In recent decades, the increasing prevalence of antibiotic-resistant gram-negative bacteria (GNB), particularly Klebsiella spp., has posed a significant threat to human health worldwide, especially in ICUs. (14) CRKP is the most commonly implicated microorganism causing nosocomial infections. (15) The emergence of MDR and XDR K. pneumoniae strains, demonstrating resistance to a range of antibiotics, including extended-spectrum cephalosporins, carbapenems, aminoglycosides, and fluoroquinolones, has introduced substantial challenges for clinicians. Therefore, it was necessary that the older antibiotics of the 1970s be revived. Colistin, an old antibiotic also known as polymyxin E, is one of the last-resort antibiotics that treat MDR CRKP. (16, 17)
Because mcr genes are located on a plasmid, they easily spread to other bacteria, thereby increasing the number of resistant strains and creating MDR and XDR species. Consequently, the prevalence of colistin resistance (CLR) due to mcr genes in GNB has become the most significant concern worldwide. (1, 18) The development of efficient tools for the rapid detection of mcr − harboring strains should be a priority to prevent the dissemination of these strains in hospital settings.
Additionally, CRKP has increased at an alarming pace in Iran, and we evaluated the occurrence of mcr-1 to mcr-10 among CRKP isolated from hospitals in Iran. The use of molecular methods, particularly pulsed-field gel electrophoresis (PFGE), has proven invaluable in comprehending the epidemiological aspects of such infections and identifying their sources. PFGE is considered the “gold standard” for bacterial typing. Currently, this technique is widely accepted as the most powerful and commonly used molecular tool for epidemiological studies on bacterial strains.(19, 20) Additionally, PFGE is crucial for obtaining important information on resistance transmission through the spread of clonal complexes worldwide. (21)
Antibiotic susceptibility testing showed that CRKP isolates exhibited resistance to multiple antibiotics; however, there was susceptibility to colistin and amikacin (Fig. 2). Our study revealed that K. pneumoniae displayed a 90% resistance rate against cefepime and an 84% resistance rate against ceftriaxone. These resistance patterns align with findings from a comparable investigation in Iran, where K. pneumoniae displayed a high resistance rate of 87.7% to ceftriaxone and 86% to cefepime. (22) Interestingly, colistin showed the lowest resistance rate at approximately 38%. Similar data were observed in research conducted in Brazil and Iran, where amikacin emerged as the second most effective antibiotic after colistin. (23, 24)
We found that all isolates were classified as XDR, with nearly half of them from urine samples (Fig. 1). Notably, the highest resistance rates were observed against imipenem and meropenem. Similar findings have recently been documented for imipenem and meropenem resistance rates. (25, 26)
In this study, out of a total of 27 urine samples collected from hospitalized patients, 21 (78%) were from female patients and 6 (22%) were from male patients. Therefore, the results of the present research, like many previous studies, show that women are more susceptible to urinary tract infections with K. pneumoniae than men. (27, 28)
Genotypic analysis of CLR was conducted through the detection of mcr genes. Among the 19 CLR isolates, three (K18, K19, and K23) were positive for the mcr-1 gene, while the remaining 16 did not harbor the mcr-1 gene. The more logical explanation for nonmcr-associated CLR in 16 out of the 19 isolates could be attributed to genetic mutations, specifically those affecting the mgrB gene. Many reports have shown that CLR in K. pneumoniae is commonly caused by chromosomal mutations. The mgrB gene, a small transmembrane protein with 47 amino acids spanning the inner membrane, plays a role in negatively regulating PhoP phosphorylation—a process vital for bacterial outer membrane lipid biosynthesis. (29, 30) Recent studies have demonstrated that nonmcr-associated CLR is frequently linked to alterations in the mgrB gene, as well as variations in the crrB, pmrB, phoQ, pmrA, and phoP genes. (29, 30)
Isolates carrying mcr genes exhibited resistance to colistin by the microdilution test (MIC ≤ 4 mg/L), and remarkably, all of these isolates displayed identical PFGE patterns, indicating their origin from a single clone. Similar data were observed in Jordan, where out of 179 CRKP isolates, only one carried the mcr-1 gene, and the prevalence was 1.1%. Additionally, another study conducted in Vietnam discovered three CRKP strains among 205 CRKP isolates that harbored the mcr-1 gene. (31) While these findings suggest a low prevalence of the mcr-1 gene among CRKP isolates, regular surveillance efforts are crucial to continually assess the epidemiology of mcr-1 among CRKP strains.
In our study, PFGE analysis showed the presence of four clusters of related strains and 16 pulsotype strains. These findings indicated a low diversity, suggesting a clonal population structure characterized by continuous exchange of K. pneumoniae strains among patients within the same and different hospital wards, as well as across various hospitals in Iran. This pattern aligns with previous epidemiological studies conducted in Iran that revealed frequent transmission of K. pneumoniae strains among patients within medical centers. Additionally, our results are also in concordance with other epidemiological studies demonstrating a polyclonal population structure of K. pneumonia. (32, 33) Within our study, multiple clones were observed to simultaneously circulate and persist, contributing to the endemic presence of K. pneumoniae within our hospital, despite the implementation of infection control measures such as hand hygiene, colonization surveillance among high-risk patients, and contact precautions.
In the present study, according to the results of PFGE analysis, in the largest cluster (Cluster C), all K. pneumoniae isolates harbored the blaOXA−48 gene. OXA-48, a class D carbapenemase gene located within a composite transposon named Tn1999, is flanked by the carbapenemase gene and facilitates the mobilization of an intervening DNA segment. Studies have shown that blaOXA-48-carrying plasmids enable both clonal and horizontal transfer, facilitating transmission between patients and healthcare staff. The presence of Cluster C suggests continuous exchange of K. pneumoniae strains not only within single hospital wards but also between different hospital wards and even across different hospitals. This emphasizes the role of widespread dissemination within a hospital setting. (34)
In Cluster B, one isolate (K4) displayed a band pattern similar to those from the ICU, implying a possible transfer between the ICU and the internal ward, which could indicate potential routes of dissemination.
Cluster A, comprising 9 urine isolates (K12, 24, 36, 48, 10, 11, 6, 7 and 9) from infectious and internal wards, 4 blood isolates (K20, 32, 5 and 8), and 1 sputum isolate, displayed a similar resistance and carbapenemase gene pattern (blaVIM). These findings strongly suggest the potential for interhospital transfer among patients within infectious, ICU, and internal wards.
Three isolates in Cluster A belonged to blood samples from the internal and CCU wards of Hospitals D and C. These isolates exhibited similar patterns of resistance, carried carbapenemase genes (VIM and KPC), and displayed identical genetic similarities. A noteworthy point in this cluster was the presence of isolates from both the CCU and internal ward with similar band patterns. Consequently, the widespread transmission of strains across various hospital wards can be attributed to medical staff and employees working outside the hospital. Therefore, if not enough attention is given to controlling these strains, we may face a high rate of possible epidemics caused by these strains in the future, which is a serious warning for physicians and the infection control team.
Notably, Cluster D isolates shared an identical antimicrobial susceptibility profile and carried the carbapenemase gene blaKPC. This suggests that these isolates were likely introduced to the ward through patients, clients, or medical staff. The genetic persistence within this cluster may have contributed to bacterial survival, colonization, and spread.
Due to the importance of studying the molecular epidemiology of K. pneumoniae, various studies have been conducted around the world. For instance, studies conducted in India on carbapenemase-positive K. pneumoniae isolates (35) and in Iran on ESBL K. pneumoniae isolates revealed five and four clusters, respectively. Notably, cluster C emerged as the dominant cluster in both studies (36), aligning with the findings of our present study.
In contrast, an Iranian study on carbapenemase-positive K. pneumoniae isolates collected from various wards of a reference hospital. Their PFGE analysis revealed 11 clusters (37) However, compared to our recent study, a significant disparity in genomic patterns was observed, likely attributed to the dispersion of samples and the diverse origins of the strains. Another study conducted in Iran analyzed 165 K. pneumoniae strains isolated from different samples; their PFGE analysis showed 17 clusters with an 80% similarity rate. (32) In this study, the genetic diversity among isolates was high; one reason for this could be the diversity of sample sources and because our samples were fewmany from diverse sources.