Introduction

Klebsiella pneumoniae (KP) is a well-established Gram-negative pathogen implicated in a broad array of clinical infections1. In recent years, it has emerged as a leading cause of hospital-acquired pneumonia, accounting for approximately 10% of all nosocomial infections, and is ranked as the second most prevalent gram-negative organism in healthcare-associated infections (2). K. pneumoniae is classified among the ESKAPE group of antibiotic-resistant pathogens—comprising Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species—recognized globally as a critical public health threat due to its formidable capacity to acquire antimicrobial resistance. As such, it is designated a Priority 1 (Critical) pathogen by the World Health Organization2. Two primary pathogenic forms of KP are recognized: classical K. pneumoniae (cKP) and the more recently characterized hypervirulent K. pneumoniae (hvKP)3,4. Although capable of causing infections similar to cKP, the hvKP pathotype demonstrated a markedly enhanced virulence profile4. Unlike its classical counterpart, hvKP predominantly affects otherwise healthy individuals within the community, rather than targeting immunocompromised hosts5. hvKp has been documented across all age groups, whereas cKp more commonly affects elderly individuals. Notably, hvKp is disproportionately reported among individuals of Asian, Pacific Islander, and Hispanic descent, while no significant ethnic predilection has been observed for cKp. A distinguishing feature of hvKp is its propensity to establish multiple infectious foci concurrently, with infections typically caused by a single microbial species, in contrast to the polymicrobial nature often associated with cKp infections3 .The first documented outbreak of hvKP dates back to 1986 and involved a series of seven cases presenting with a distinct clinical triad of liver abscesses in the absence of biliary disease, accompanied by septic endophthalmitis, in previously healthy individuals6. Of particular concern is the propensity of hvKP to cause severe invasive infections, including central nervous system involvement and ocular complications, both of which require urgent medical intervention because of their high mortality rates7. Key virulence determinants of hvKP include excessive capsule production and siderophore synthesis, such as aerobactin8. Historically, cephalosporins and carbapenems have been the mainstay therapies for serious infections caused by Enterobacterales such as K. pneumoniae. However, their clinical efficacy has been significantly compromised by the widespread dissemination of genes encoding extended-spectrum β-lactamases (ESBLs) and carbapenemases—enzymes that mediate resistance to these critical antibiotic classes2. Recent epidemiological trends reveal the emergence of antibiotic-resistant hvKp strains, particularly in low- and middle-income countries (LMICs) such as Vietnam and nations across Southeast Asia. In Vietnam, K. pneumoniae is not only a leading cause of hospital-acquired infections but also a predominant ESBL-producing Enterobacterales species. A study examining 19 K. pneumoniae isolates from Vietnam identified all as antimicrobial-resistant (AMR), with five characterized as hvKp9. Similar trends have been observed in southeastern Iran. A study conducted in Kerman analyzed 146 clinical K. pneumoniae isolates, of which 22 (15.1%) were identified as hvKp strains exhibiting multidrug resistance10. Increasingly, hvKP strains have acquired mobile genetic elements harboring antibiotic resistance genes, mirroring the multidrug resistance trends observed in cKP4. This convergence has led to the emergence of multidrug-resistant hvKP strains, particularly in hospital environments where drug-resistant cKP strains have integrated hvKP-specific virulence factors11. Phenotypically, hvKP strains often display a hypermucoviscous appearance regulated by rmpA and rmpA2, which govern the mucoid phenotype12,13,14. The widely employed string test is a practical method to identify strains with this characteristic viscous profile (12). Genetically, hvKP exhibits a broader virulence repertoire than cKP, including the enhanced expression of rmpA, siderophores, and a robust capsular polysaccharide layer13,15. Recent investigations have identified aerobactin as a key virulence determinant specific to hvKp, implicated in both pathogenicity and strain differentiation. When co-present with salmochelin and rmpA2, or in association with the hypermucoviscosity phenotype, aerobactin plays a pivotal role in disease pathogenesis. The five most reliable biomarkers currently employed for hvKp identification include iucA, iroB, peg-344, rmpA, and rmpA2—markers that together offer high diagnostic precision16. Eight capsular serotypes have thus far been associated with hvKP: K1, K2, K5, K16, K20, K54, K57, and KN117. Among these, serotypes K1 and K2 are most frequently linked to hvKP-induced liver abscesses18,19. rmpA/A2 genes are rarely found in non-K1/K2 strains, suggesting a distinct genetic and pathogenic signature7,17,19. In healthcare environments, extensively drug-resistant (XDR) cKP strains are increasingly found in long-term care settings such as hospitals and intensive care units (ICUs)4,20,21. Transmission within these facilities often occurs via healthcare personnel, particularly owing to suboptimal hand hygiene practices or contaminated medical devices15. Thus, rigorous infection prevention protocols remain critical for controlling nosocomial spread22. Convergence of hypervirulence and multidrug resistance through the acquisition of hvKP plasmids by XDR cKP strains is a growing clinical and public health concern7,23. These hybrid strains significantly increase the risk of adverse outcomes, elevate patient mortality rates, and contribute to escalating healthcare costs24. Although the exact community transmission routes for hvKP remain poorly defined, suspected pathways include contaminated food or water, interpersonal contact (e.g., among household members or sexual partners), and potentially zoonotic sources20. While hvKP is primarily associated with community-acquired infections, reports of healthcare-associated hvKP infections are on the rise, underscoring its expanding epidemiological footprint23. Given the rapid progression and increasing antimicrobial resistances in hvKP infections, prompt and accurate diagnosis is paramount to guide effective treatment strategies1,23,25. This study was undertaken to investigate the prevalence of hypervirulent K. pneumoniae strains at Imam Reza Hospital in Mashhad, Iran, from March to May 2024.

Materials and methods

Sample collection

This investigation was conducted using K. pneumoniae isolates obtained over a three-month period (March to May 2024) from the microbiology laboratory of Imam Reza Hospital in Mashhad. Informed consent forms were obtained from the patients. A total of 187 isolates derived from both outpatient and inpatient clinical specimens from various hospital departments were included to assess the prevalence of hvKP. The isolates originated from a range of specimen types including blood, urine, body fluids, respiratory secretions, and other clinical samples. Hypervirulent strains were preliminarily identified based on their hypermucoviscous phenotype by using a string test. All isolates were preserved at − 70 °C until PCR analysis.

String test

To evaluate mucoviscosity, after the culture of all the isolates on blood agar and incubation at 37 °C for 24 h, fresh colonies were gently touched with an inoculating loop and pulled vertically to assess viscosity. All isolates in this study were measured using a calibrated, standardized precision ruler to ensure consistency across phenotypic assessments. A positive result was defined as the formation of a mucoid string measuring greater than 5 mm in length14.

DNA extraction

DNA was extracted from all isolates by the boiling method26. Briefly, a bacterial colony was suspended in 300 µL of TE (Tris-EDTA) buffer and heated at 95 °C for 15 min. After centrifugation at 14,000 rpm for 10 min, the DNA-containing supernatant was aseptically transferred to a new sterile microtube. DNA purity and concentration were subsequently evaluated using a spectrophotometer based on optical density measurements. Virulence genes, including rmpA, rmpA2, and iucA, were screened by PCR.

Primer selection

Primers targeting rmpA and iucA were selected based on previously validated sequences reported in the literature that aligned with the corresponding target regions. Specific primer sets used in this study, noted for their high specificity, were obtained from a reference source27,28. Primer sets for rmpA2 gene were designed for this study. Detailed nucleotide sequences of the primers used are listed in Table 1.

Table 1 Nucleotide sequences of primers used in this study.
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Primer preparation

A concentrated stock solution of primers that synthesized by SinaClon (Iran) was prepared to a final concentration of 100 pmol/µL following the manufacturer’s guidelines. Working aliquots at a concentration of 10 pmol/µL were prepared from the stock and stored at − 20 °C.

PCR amplification and gel electrophoresis

PCR amplification was conducted in a total reaction volume of 25 µL using a commercial premixed master mix (ParsTous, Iran). Each reaction was subjected to 35 thermal cycles under standard amplification conditions and was optimized for the target genes. Amplified PCR products were subjected by electrophoresis on a 1.5% agarose gel using a 100 bp DNA ladder as a molecular size reference to verify the amplicon length.

Antibiotic susceptibility testing and ESBL detection

Antimicrobial susceptibility testing was carried out using the standard disk diffusion method to evaluate the resistance profiles against a panel of clinically relevant antibiotics, including amikacin (30 µg), gentamicin (10 µg), imipenem (10 µg), ceftazidime (30 µg), cefazolin (30 µg), cefepime (30 µg), cefotaxime (30 µg), Trimethoprim/Sulfamethoxazol (1.25/23.75 µg), and ciprofloxacin (10 µg)29. The production of extended-spectrum β-lactamases (ESBLs) was assessed in accordance with guidelines established by the Clinical and Laboratory Standards Institute (CLSI 2024)30.

Results

Demographic characteristics of patients

In this study, 187 clinical isolates of K. pneumoniae were obtained from patients admitted to the Imam Reza Hospital, Mashhad, Iran. The patients comprised 93 males (49.7%) and 94 females (50.3%), with a mean age of 59.1 ± 1.4 years (mean ± SE). hvKp strains were identified using a positive string test, in combination with PCR confirmation of the iucA gene. Eighteen isolates (9.63%) met the criteria for the hvKp classification. The distribution of the K. pneumoniae isolates across various clinical specimens is summarized in Table 2. Urine samples accounted for the largest proportion of isolates (n = 91), comprising 42.7% of classical strains (cKp) and 4.81% of hypervirulent strains. Statistically, hvKp and cKp isolates differed significantly across most sample types, except for ascitic fluid and peritoneal abscesses. There was no significant age difference between patients infected with hvKp and cKp strains (p ≥ 0.05). Table 3 outlines the microbiological and genetic profiles of hvKp isolates. Virulence genes rmpA, rmpA2, and iucA were detected by PCR, and their presence was significantly associated with the hvKp isolates (p < 0.05). The statistical analysis further supported the role of these genetic markers as key differentiators between the hypervirulent and classical K. pneumoniae strains.

Table 2 Distribution of K. pneumoniae isolates across clinical sample types.
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Table 3 Microbiological and genetic characteristics of HvKp isolates.
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Comparison of genetic characteristics between HvKp and cKp

The antimicrobial susceptibility of all isolates was evaluated using commonly used antibiotics, including amikacin, gentamicin, cefepime, ceftazidime, ciprofloxacin, trimethoprim-sulfamethoxazole, imipenem, cefotaxime, and cefazolin. ESBL production was assessed using a combination disk synergy test with ceftazidime and clavulanate. As shown in Table 4, the statistical revealed no significant differences in the resistance profiles between the hvKp and cKp strains (p > 0.05). However, hvKp isolates exhibited lower overall resistance rates than their classical counterparts.

Antibiotic resistance patterns and ESBL production in HvKp and cKp isolates

Antimicrobial susceptibility testing confirmed the resistance profiles of all isolates against the aforementioned antibiotics. The ESBL production was evaluated using a combination-disk approach. Consistent with previous findings, statistical analysis did not demonstrate significant differences in antimicrobial resistance between the hvKp and cKp strains (p > 0.05). Nonetheless, hvKp isolates seems to be tended to display reduced resistance levels relative to cKp strains.

Table 4 Antibiotic resistance and ESBL production in HvKp and cKp Isolates.
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Distribution of HvKp and cKp in different hospital departments

The distribution of the hvKp and cKp isolates across different hospital wards is presented in Table 5. The highest number of hvKp cases was recorded in the ICU (n = 7; 18.7%), followed by the internal medicine department (n = 4; 18.4%). Despite these trends, statistical analysis did not reveal a significant association between the type of isolate and the hospital department (p = 0.598).

Table 5 Distribution of HvKp and cKp isolates in hospital Departments.
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Discussion

The emergence of hvKp as a potent clinical pathogen has raised significant concerns, primarily because of its ability to cause invasive and often life-threatening infections, even in immunocompetent individuals7,25. Distinct from classical K. pneumoniae, hvKp possesses unique phenotypic and genetic characteristics that endow it enhanced virulence and metastatic potential4. Although traditionally associated with community-acquired infections, hvKp is increasingly being reported in nosocomial settings, where it may either acquire resistance determinants or evolve from MDR cKp strains via horizontal gene transfer1,21,25. This concern is particularly acute in ICUs, which serve as critical reservoirs for MDR K. pneumoniae strains20. The potential for hvKp to disseminate within healthcare environments underscores the urgent need to implement rigorous infection control measures to reduce its transmission rates15. In the current study, the hypermucoviscous phenotype, observed in K. pneumoniae isolates was significantly more prevalent among the hvKp strains. This observation aligns with findings from a study by Alharbi et al. in Egypt, which reported a hypermucoviscous phenotype in 22.5% of isolates31. Discrepancies in the prevalence rates may reflect differences in geographic location, sample size, and local epidemiological factors. The overall prevalence of hvKp in our study was 9.63%, consistent with other reports indicating rates between 7.8% and 25.4%32,33,34. Our molecular analysis revealed a strong association between hvKp and the virulence genes rmpA and rmpA2, which are implicated in capsule overproduction and the hypermucoviscous phenotype. These findings are in line with those of Liu et al. (2019), who also reported significant enrichment of these genes in hvKp isolates compared to cKp isolates35. Similarly, iucA, was found to be significantly associated with hvKp in our study, corroborating previous observations by Hsieh et al. regarding its role in facilitating iron acquisition and contributing to hvKp pathogenesis36. Regarding antimicrobial susceptibility, we found no statistically significant differences in the resistance rates between the hvKp and cKp isolates. Nevertheless, hvKp strains demonstrated relatively lower levels of resistance overall. This is supported by the findings of Lam et al., which noted hvKp is less likely to harbor antimicrobial resistance plasmids, possibly because of the physical barrier posed by capsule overproduction, which may limit plasmid uptake8. Interestingly, although our data showed a slightly higher prevalence of ESBL production among hvKp isolates, the difference was not statistically significant (p = 0.36). This is consistent with results from El-Mahdy et al. (2018) in Egypt, which reported no significant variation in resistance patterns between hvKp and cKp strains37. The increasing global threat of carbapenem-resistant hvKp has been highlighted in multiple studies7,17,25. For instance, Huang et al. (2016) noted the progressive development of hvKp strains resistant to carbapenems, polymyxins, tigecycline, and ESBL inhibitors, posing substantial clinical and public health challenges38. Li et al. postulated that the emergence of carbapenem resistance in hvKp strains may be attributed to the horizontal acquisition of resistance plasmids39. These often carry multiple genes—such as the ESBL gene blaCTX-M-14, the AmpC-type β-lactamase blaDHA-1, the 16SrRNA methylase armA, and the quinolone resistance gene qnrB. Given the typically limited baseline resistance profiles in hvKp, the acquisition of such plasmids renders certain hvKp strains simultaneously hypervirulent and extensively drug-resistant. Moreover, in carbapenem-resistant organisms lacking known carbapenemase genes, the loss of porin channels has been proposed as a key mechanism contributing to reduced antibiotic permeability and resistance40. The origins of resistance in hvKp isolates identified in our study remain uncertain, emphasizing the need for further research to clarify whether these strains acquired resistance gradually or through horizontal gene transfer. Demographic analysis revealed no significant age difference between patients infected with hvKp and those with cKp (p = 0.504). However, underlying conditions such as diabetes mellitus and renal failure emerged as significant risk factors for hvKp infection (p = 0.025), reflecting previous reports that identified diabetic individuals as particularly susceptible37,41. Hyperglycemia and vascular dysfunction in diabetic patients likely contribute to increased bacterial dissemination and metastatic complications during hvKp bacteremia3. Supporting this, in vitro studies by Lin et al. demonstrated that hyperglycemia impairs neutrophil phagocytic activity against hvKp but has no significant effect on cKp clearance42.

Conclusion

This study provides an overview on the prevalence, genetic characteristics, and antibiotic resistance profiles of hvKp strains isolated from patients at Imam Reza Hospital in Mashhad, Iran. hvKp accounted for 9.63% of all K. pneumoniae infections, with isolates showing a robust association with the key virulence determinants rmpA, rmpA2, and iucA. These markers underscore the enhanced virulence of the pathogen, particularly through mechanisms such as capsule overexpression and increased iron acquisition. Although traditionally regarded as a community-acquired organism, the identification of hvKp in hospital environments, particularly ICUs, signals an evolving epidemiological landscape and raises concerns regarding nosocomial transmission. No statistically significant differences in antibiotic resistance were detected between hvKp and cKp strains. These findings emphasize the critical importance of robust infection prevention protocols, targeted antimicrobial stewardship, and molecular surveillance to mitigate the spread of hvKp, particularly in high-risk hospital units. Future research should prioritize investigations into the genomic evolution of hvKp, its environmental and clinical reservoirs, and host-related factors that contribute to disease severity. Advances in novel therapeutic strategies may ultimately prove essential in reducing the burden of hvKp infections on global health systems.