Klebsiella pneumoniae is a member of the Enterobacteriaceae and is found in various habitats, including water, soil, and the intestines and oropharynx of humans and animals (1, 2). There are two main pathotypes of K. pneumoniae, distinct phenotypically, clinically, and genetically: classical K. pneumoniae (C-K. p) and hypervirulent K. pneumoniae (hv-K. p) (3, 4). Phenotypically, hv-K. p shows hypermucoviscous colonies on an agar plate, which can be demonstrated by the string test (>5mm) (5). Clinically, unlike C-K. p, which generally causes hospital-acquired infections in immunocompromised individuals, hv-K. p is implicated in serious community-acquired infections in young and healthy individuals. Initially, hv-K. p was reported in Taiwan in the 1980s in cases of community-acquired liver abscesses; subsequently, infections have been reported in the Middle East (6, 7, 8), India (9), China (10), Europe (11), and America (12). hv-K.p hepatic infections tend to metastasize to other body sites, a clinical feature not previously known in C-K. p or other Enterobacteriaceae (13) Moreover, many reports worldwide have documented extrahepatic infections caused by hv-K. p, including urinary tract infections, pneumonia, kidney abscesses, and septic arthritis (14, 15). At the genomic level, hv-K.p harboring several virulence-associated genes, including crampA, rampA2, iroB, and iucA within a large virulence plasmid termed pLVPK, enables it to be more virulent than the classical pathotypes. This cardinal difference has been employed to differentiate hv-K.p from C-K.p in clinical samples (16, 17). Most previous studies investigated the prevalence of hv-K. p in human clinical samples (4, 18, 19, 20). However, little is known about the prevalence of hv-K. p in non-human sources. It has been hypothesized that non-clinical sources, particularly animals, represent an important reservoir of diverse clones of K. pneumoniae that contribute to inter-species transmission between humans and animals (21). Poultry and the poultry industry, in particular, provide a thriving ground for propagating and spreading pathogens in communities (22). Studies investigating the occurrence, prevalence, and antibiogram profile of hv-Kp in nonclinical sources might provide valuable information that aids in drawing a complete epidemiological figure of that virulent pathotype of K. pneumoniae. Therefore, this study investigated the occurrence and antibiogram profile of hv-K. p in broiler chickens at chicken slaughter shops, representing the final stage in the poultry industry.

Although four decades have passed since hv-K. p was first recognised in Taiwan, several aspects of its epidemiology remain unexplored (15). One such gap is the potential role of animals as reservoirs contributing to hv-K. p transmission in communities. To our knowledge, this is the first study in Iraq and the Middle East to explore the occurrence of hv-K. p in broiler chickens and highlight chicken slaughter shops as a potential public health risk.

This study revealed a K. pneumoniae prevalence of 29.5% in broiler chickens, consistent with a previous study from northern Iraq (24). This prevalence aligns with similar findings from healthy broiler chickens in Ethiopia, 22% (25), Indonesia, 22.5% (26), and Norway, 25.8% (27). Also, our finding is concordant with the results of a study conducted in Bangladesh, where 34.7% of chicken meat samples were positive (28). However, our prevalence was lower than those reported by Kahin et al. in Ethiopia (29), and Chika et al. in Nigeria (60%) (30). Conversely, our finding exceeded those from Egypt, 15% (31); China, 14.5% (32) and another study in Indonesia, 7.8% (33). This discrepancy may be due to differences in the geographical location, sample types, culturing techniques, and poultry farming practices (25).

The identification of hv-K.p poses a challenge due to limitations of phenotypic methods. However, molecular detection of hypervirulence-associated genes such as crmpA, rmpA2, iroB, iucA, and peg-344, has proven be a highly specific approach with up to 96% accuracy (17). Using this method, we found a 26.9% prevalence of hv-K.p in broiler chickens. Unfortunately, data on hv-K.p. prevalence in chickens are scarce, limiting direct comparison. However, our findings are comparable to a previous study from China (31). Other studies have detected hv-K.p in various animal sources, including oysters, 62.5% (34), pork,16.7% (35), and farm animals, 24.2% (36). Additionally, hypervirulent multidrug-resistant K. pneumoniae has been recorded in captive marmosets in Brazil (37), sea lions in California were identified as a potential zoonotic source (38). Our prevalence also parallels findings in human clinical isolates: Iraq, 43.3% (7); Iran, 32.2% (20), and Sudan, 31.6% (39). However, it was notably higher than that reported in European clinical isolates, including Italy, 3.7% (40) and Germany, 7.6% (41).

Among the hypervirulence-associated genes, the most frequently detected gene in our study were iucA, followed by crmpA, iroB, rmpA2, and peg-344. Our findings regarding the icuA gene, which encodes aerobactin, align with previous studies showing it as a dominant siderophore gene in 90% of hv-K.p strains and rarely present in C-Kp (3, 42). Asian studies have similarly reported iucA prevalence ranging from 60-90% (43). The _crmpA gene involved in hypermucoviscosity and virulence (3) was detected in 74.3% of our isolates, consistent with other reports (34, 44). Meanwhile, the peg-344 gene, a metabolic transporter protein, was the least frequently detected, consistent with the report from Sanikhani (20). The variation in gene detection frequencies may be due to incomplete virulence plasmid (pVLK) in some isolates (16).

A statistical significance difference (P≤0.05) was observed between the string test (19.2%) and the molecular method (26.9%) for detecting of hv-K.p, with the string test demonstrate low sensitivity 57.1%. This is consistent with the previous studies that reported string test sensitivity ranging from 50% to 90% (17, 45, 46). In our data 14.3% of the isolates that were negative string tests were positive for hypervirulence genes and five isolates of C-Kp tested positive by string test. These finding reinforce the notion that some hv-K.p strains may falsely appear hypermucoviscosity (18, 19, 47). Moreover, as highlighted by Catalán-Nájera et al. (48) that hypermucoviscosity and hypervirulence are distinct and should not be used interchangeably. The expression of hypermucoviscosity can also be influenced by the growth conditions and the type of agar medium used (42, 49).

Regarding the antibiotic resistance, our finding of relatively low resistance rate align with previous studies, indicating that hv-K.p strains generally exhibit low resistance rate compared to those of the classical pathotypes (43, 44). Non-clinical settings: animals often display lower resistance rate than human clinical samples (50). However, remarkable resistance rates were found against gentamicin, azithromycin, and ceftriaxone, with similar findings also being obtained previous reports (31, 51, 52). The widespread use of gentamicin and erythromycin in poultry farms at high doses likely contributes to this trend (53). In our study, 60% of isolates exhibited multi-drug resistance, which is lower than 100% reported by others (33, 53), and the 88.9% observed in animal isolates (50). This low rate may be due to our isolates being predominantly hypervirulent pathotypes as multi-drug resistant phenotype is generally more prevalent among classical strains pathotypes (4). Notably, we detected a high prevalence (45.7%) carbapenem-resistant hv-K.p (CR-hv-K.p). While, this contrast with some studies (54), it along with other that reported CR-hv-K.p ranging from 31.8%- 55.9% (7, 31, 34), and even a higher rate in China (64.1%) (54). The presence of CR-hv-K.p in chickens strongly suggest human to animals transmission, as carbapenem such as imipenem or meropenem are not used in poultry. Although K. pneumoniae is not traditionally considered a zoonotic threat, its ability to acquire and transfer the resistance and virulence plasmids through horizontal and vertical gene transfer, necessitate reconsideration of it zoonotic potential. A key strength of this study is that it is the first to investigate hv-K.p in chicken within this region, a relevant focus given the high consumption of chicken meat due to its affordability compared to other meats. Moreover, unlike previous studies that depended on string test or screened only one or two hypervirulence- associated genes, we evaluated all five proposed hypervirulence associated genes and compare molecular method to the string test in defining hypermucoviscosity. The relatively large sample size further enhances the reliability of our findings. However, this study has limitations, we were unable to obtain clinical isolate from hospitals preventing the assessment the genetic relatedness between chicken and clinical isolates. moreover, since samples were taken from intestinal swabs of slaughtered chickens, data on the clinical status and demographic characteristics, including age, sex, and breed, were not considered in this study.