Klebsiella pneumoniae is a Gram-negative bacterium that colonizes the human gut and mucosal surfaces, but it can cause severe, life-threatening infections including pneumonia, bloodstream infections, meningitis, and liver abscesses. The bacterium has long been a fixture of hospital-acquired infection surveillance because of its remarkable capacity to acquire and disseminate antibiotic resistance genes via mobile genetic elements, particularly plasmids. Carbapenems, a class of last-resort beta-lactam antibiotics reserved for the most serious multidrug-resistant infections, have historically been the therapeutic mainstay for treating serious K. pneumoniae infections. The emergence of strains resistant to these agents has therefore been recognized by the World Health Organization as a critical public health threat requiring urgent global attention.

The research team, led by Montaser M. Y. Amro, Aysegul Bostanci, and Buket Baddal of Near East University’s Faculty of Medicine and DESAM Research Institute, set out to determine the prevalence of beta-lactamase genes and hypervirulence-associated markers among clinical carbapenem-resistant K. pneumoniae isolates circulating in Cyprus. Their methodology combined classical microbiological phenotyping with targeted molecular detection of resistance and virulence determinants, providing a comprehensive snapshot of what is circulating in a single tertiary care facility.

The isolates were first identified to the species level and subjected to antimicrobial susceptibility testing using the VITEK-2 automated system, a widely adopted platform in clinical microbiology laboratories. Carbapenem resistance was confirmed using the Modified Hodge test, a phenotypic assay in which a carbapenem-susceptible indicator strain is used to detect carbapenemase production by the test isolate. The results were striking: 75 of the 96 isolates, or 78.1 percent, were confirmed as carbapenem-resistant K. pneumoniae by the Modified Hodge test. This figure alone underscores the substantial burden of carbapenem resistance within the hospital’s patient population.

Molecular characterization of the carbapenemase genes revealed a clear predominance of blaOXA-48, which was detected in 84.0 percent of the carbapenem-resistant isolates. The OXA-48-type carbapenemases, originally described in Turkey and now widespread across Europe, the Middle East, and North Africa, hydrolyze carbapenems and penicillins but spare extended-spectrum cephalosporins. Their dominance in this Cypriot cohort is consistent with regional epidemiological patterns documented by the European Centre for Disease Prevention and Control, which has repeatedly highlighted OXA-48 as the most prevalent carbapenemase in Enterobacterales across the EU and EEA.

The remaining carbapenemase determinants were less frequent but clinically significant. The metallo-beta-lactamase gene blaIMP was found in 8.0 percent of isolates, blaVIM in 4.0 percent, and blaNDM in 2.7 percent. New Delhi metallo-beta-lactamases are particularly feared because they hydrolyze nearly all beta-lactam antibiotics, including carbapenems, and are often embedded in plasmids that carry additional resistance determinants. Perhaps most concerning was the detection of co-existing carbapenemase genes in eight isolates. These strains harbored combinations of blaOXA-48 with either blaNDM, blaIMP, or blaVIM, meaning they carry both a serine carbapenemase and a metallo-beta-lactamase on the same genetic background. Such co-carriage severely constrains therapeutic options and increases the risk that treatment with any single beta-lactam-beta-lactamase inhibitor combination will fail, as the two enzyme classes have complementary hydrolysis spectra that can compensate for each other’s weaknesses.

The hypervirulence component of the investigation is what elevates this study from a routine resistance survey to a report of genuine epidemiological significance. Hypervirulent K. pneumoniae is a distinct pathotype characterized by enhanced ability to cause invasive, metastatic infections in otherwise healthy individuals. Classical hypervirulent strains were first recognized in East Asia, where they caused dramatic cases of community-acquired liver abscess with metastatic spread to the eyes, central nervous system, and other sites. These strains typically overproduce capsular polysaccharide, giving colonies a hypermucoviscous appearance, and produce additional iron-scavenging systems that enhance survival within the host.

To assess hypervirulence potential, the researchers performed the string test, a simple phenotypic assay in which a bacterial colony is touched with an inoculation loop and the resulting string is measured; a positive result, defined as a string extending more than 5 millimeters, indicates hypermucoviscosity associated with hypervirulent potential. They also used conventional polymerase chain reaction to screen for three hypervirulence-associated genes: iucA, which encodes a key enzyme in aerobactin synthesis; peg-344, a metabolic transporter gene associated with hypervirulent strains; and iroB, involved in salmochelin siderophore synthesis.

Of the 75 carbapenem-resistant isolates, 32, or 42.7 percent, exhibited the hypermucoviscous phenotype on string testing. This is a remarkably high proportion for a carbapenem-resistant population and suggests that hypervirulence traits are well established within the resistant strains circulating at the hospital. At the genetic level, the aerobactin synthesis gene iucA was detected in 44 isolates, representing 58.7 percent of the carbapenem-resistant cohort. Aerobactin is a siderophore, an iron-chelating molecule that allows the bacterium to scavenge iron from its host, and it is widely regarded as one of the most reliable molecular markers of hypervirulent K. pneumoniae. Previous studies have shown that aerobactin-positive strains exhibit significantly enhanced virulence in experimental infection models compared to aerobactin-negative strains.

Interestingly, the peg-344 and iroB genes were not detected in any of the examined isolates. This finding suggests that the Cypriot strains possess a partially assembled hypervirulence genetic repertoire rather than the complete complement typically found in classical hypervirulent K. pneumoniae clones. It also highlights an ongoing debate within the field regarding the prudent use of the term “hypervirulence” when applied to carbapenem-resistant isolates, as some researchers have cautioned that partial virulence gene carriage may not equate to the clinical severity associated with classical hypervirulent strains. Nevertheless, the combination of carbapenem resistance with aerobactin production and a hypermucoviscous phenotype in nearly half of the resistant isolates represents a convergence of traits that has historically been associated with worse patient outcomes, including higher mortality rates in bloodstream and intra-abdominal infections.

The convergence of hypervirulence and carbapenem resistance in a single strain is a relatively recent phenomenon in the evolution of K. pneumoniae. For many years, these two traits appeared to occupy separate evolutionary niches: classical multidrug-resistant hospital strains tended to be less virulent, while hypervirulent community strains remained susceptible to most antibiotics. However, reports from China and increasingly from Europe have documented the emergence of strains that carry both resistance determinants and virulence plasmids, often through horizontal transfer of virulence plasmids into resistant backgrounds or acquisition of resistance plasmids into virulent clones. The Cypriot findings add the eastern Mediterranean island to the growing list of regions where this convergence has been documented.

The clinical implications are substantial. Carbapenem-resistant K. pneumoniae infections already carry high mortality because of limited treatment options, typically restricted to combinations of tigecycline, colistin, ceftazidime-avibactam, and meropenem in various permutations. Adding hypervirulence traits to this picture could make infections even more difficult to manage, particularly in vulnerable patient populations such as the elderly, immunocompromised individuals, and those with indwelling medical devices. The presence of metallo-beta-lactamases in some isolates further complicates the therapeutic landscape, as these enzymes are not inhibited by newer beta-lactamase inhibitors such as avibactam.

The Cyprus study also carries regional significance given the island’s position as a crossroads between Europe, the Middle East, and North Africa, and given its proximity to countries with high endemic rates of carbapenem resistance. Cross-border movement of resistant organisms through medical tourism, patient transfer, and population mobility is well documented, and Cyprus’s role as both a destination and a transit point makes it a potentially important location for monitoring the spread of these convergent strains.

The authors of the study emphasize that this is the first report describing carbapenem-resistant K. pneumoniae isolates with hypervirulence-associated characteristics in Cyprus and stress the need for continuous molecular surveillance and stringent infection control measures to prevent further dissemination within the healthcare environment. Their findings serve as a stark reminder that the evolution of dangerous bacterial pathogens does not respect national boundaries and that vigilant, genomics-informed surveillance remains one of the most effective tools available for early detection and containment of emerging threats. As antimicrobial resistance continues to climb globally, studies such as this one provide critical baseline data that will inform future monitoring, infection prevention strategies, and therapeutic decision-making in the region and beyond.