers are effective in impeding the spread of ESBL-encoding genes, but not entirely impenetrable.

Characterization of Acinetobacter baumannii-calcoaceticus complex isolates and microbiological outcome for patients treated with sulbactam-durlobactam in a phase 3 trial (ATTACK).

Acinetobacter baumannii-calcoaceticus complex (ABC) causes severe, difficult-to-treat infections that are frequently antibiotic resistant. Sulbactam-durlobactam (SUL-DUR) is a targeted ß-lactam/ß-lactamase inhibitor combination antibiotic designed to treat ABC infections, including those caused by multidrug-resistant strains. In a global, pathogen-specific, randomized, controlled phase 3 trial (ATTACK), the efficacy and safety of SUL-DUR were compared to colistin, both dosed with imipenem-cilastatin as background therapy, in patients with serious infections caused by carbapenem-resistant ABC. Results from ATTACK showed that SUL-DUR met the criteria for non-inferiority to colistin for the primary efficacy endpoint of 28-day all-cause mortality with improved clinical and microbiological outcomes compared to colistin. This report describes the characterization of the baseline ABC isolates from patients enrolled in ATTACK, including an analysis of the correlation of microbiological outcomes with SUL-DUR MIC values and the molecular drivers of SUL-DUR resistance.

In vitro activity of meropenem-vaborbactam plus aztreonam against metallo-ß-lactamase-producing Klebsiella pneumoniae.

We evaluated the in vitro activity of meropenem-vaborbactam plus aztreonam (MEV-ATM) against 140 metallo-ß-lactamase (MBL)-producing Klebsiella pneumoniae isolates. Among them, 25 isolates (17.9%) displayed minimum inhibitory concentrations (MIC) ≥ 8 µg/mL, while 112 (80.0%) had MIC ≤ 2 µg/mL. Genomic analysis and subsequent gene cloning experiments revealed OmpK36 134-135GD-insertion and increased carbapenemase gene (blaNDM-1 and blaOXA-48-like) copy numbers are the main factors responsible for MEV-ATM non-susceptibility. Notably, MEV-ATM is actively against aztreonam-avibactam-resistant mutants due to CMY-16 mutations.