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.

Pseudomonas aeruginosa is an environmental bacterium that can also become a formidable opportunistic pathogen, particularly in hospitals. It is associated with pneumonia, bloodstream infections, urinary tract infections and wound infections, with the greatest risks affecting people whose immune defenses are weakened or who require invasive devices such as ventilators and catheters. Treatment is difficult because the species naturally resists many antibiotics and can rapidly acquire additional resistance mechanisms. Carbapenems, a class of broad-spectrum antibiotics often reserved for serious infections, are among the drugs used when other treatments fail. KPC enzymes can destroy carbapenems before they reach their bacterial targets, converting an already difficult pathogen into a multidrug-resistant one.

The enzyme is produced from the blaKPC gene, which is frequently carried on plasmids or other mobile genetic structures. Plasmids are independent DNA molecules that can move between bacteria, sometimes transferring resistance genes across otherwise unrelated strains. This mobility makes KPC resistance especially important for genomic surveillance: investigators must determine not only whether a bacterium is resistant, but also whether the resistance is spreading through a successful bacterial clone, through transferable DNA, or through both processes at once. The new study, led by researchers in China and published in BMC Genomics, combined these perspectives by examining the chromosomes, plasmids, resistance genes and evolutionary relationships of a worldwide collection of KPC-producing P. aeruginosa genomes.

To build the dataset, the researchers first generated a complete genome for a clinical ST463 isolate known as PA328 using hybrid sequencing. This approach combines sequencing technologies with different strengths. Short-read sequencing can accurately identify individual DNA bases, while long-read sequencing helps assemble repetitive regions and resolve large structural features such as plasmids, genomic islands and rearrangements. A complete reference genome can therefore provide a more reliable framework for comparing fragmented public assemblies. The PA328 genome was analyzed alongside 655 publicly available KPC-producing P. aeruginosa genomes using comparative genomics, phylogenomic reconstruction, pangenome analysis, recombination screening and Bayesian temporal modelling.

The resulting evolutionary analysis divided the bacteria into eight major phylogenetic lineages. A phylogenetic tree reconstructs relationships from patterns of shared and differing mutations, allowing researchers to estimate which isolates are closely related and which represent more distant branches. These eight groups had different combinations of sequence types and geographic distributions rather than forming one globally mixed population. ST463 was particularly prominent in China, ST282 was associated mainly with the United States, and ST654 was linked to Chile. Such geographic clustering does not by itself prove that a lineage originated in a particular country or that transmission occurred directly between patients, but it indicates that local healthcare systems, antibiotic exposure, bacterial introductions and infection-control conditions may be influencing which clones become established.

ST463 was the dominant sequence type in the full collection, representing 43.6 percent of the genomes. Sequence types are defined through multilocus sequence typing, a method that classifies bacteria according to DNA variation in a set of housekeeping genes. They are useful for tracking major clones, but they do not capture every important difference in a bacterial genome. Two isolates with the same sequence type may still differ in plasmids, resistance genes, virulence-associated regions or recently acquired DNA. The study’s broader genomic comparisons therefore add detail to the ST463 signal, showing how a successful lineage can diversify while retaining a recognizable evolutionary backbone. The overwhelming prevalence of blaKPC-2, detected in 99.5 percent of the genomes, suggests that this particular resistance determinant has become a defining feature of the sampled KPC-producing population.

The investigators also searched the assemblies for potentially important mutations and disrupted genes. Their screening identified candidate frameshift calls in genes annotated as participating in metabolism, iron acquisition, transport and functions associated with virulence. A frameshift occurs when inserted or deleted DNA shifts the three-letter reading frame used to translate a gene into a protein. The resulting protein may be shortened or otherwise altered, potentially changing bacterial behavior. However, assembly-based predictions can be misleading when sequencing errors, repetitive DNA or mixed populations create false signals. The authors therefore present these findings as candidates requiring confirmation with raw read-level data and experimental studies. In other words, the analysis points to biological clues, but does not yet establish that each predicted frameshift changes virulence, antibiotic susceptibility or the ability to survive in a host.

The study found descriptive patterns in the way antibiotic-resistance genes and mobile genetic elements occurred together. These elements include plasmids, transposons, integrative structures and other pieces of DNA capable of moving within or between genomes. When resistance genes repeatedly appear in the same genetic neighbourhood, they may be inherited together or transferred as a package, potentially allowing exposure to one antibiotic to help maintain resistance to several others. The researchers did not frame every observed association as proof of direct gene transfer, because co-occurrence can arise through shared ancestry as well as recent mobility. Nevertheless, the patterns emphasize why routine surveillance based only on bacterial species or resistance phenotype can miss the pathways connecting outbreaks and regions.

Within ST463, the analysis identified what the researchers operationally defined as a high-recombination region, or HRR. Recombination is the exchange or replacement of DNA between related organisms, and it can accelerate bacterial evolution by bringing together genetic segments that arose in different lineages. The ST463 HRR contained genes annotated as being involved in secretion systems, biofilm formation, metabolism and mobile genetic elements. Secretion systems act as molecular machines that export proteins or other substances and can contribute to interactions with host cells. Biofilms are structured microbial communities enclosed in a self-produced matrix; they can attach to surfaces, resist environmental stress and reduce the effectiveness of antibiotics or immune responses. The presence of these gene categories in a recombination-rich region makes the area a plausible target for further investigation, although its precise effects remain untested.

A comparison between plasmid sequences and the ST463 HRR revealed partial similarity, suggesting a possible evolutionary connection between mobile DNA and the recombination-rich chromosomal region. The result could reflect past exchange, shared ancestral sequences or related mobile genetic structures, but the available evidence cannot determine which explanation is correct. Establishing the direction and timing of such events would require more complete genomes, carefully validated read mappings and laboratory experiments. The researchers also used Bayesian temporal analysis to estimate that the most recent common ancestor of the sampled Chinese ST463 population existed around 2005. A time to the most recent common ancestor is an estimate of when the sampled genomes last shared a common ancestral population, not a date marking the emergence of the species or the first appearance of KPC resistance. It can nevertheless help investigators place the expansion of a lineage within the history of antibiotic use, healthcare practices and infection-control interventions.

The authors describe the work as a genomic resource for surveillance rather than a final explanation of KPC-producing P. aeruginosa evolution. The collection is substantial, but public genomes are not a perfect mirror of global infections: countries, hospitals and outbreak settings that sequence more intensively will be overrepresented, while unsampled regions may contain additional lineages. Genome assemblies can also vary in quality, and the study’s candidate mutations and plasmid relationships need confirmation. Even with those limitations, the eight-lineage framework offers an actionable way to organize future monitoring. Hospitals and public-health laboratories could use whole-genome sequencing to identify whether a new resistant isolate belongs to a known high-risk lineage, carries a familiar mobile resistance structure or contains unusual combinations of genes. As KPC-producing P. aeruginosa continues to challenge last-resort treatment, distinguishing the bacterial clone from the resistance gene—and tracking how both move—may be essential for detecting outbreaks before they become international problems.