Superbug With Multiple Carbapenemases Shows Genomic Diversity but New Drug Combinations Hold the Line

A nationwide study of Italian hospitals has delivered one of the most detailed portraits yet of Klebsiella pneumoniae strains that carry not just one but multiple carbapenemase enzymes, the bacterial weapons that dismantle last-resort carbapenem antibiotics. The investigation, conducted by the MULTI-KLEB Study Group and published in Molecular Biology Reports, combined whole-genome sequencing with extensive laboratory susceptibility testing to answer two urgent questions: how genetically diverse these feared pathogens are, and whether the newest generation of antibiotic combinations can still stop them. The findings offer both a warning about the relentless spread of resistance genes and a measure of reassurance that several novel agents retain striking potency.

The scale of the surveillance effort was considerable. Between 2022 and 2023, the researchers collected 414 non-duplicate carbapenemase-producing K. pneumoniae isolates from 14 Italian hospitals, drawing from bloodstream infections, the most clinically consequential setting for these organisms. Within this collection, isolates producing more than one carbapenemase, designated multi-carbapenemase-producing K. pneumoniae or MCP-Kp, accounted for 2.4 percent of all carbapenemase producers. That figure may appear modest, but it represents a category of pathogen that clinicians dread: bacteria equipped with several distinct enzymes capable of degrading nearly every beta-lactam antibiotic in the pharmacopoeia, leaving treatment options perilously thin.

Genomic sequencing revealed that the MCP-Kp population was emphatically polyclonal, meaning it did not descend from a single successful clone sweeping through hospitals. Instead, the researchers identified six distinct multilocus sequence types among the multi-carbapenemase isolates: ST512, ST147, ST383, ST35, ST320, and ST6668. Several of these lineages, such as ST512 and ST147, are internationally recognized high-risk clones with a documented history of hospital outbreaks, while ST383 has recently been reported as spreading through central Italy. The presence of multiple unrelated lineages carrying multiple carbapenemases points strongly to horizontal gene transfer as the dominant force assembling these resistance arsenals, rather than simple clonal expansion.

The genomic architecture of these isolates underscored that conclusion. The strains displayed heterogeneous capsule loci, the gene clusters that determine the polysaccharide coat surrounding the cell, alongside a diverse array of plasmid replicons, the genetic backbones on which resistance genes hitch rides between bacterial cells. Each isolate carried an extensive repertoire of beta-lactamase genes, and the researchers documented porin alterations, changes to the outer membrane channels through which antibiotics enter the cell, that further reduce drug susceptibility. Beyond beta-lactam resistance, widespread non-beta-lactam resistance determinants were detected, confirming that these bacteria resist entire classes of antimicrobials simultaneously. Two isolates carried disruptions in the mgrB gene, a well-characterized mechanism that confers resistance to colistin, an older antibiotic often reserved as a treatment of last resort.

Perhaps the most striking genomic finding was the sheer variety of carbapenemase combinations. The MCP isolates displayed six distinct pairings of carbapenemase enzymes, reflecting the modular way bacteria acquire resistance. Carbapenemases fall into different enzymatic families: class A enzymes such as KPC, class B metallo-beta-lactamases such as NDM, which are impervious to all currently available beta-lactamase inhibitors, and class D enzymes of the OXA-48-like family. When a single strain co-produces enzymes from different classes, the resulting phenotype approaches true pan-beta-lactam resistance, because each family covers the weaknesses of the others. This combinatorial logic explains why multi-carbapenemase organisms are considered an evolutionary endpoint of the resistance arms race and why their emergence is monitored as a sentinel event in antimicrobial resistance surveillance.

Against this formidable background, the susceptibility testing produced results that will interest clinicians worldwide. The MCP-Kp isolates exhibited high-level resistance to most beta-lactam antibiotics and to first- and second-generation beta-lactam/beta-lactamase inhibitor combinations, the older pairings such as piperacillin-tazobactam and ceftazidime-avibactam alone. However, three newer agents held firm. Aztreonam/avibactam, a combination that pairs the only monobactam active against gram-negative bacteria with an inhibitor that neutralizes class A and class C enzymes while aztreonam itself evades metallo-beta-lactamases, inhibited 100 percent of the isolates. Cefepime/zidebactam, which combines a cephalosporin with a dual-action protein synthesis inhibitor that also acts as a beta-lactamase-resistant binding agent, likewise achieved 100 percent susceptibility. Cefiderocol, a siderophore cephalosporin that hijacks bacterial iron-uptake systems to shuttle the drug across the outer membrane, retained activity against 80 percent of the isolates.

The synergy experiments added a second layer of therapeutic insight. For aztreonam-resistant, metallo-beta-lactamase-producing isolates, the researchers tested combinations pairing aztreonam with ceftazidime/avibactam, meropenem/vaborbactam, or cefepime/enmetazobactam. In every such isolate, the combination produced consistent synergy. The mechanistic rationale is elegant: the beta-lactamase inhibitor component suppresses the non-metallo enzymes, such as KPC or OXA-48-like carbapenemases, that would otherwise destroy aztreonam, while the metallo-beta-lactamase itself is intrinsically unable to hydrolyze aztreonam. This division of labor restores the efficacy of a drug that would fail as monotherapy. The strategy has clinical precedent, with observational studies of ceftazidime/avibactam plus aztreonam in bloodstream infections caused by metallo-beta-lactamase-producing Enterobacterales showing encouraging outcomes, and the present findings extend that evidence to the multi-carbapenemase subset.

The study’s conclusions about how this resistance emerged carry important implications for control. The polyclonal population structure, the diversity of plasmid replicons, and the heterogeneous capsule loci all indicate that plasmid-mediated dissemination and horizontal gene transfer, rather than a single outbreak strain, are driving the accumulation of multiple carbapenemases in Italian hospitals. This means that infection control measures targeting one clone will not suffice; the resistance genes themselves are mobile and can be exchanged across lineages. K. pneumoniae has long been described as a major worldwide source and shuttle of antibiotic resistance, and these data illustrate that role in real time. Surveillance systems that combine genomic typing with susceptibility testing, as this study did, are therefore essential for detecting the assembly of multi-carbapenemase strains before they become established.

The therapeutic message is cautiously optimistic but nuanced. Aztreonam/avibactam and cefepime/zidebactam emerge as the most reliable options against this collection of extensively resistant isolates, with cefiderocol as a valuable but imperfect alternative given the 20 percent of strains it failed to inhibit. For laboratories without access to the newest agents, the demonstrated synergy of aztreonam with inhibitor-protected beta-lactams offers a rational combination strategy, though synergy testing requires specialized methods and clinical validation. The authors note that the preserved activity of these agents underscores promising therapeutic strategies for managing infections caused by these highly resistant pathogens. As multi-carbapenemase organisms continue their global spread, documented now in Europe, the Americas, and the Middle East, studies of this kind provide the genomic and phenotypic groundwork on which treatment guidelines and stewardship policies will increasingly depend.

Subject of Research: Genomic characterization and antimicrobial susceptibility of multi-carbapenemase-producing Klebsiella pneumoniae bloodstream isolates

Article Title: Multi-carbapenemase-producing Klebsiella pneumoniae: Genomic characterization and In Vitro evaluation of cefiderocol and novel β-lactam/β-lactamase inhibitor combinations

Article References: Cornacchia, A., Comini, S., Pomilio, F., Quirino, A., Paglietti, B., Greco, R., Costa, C., Boattini, M., Bianco, G., on behalf of the MULTI-KLEB Study Group, Lupo, L., Ambretti, S., Gerardino, A., Degl’Innocenti, L., Ranieri, S. C., Fasciana, T., Mazzariol, A., Quirino, A., Farina, C., … Sacco, F. (2026). Multi-carbapenemase-producing Klebsiella pneumoniae: Genomic characterization and In Vitro evaluation of cefiderocol and novel β-lactam/β-lactamase inhibitor combinations. Molecular Biology Reports, 53(1), Article 1699. https://doi.org/10.1007/s11033-026-12798-4

Image Credits: AI Generated

DOI: 10.1007/s11033-026-12798-4

Keywords: Klebsiella pneumoniae, multi-carbapenemase, antimicrobial resistance, cefiderocol, aztreonam/avibactam, cefepime/zidebactam, metallo-beta-lactamase, whole-genome sequencing, plasmid-mediated resistance, bloodstream infections, beta-lactamase inhibitors, hospital surveillance