Molecular Characterization and Prevalence of Hypervirulent and Multidrug-Resistant Klebsiella pneumoniae in Mastitis of Bactrian Camels from Xinjiang, China

Molecular Characterization and Prevalence of Hypervirulent and Multidrug-Resistant Klebsiella pneumoniae in Mastitis of Bactrian Camels from Xinjiang, China | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Molecular Characterization and Prevalence of Hypervirulent and Multidrug-Resistant Klebsiella pneumoniae in Mastitis of Bactrian Camels from Xinjiang, China Meiting Niu, Ling Zhao, Mengzhe Hou, Yu Han, Jiabin Zhang, Maimaiti Tuniyazi, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8650943/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract Aims The escalating prevalence of multidrug-resistant (MDR) and hypervirulent (hv) Klebsiella pneumoniae ( K. pneumoniae ) strains represents a significant threat to both animal and human health globally. In Xinjiang, China, where Bactrian camels are an economically important livestock and human interaction with camel products is increasing, this study aimed to characterize the virulence, antibiotic resistance, and potential public health implications of K. pneumoniae isolates associated with camel mastitis. Results A total of 14 K. pneumoniae strains were isolated from camel milk, milker hand swabs, and environmental samples. Antimicrobial susceptibility testing revealed widespread MDR, with 100% of isolates resistant to β-lactams, aminoglycosides, macrolides, lincosamides, and glycopeptides. PCR analysis identified multiple acquired resistance genes. Multilocus sequence typing (MLST) revealed diverse sequence types, including ST42, ST4089, ST297, and ST495, with ST495 being the dominant type. Virulence gene profiling confirmed the presence of various virulence-associated genes. Pathogenicity assays in mice and rabbits demonstrated high virulence in ST495:KL121 strains. Notably, ST495 was isolated from camels, dairy workers, and environmental samples, indicating a potential transmission cycle between these reservoirs. Conclusion This study underscores the presence of highly MDR and potentially virulent K. pneumoniae strains linked to camel mastitis in Xinjiang. The identification of ST495:KL121 strains, exhibiting both hypervirulence and multidrug resistance, suggests the emergence of "superbug" characteristics and poses a substantial public health risk. These findings emphasize the critical need for intensified surveillance and robust control measures targeting K. pneumoniae in camel farming environments within the region to effectively mitigate the impact on both animal and human health. Klebsiella pneumoniae camel mastitis virulence antimicrobial resistance molecular characterization public health risk Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction K. pneumoniae is a flagellated, encapsulated bacterium ubiquitous in natural environments and recognized as an opportunistic pathogen. It primarily infects immunocompromised individuals, causing a range of serious human and animal diseases, including pneumonia, bacteremia, urinary tract infections, and liver abscesses(Wusiman et al., 2024 ) (Chen et al., 2023 ; Dixon et al., 2022 ; Raza et al., 2023 ). Notably, the mortality rate for pneumonia caused by K. pneumoniae is approximately 50% (Martin and Bachman, 2018 ). In camels, it is also a causative agent of mastitis, a significant zoonotic disease that poses a serious threat to both public health and animal production (Chen et al., 2021 ). Compared to bovine mastitis, research on the etiology of camel mastitis is relatively limited, with existing studies indicating bacterial infection as a primary cause (Aqib et al., 2022 ). Environmental mastitis can be induced by various bacteria, among which K. pneumoniae is one of the most clinically significant pathogens (Mao et al., 2025 ), widely present on animal mucosal surfaces and in the environment. In recent years, the convergence of carbapenem resistance (CR) and hypervirulence (hv) phenotypes has given rise to strains combining heightened pathogenicity with multidrug resistance, often termed "highly virulent and highly resistant" strains. The global dissemination of ST11 CR-HvKP, in particular, constitutes a "superbug" threat (Zhu et al., 2023 ) (Zhang et al., 2025 ) and represents one of the most severe challenges in current global public health. Clinically relevant lineages of K. pneumoniae can be isolated from diverse animal and environmental sources, revealing their potential for cross-species transmission (Zhu et al., 2024 ). Whole-genome analyses have confirmed close phylogenetic relationships between clinical isolates and those from retail meat products (Abo-Shama et al., 2025 ), bovine mastitis cases (Holt et al., 2015 ), and environmental samples (Runcharoen et al., 2017 ). Of particular concern is the situation in Southern Xinjiang, where camels are a distinctive economic livestock species, and products like camel milk are in increasing contact with humans. However, research on the virulence and drug resistance of K. pneumoniae from camel hosts in this specific regional context remains scarce. Furthermore, exacerbated by antibiotic misuse, K. pneumoniae employs diverse antimicrobial resistance mechanisms. The increasing frequency of drug-resistant isolates complicates the treatment of K. pneumoniae infections (Kot and Witeska, 2024 ). The emergence of MDR strains has intensified this therapeutic challenge. Resistance in K. pneumoniae is often associated with transferable plasmids carrying resistance and virulence genes, which can enhance bacterial survival in hostile environments (Da Silva and Mendonça, 2012 ). The diagnostic criteria and strain definition for hypervirulent K. pneumoniae (HvKP) remain subjects of ongoing debate (Kochan et al., 2023 ; Wang et al., 2024 ). Current detection methods primarily rely on screening for specific virulence gene markers. Genes such as iroB , iucA , peg-344 , rmpA , and rmpA2 are considered reliable biomarkers for identifying HvKP (Russo et al., 2018 ). Key virulence factors include capsular polysaccharide (CPS), lipopolysaccharide (LPS), allantoin metabolism, fimbriae, urease, and iron acquisition systems (Li et al., 2014 ). Among the identified serotypes, K1 and K2 are regarded as the most virulent. The capsule, an extracellular polysaccharide matrix enveloping the bacterium, impedes phagocytosis, inhibits antimicrobial peptide activity, blocks complement components to prevent complement-mediated lysis and opsonization, and suppresses the acute activation of immune responses (Paczosa and Mecsas, 2016 ). LPS, composed of lipid A, core oligosaccharide, and O antigen, functions as both an immune activator and a virulence factor. Lipid A can dampen host inflammatory responses, while the diversity of the O antigen aids in immune evasion (Choby et al., 2020 ; Clegg and Murphy, 2016 ). Type I and Type III fimbriae are membrane-associated structures consisting of structural and adhesive subunits, encoded by specific gene clusters ( fim and mrk , respectively) (Araújo et al., 2018 ; Huang et al., 2009 ). Allantoin, a product of purine catabolism, can be degraded by K. pneumoniae as a source of nitrogen and carbon. This metabolic pathway involves several enzymes, with the allS gene playing a key role in its activation (Chou et al., 2004 ). Among the siderophores secreted by K. pneumoniae , enterobactin is encoded by the entA, B, C, D, E , and F gene cluster. The entB gene is frequently used as a marker in various studies, as this siderophore is prevalent in both classical and hypervirulent strains (Bachman et al., 2011 ). This study isolated 14 strains of K. pneumoniae from camel milk, milker, and environmental samples collected from several farms in Xinjiang, China. The isolates were analyzed for antimicrobial resistance, pathogenicity, and the presence of resistance and virulence genes. These findings aim to inform precise treatment strategies for K. pneumoniae infections in farmed camel populations and to highlight potential public health risks. 2. Materials and Methods 2.1. Sample Collection From October 2024 to October 2025, a total of 510 samples were randomly collected from an intensive camel farm in Keping County, Aksu Prefecture, Xinjiang, China（Fig1. A）. These included 480 camel milk samples, 10 soil samples, 10 water samples, and 10 hand swab samples from milkers. Among the camel milk samples, 81 subclinical mastitis and 20 clinical mastitis samples, identified using the LMT diagnostic kit, were selected for this study. 2.2. Isolation and Purification of K. pneumoniae Under aseptic conditions, milk samples were inoculated into Brain Heart Infusion (BHI) broth (Qingdao Hope Bio-technology Co., Ltd., China) and incubated overnight at 37°C with shaking at 180 rpm. Genomic DNA was extracted from the milk samples using the boiling method and used as a template. Initial screening for K. pneumoniae was performed via PCR amplification targeting the species-specific K. pneumoniae hemagglutinin type 3 fimbrial gene ( Khe ). The PCR protocol was as follows: initial denaturation at 95°C for 3 min; 30 cycles of denaturation at 94°C for 25 s, annealing at 61°C for 25 s (The primer sequences and related information are detailed in Table 1)(Wan et al., 2025b), and extension at 72°C for 1 min; followed by a final extension at 72°C for 10 min. Milk samples yielding positive PCR results were then streaked onto BHI agar plates and incubated at 37°C overnight. Single colonies were selected and repeatedly streaked onto fresh BHI plates for purification until uniform colony size and morphology were achieved. Colony characteristics, including size, morphology, and color, were recorded. Gram staining (Beijing Solarbio Science & Technology Co., Ltd., China) was performed for microscopic examination to confirm bacterial morphology and Gram reaction. 2.3. Biochemical Identification Biochemical identification of the isolated strains was conducted using a biochemical test tube kit (Hangzhou Microbial Reagent Co., Ltd., China), following the manufacturer's instructions. 2.4. PCR Confirmation and 16S ribosomal Ribonucleic Acid Gene ( 16S rRNA ) Sequencing Genomic DNA from bacterial isolates was extracted using the boiling method and served as the PCR template. The PCR protocol was: initial denaturation at 94°C for 5 min; 30 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s (The primer sequences and related information are detailed in Table 1), and extension at 72°C for 30 s; followed by a final extension at 72°C for 10 min. PCR products were verified by 1% agarose gel electrophoresis. The 16S rRNA gene of the isolates was amplified using universal bacterial primers. The purified PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The assembled full-length sequences were compared against the NCBI database using BLAST，and the sequence has been uploaded to GenBank (SUB15995834: X975972 - PX975985). All primers were synthesized by Youkang Biotechnology Co., Ltd. (Xinjiang, China). 2.5. Capsular serotyping and MLST analysis The wzi gene sequencing method was used to determine the capsular polysaccharide type of the isolates, while the MLST method was employed to identify the sequence type (ST) of the isolates as described in (Brisse et al., 2013), primers for the wzi gene of K. pneumoniae and seven housekeeping genes ( Glyceraldehyde-3-phosphate dehydrogenase A : gapA , Translation initiation factor IF-2 : infB , Malate dehydrogenase: mdh , Phosphoglucose isomerase: pgi , Phosphate limitation-inducible outer membrane porin E: phoE , RNA polymerase beta subunit: ropB , and TonB protein: tonB ) were designed and subjected to PCR amplification. The primer sequences and related information are detailed in Table 2. PCR reaction program: 95 °C for 3 min for pre-denaturation; 94 °C for 25s for denaturation; 60 °C for 25s for annealing; 72 °C for 1 min for extension, set for 30 cycles; followed by a final extension at 72 °C for 5 min. Use 1% agarose gel electrophoresis to validate the PCR products. The PCR positive products were sequenced using the Sanger method, with the sequencing performed by General Biologicals (Shanghai, China). Submit the wzi gene sequence to the online (https://bigsdb.pasteur.fr/klebsiella/). The capsular serotypes of the isolates were obtained, and 7 housekeeping gene sequences were uploaded to MLST network database, and the ST types of the isolates were obtained. The wzi gene and housekeeping genes sequences have been uploaded to GenBank (PX991553 - PX991664). 2.6. Quantitative biofilm production assay The ability of K. pneumoniae to form biofilm was determined by crystal violet staining K. pneumoniae isolates were inoculated with LB solid medium by four-zone method and cultured at 37 °C for 12 h. A single colony was selected and inoculated in LB liquid medium, and cultured at 37 °C and 180 r/min until the concentration of the liquid reached 0.5 mL. 200 µL of the culture medium was transferred to the sterile 96-well plate and incubated at 37 °C for 48 h. After culture, the medium was discarded, washed 3 times with PBS buffer, and fixed with methanol solution (200 µL/Well) for 15 min. PBS buffer was washed three times, 96-well cell plates were naturally dried and then stained with 0.1% crystal violet staining solution (200 µL/Well) for 10 min at room temperature, and a large amount of sterile distilled water was used for multiple washings until colorless, after decolorization with anhydrous ethanol (200 µL/well), the absorbance at 570 nm was measured by Enzyme-linked Immunosorbent Assay Reader. The critical ODc values (ODc=the mean of the blank control wells) were used to determine the results: OD ≤ ODc was considered to have no ability to form membrane, ODc < OD ≤ 2ODc was considered to have weak ability to form membrane, and 2ODc 4ODc is the strong ability of film formation. 2.7. Antimicrobial Susceptibility Testing and Resistance Gene Detection The antimicrobial susceptibility of the isolates was determined using the disk diffusion method against the following antibiotics: β-lactams (Penicillin, Ampicillin, Piperacillin, Cefuroxime sodium, Ceftriaxone, Cephalexin, Cefoperazone, Ceftazidime, Cefazolin), aminoglycosides (Gentamicin, Kanamycin, Streptomycin, Amikacin), macrolides (Erythromycin), lincosamides (Lincomycin), glycopeptides (Vancomycin), tetracyclines (Tetracycline, Minocycline, Doxycycline), and polypeptides (Polymyxin B)(CLSI, 2022). Additionally, 16 antimicrobial resistance genes from six classes were detected by PCR in the 14 K. pneumoniae isolates (Candan and Aksöz, 2015; Tabaran et al., 2017; Wu et al., 2022): β-lactams ( Temoneira β-lactamase: bla TEM , Cefotaxime-hydrolyzing β-lactamase: bla CTX , New Delhi metallo-β-lactamase : bla NDM , Oxacillinase-48: bla OXA-48 , Active on imipenem β-lactamase : bla IMP , Verona integron-encoded metallo-β-lactamase : bla VIM , Verona integron-encoded metallo-β-lactamase : bla DHA , Klebsiella pneumoniae carbapenemase : bla KPC ), aminoglycosides ( Aminoglycoside adenylyltransferase : aadA ), tetracyclines ( Tetracycline resistance protein B : tetB ), chloramphenicol ( Chloramphenicol resistance protein A : cmlA , Florfenicol/Chloramphenicol resistance protein : floR ), sulfonamides ( Sulfonamide-resistant dihydropteroate synthase 2 : sul2 ), and quinolones ( ParC subunit of topoisomerase IV : parC , Gyrase A subunit : gyrA , Gyrase B subunit : gyrB ). The PCR conditions were the same as described in section 2.2, and the specific primer information is detailed in Table 3. 2.8. Virulence Gene Detection Based on previous reports (Alcántar-Curiel et al., 2013; Candan and Aksöz, 2015; Lan et al., 2019; Russo et al., 2018), 12 virulence genes were detected in the K. pneumoniae strains via PCR. These included genes associated with lipopolysaccharide ( Uridine diphosphate galacturonate 4-epimerase : uge , Wall polysaccharide biosynthesis protein G : wabG ), fimbriae ( Fimbrial adhesion subunit H : fimH , Mrk fimbriae adhesion protein D : mrkD ), iron transport ( Enterobactin synthetase component B : entB , Klebsiella ferric uptake system : kfu , Iron-regulated outer membrane protein N : iron , Iuc aerobactin synthetase A : icuA ), urease ( Urease alpha subunit : ureA ), and allantoin metabolism ( Allantoin metabolism regulatory protein S : allS ). The PCR reaction system and operational procedures followed the standard protocol outlined in section 2.2. Specific primer sequences and related information are provided in Table 4. 2.9 String test K. pneumoniae isolates were streaked onto MacConkey agar plates and incubated at 37 °C for 12 h. Using an inoculation loop, a single colony was gently touched and slowly lifted to observe the formation of a string-like growth. The length of the slime string was measured and recorded. Isolates were classified as mucoid if they produced a string and as Hv if the string length exceeded 5 mm (Kumabe and Kenzaka, 2014). 2.10. Pathogenicity Assays in Mice and Rabbits Thirty-two non-specific pathogen-free Kunming mice and six (6W) New Zealand white rabbits were all purchased from the Animal Experiment Center of Xinjiang Medical University (animal ethics committee approval number: No. PA20251024017). The virulence of the strains was assessed using the "string test," PCR detection of virulence genes, a mouse infection model, and a New Zealand White rabbit infection model. Strains exhibiting strong adhesion in the string test and concurrently carrying a strong virulence gene profile via PCR were selected for the mouse intraperitoneal infection experiment (200 µL, 10^6 CFU/mouse). Strains demonstrating high pathogenicity in the mouse infection assay were subsequently used for the New Zealand White rabbit intratracheal challenge (500 µL, 10^7 CFU/rabbit). Following the challenge, animals were monitored daily. Any animal exhibiting a body weight loss of 30% or more was humanely euthanized. All remaining surviving animals were euthanized at the end of the study on day 7. Lung and liver tissues from the experimental rabbits were harvested, processed, and subjected to hematoxylin and eosin (H&E) staining. The euthanasia procedure involved an overdose of pentobarbital sodium administered after deep anesthesia. For mice, anesthesia was induced by intraperitoneal injection of ketamine (90 mg/kg) and sevoflurane (6 mg/kg), followed by euthanasia with pentobarbital sodium (150 mg/kg, intraperitoneal). For rabbits, anesthesia was induced by intramuscular injection of ketamine (40 mg/kg) and sevoflurane (6 mg/kg), and euthanasia was subsequently performed by intravenous injection of pentobarbital sodium (100 mg/kg) via the marginal ear vein. The absence of vital signs was verified in all animals to ensure death. 2.11. Statistical Analysis Statistical analyses were performed using GraphPad Prism 8.0. Comparisons between groups were conducted via Two-way ANOVA or Student's t-tests, as appropriate. Data are presented as mean ± standard deviation (SD). Statistical significance was defined as P < 0.05, ** P < 0.01, and *** P < 0.001. 3. Results 3.1. Isolation Rate and Identification of Klebsiella pneumoniae PCR analysis revealed that K. pneumoniae was detected in 21.78% of the 101 mastitic camel milk samples collected, with an isolation rate of 11.88%. Among 10 hand swab samples from dairy workers, the detection rate was 20% (2/10) and the isolation rate 10% (1/10). In soil samples, both detection and isolation rates were 10% (1/10), while no K. pneumoniae was detected in any of the 10 water samples (Fig. 1 B). Agarose gel electrophoresis of PCR-positive samples showed a clear band at 286 bp (Fig. 1 C). On BHI agar, the isolates formed large, moist, mucoid, raised, and grayish-white colonies. Gram staining revealed short, plump, pink-colored rods, occurring singly, in pairs, or in short chains (Fig. 1 D). Biochemical profiling indicated that all isolates were positive for glucose fermentation with gas production, lysine decarboxylase, citrate utilization, lactose fermentation, and Voges–Proskauer test, but negative for hydrogen sulfide production, ornithine decarboxylase, indole production, dulcitol fermentation, phenylalanine deaminase, and urease. These biochemical characteristics are consistent with those of K. pneumoniae as described in Bergey’s Manual of Determinative Bacteriology (data not shown). Furthermore, 16S rRNA gene sequencing confirmed the species identity, with sequence homologies ranging from 99.57% to 100% compared to reference K. pneumoniae sequences in the GenBank database. 3.2. Sequence Typing of Klebsiella pneumoniae The STs identified in this study included ST42, ST4089, ST297, and ST495. Specifically, there were two isolates of ST42, two of ST4089, two of ST297, and eight of ST495 (Fig. 2 A, B). ST495 was isolated not only from camels but also from the hands of dairy workers (KP60) and soil samples (KP289). ST4069 clusters within the same clade as ST11, a hypervirulent sequence type commonly prevalent in China, whereas ST495, the most frequently isolated sequence type in this study, exhibits close phylogenetic relatedness to ST23. In contrast, ST297 was more distantly related (Fig. 2 C). Based on wzi gene sequencing, the 14 K. pneumoniae isolates were classified into four distinct wzi allele types (Fig. 2 D). Ten isolates of ST4069 and ST495 all exhibited the wzi122 allele, corresponding to serotype KL121. The two ST42 isolates carried the wzi464 allele, and the two ST297 isolates carried the wzi475 allele. Phylogenetic analysis indicated substantial genetic divergence among these three wzi allele types. 3.3. Antimicrobial Resistance Results All 14 isolates (100%, 14/14) exhibited resistance to β-lactams (penicillin, ampicillin), aminoglycosides (kanamycin), macrolides (erythromycin), lincosamides (lincomycin), and glycopeptides (vancomycin). In contrast, susceptibility to tetracyclines was generally observed: susceptibility to tetracycline was 71.43% (10/14), intermediate susceptibility 14.29% (2/14), and resistance 14.29% (2/14); for minocycline, susceptibility was 28.57% (4/14) and intermediate susceptibility 71.43% (10/14); for doxycycline, susceptibility was 57.14% (8/14), intermediate susceptibility 28.57% (4/14), and resistance 14.29% (2/14). Susceptibility to the β-lactam agent ceftriaxone was 71.43% (10/14). All isolates (100%) displayed intermediate susceptibility to polymyxin B (Fig. 3 ). PCR-based screening identified nine distinct antimicrobial resistance genes, which were categorized into four classes: β-lactams ( bla TEM , bla CTX , bla NDM , bla VIM ), aminoglycosides ( aadA ), quinolones ( parC , gyrA , gyrB ), and sulfonamides ( sul2 ). The detection rates for bla VIM (β-lactams), gyrA , and gyrB (quinolones) were 100%; bla CTX , bla NDM (β-lactams), and aadA (aminoglycosides) were detected in 86% of isolates; bla TEM (β-lactams) was present in 43% of isolates; while parC (quinolones) and sul2 (sulfonamides) showed relatively low detection rates, each at 14%. The resistance gene combination gyrA + gyrB + bla VIM was the most prevalent, occurring in 100% of the isolates. In contrast, the combinations gyrA + gyrB + bla VIM + bla CTX + aadA + bla NDM + parC , gyrA + gyrB + bla VIM + bla CTX + bla NDM , and gyrA + gyrB + bla VIM + bla CTX + bla NDM + aadA + sul2 were the least prevalent, each detected in only 14% of the isolates. 3.4. Virulence Testing Results Through polymerase chain reaction (PCR) analysis, a total of 10 virulence genes were identified, which were categorized into five functional groups: fimbria-associated genes ( mrkD , fimH ), urease gene ( ureA ), siderophore genes ( kfu , entB , iroN ), lipopolysaccharide synthesis-related genes ( wabG , uge ), and capsule-associated genes ( magA , rmpA ). The detection rates of mrkD , ureA , kfu , entB , and wabG were the highest, each reaching 100%. The capsule gene magA was present in 86% of the isolates, while the siderophore gene iroN was detected in 71%. The fimbrial gene fimH and the lipopolysaccharide gene uge showed relatively lower detection rates, both at 43%. In contrast, the rmpA gene had the lowest prevalence, detected in only 14% of isolates (Fig. 4 A, B, E). A conserved combination of virulence genes— wabG + kfu + ureA + entB + mrkD —was universally present across all isolates, with 100% coverage (Fig. 4 B). In the string test, 12 out of 14 isolates exhibited a positive phenotype, consistent with biofilm-forming capability (Fig. 4 D). Further integration of virulence gene profiles, antimicrobial resistance genes, and STs revealed that ST495 strains KP25 and KP386 both carried the rmpA virulence gene. Notably, KP386 also harbored the most diverse array of carbapenem resistance genes, suggesting a potentially greater public health risk. In a murine intraperitoneal infection model, all tested isolates exhibited lethal effects. Strains KP99 and KP386 resulted in 100% mortality (Fig. 5 A); both belonged to ST495 and carried the rmpA virulence gene. Furthermore, in a rabbit intratracheal challenge assay, strain KP386 demonstrated high pathogenicity, causing pulmonary infection and liver abscesses (Fig. 5 B). Compared with the blank control, hematoxylin-eosin (HE) staining revealed significant pathological lesions in the lungs and liver of KP386-infected mice, as indicated by arrows (Fig. 5 C). In summary, K. pneumoniae isolates obtained from camels in Xinjiang frequently harbor multiple virulence genes and include strains with high pathogenic potential. To evaluate the risk of cross-species transmission between animals and humans, further epidemiological surveillance in this region is warranted. 4. Discussion In 1882, K. pneumoniae was first isolated from the lungs of a patient who died of pneumonia. Initially regarded as an opportunistic pathogen in the natural environment, it did not receive widespread societal attention (Bengoechea and Sa Pessoa, 2019 ). However, this bacterium has since spread extensively among humans, the environment, and animals, posing a potential threat to public health and the livestock industry (Giri et al., 2021 ). In this study, the incidence of mastitis in Bactrian dairy camels was detected at 21% (101/480), slightly lower than the 25% incidence of clinical mastitis reported in organized camel herds in Bikaner, Rajasthan, India (Ranjan et al., 2021 ). This difference might be attributed to the early developmental stage of the camel dairy industry in Keping County, Xinjiang, where environmental pathogen loads may be lower. The detection rate of K. pneumoniae in mastitic camel milk samples in this study was 21.78%, similar to the rate found in Libyan camel milk (1/5; 20%) (Azwai et al., 2024 ) and slightly lower than the proportion in Kuwaiti camel milk (12/33; 36%) (Rahmeh et al., 2024 ). The isolation rate of K. pneumoniae varies from 4.55% to 36.59% in pigs from certain regions of China (Córdova-Espinoza et al., 2023 ; Hou et al., 2024b ; Rui et al., 2023 ; Wan et al., 2025a ). Nobrega et al. detected K. pneumoniae in most milk samples from 25 lactating dairy cows (Nobrega et al., 2021 ). Furthermore, we also detected K. pneumoniae in samples from workers' hands and soil, consistent with previous reports from dairy farms indicating that sources of K. pneumoniae include organic bedding materials like wood by-products, cow manure, and dairy products (Cheng et al., 2021 ). This further highlights the severe challenge posed by K. pneumoniae to global public health. As of now, the Pasteur MLST database lists over 7,000 STs of K. pneumoniae globally. ST495 has been isolated from human blood, sputum, and the environment; ST297 from cattle (rumen, milk), seawater, and humans (blood, urine); ST42 from humans (blood, urine, sputum, feces); and ST4089 from human urine. Relevant literature reports that ST495 was isolated from a Greek hospital in 2011 (Giakkoupi et al., 2011 ). Additionally, ST42 has been detected in samples from pig and dairy farms in Xinjiang (Hou et al., 2024a), in rivers receiving wastewater treatment plant effluent in 2024 (Swain et al., 2024 ), in the feces of a patient with a liver abscess in 2019 (Kim et al., 2020 ), in a tertiary care hospital in North India in 2021 (Nirwan et al., 2021 ), and in a high-complexity hospital in Ecuador (Reyes et al., 2021 ). Reports on ST297 and ST4089 are relatively scarce. In this experiment, 14 strains were isolated, all exhibiting multidrug resistance. ST495 was the predominant type in the camel farm. The study indicates that the four K. pneumoniae types ST495, ST297, ST42, and ST4089 all pose a risk of transmission among humans, the environment, and camels, constituting a serious threat to public health. However, several recent reports from Xinjiang identified ST161 from bedding in a Urumqi dairy farm, and ST5387 from cloacal swabs of healthy laying hens and environmental fecal samples from a chicken farm. ST37 and ST967 K. pneumoniae strains were isolated from pig farms (Wan et al., 2025b ), suggesting significant differences in prevalent STs across different regions of Xinjiang. Ten isolates of ST4089 and ST495 all exhibited the wzi122 allele, corresponding to serotype KL121. In this study, the CC17 clonal complex (ST495) was identified among CRKP strains, and the expression of NDM-1 was observed in camel-derived isolates, aligning with previous reports of CC17-associated NDM-1 expression in neonatal infections(Yin et al., 2023 ). Notably, these ST495 strains exhibited the KL121 capsular serotype, suggesting potential acquisition of virulence plasmids. Plasmids play a pivotal role in the dissemination of both antimicrobial resistance and hypervirulence in K. pneumoniae (Dunn et al., 2019 ). Biofilm formation is a crucial mechanism of drug resistance in K. pneumoniae . The most important surface structures during K. pneumoniae biofilm formation are type 3 fimbriae and CPS. Fimbriae mediate stable adhesion, while CPS ultimately influences biofilm structure and intercellular communication (Clegg and Murphy, 2016 ). In this study, among the 14 isolated K. pneumoniae strains, the detection rate for the fimH gene was 42.86% (6/14), primarily as ST495 strains generally did not carry the fimH gene. The detection rate for the mrkD gene was 100% (14/14), for the capsular gene magA was 86% (12/14), and for rmpA was 14% (2/14). This differs from another study on K. pneumoniae in pigs from Xinjiang, where among 21 isolated strains, the detection rates for fimH and mrkD were 100%, but no capsular genes ( rmpA and magA ) were detected (Wan et al., 2025b ). Among these 14 strains, 6 exhibited strong biofilm-forming ability, and 6 had moderate ability, aligning with the multidrug resistance observed in susceptibility tests. Concurrently, the high detection rate of multiple resistance genes indicates that treating infections caused by these bacteria would be challenging. In this study, resistance genes for β-lactams, aminoglycosides, sulfonamides, and quinolones were detected in the K. pneumoniae isolates, with varying degrees of resistance, likely related to drug usage practices within the farm. The resistance mechanisms of K. pneumoniae are complex and diverse; the most common involves acquiring plasmids carrying resistance genes, while another involves the accumulation of gene mutations (Cheng et al., 2020 ). Notably, the dissemination of these resistance genes heavily relies on mobile genetic elements (Koudoum et al., 2025 ). These plasmids and transposons can not only spread among human pathogens but also cross species barriers, widely disseminating among animal and environmental microbes, ultimately leading to a surge and persistent prevalence of multidrug-resistant strains (Pitout et al., 2019 ). Tandem repeat-mediated mutagenesis is a significant, previously underestimated genetic mechanism driving the rapid diversification and adaptive evolution of bla KPC enzymes under antibiotic selection pressure (Ye et al., 2025 ). A limitation of our study is the lack of testing for resistance genes in the environment, which will be addressed in future work. With the intensification of animal farming, the extensive use of antimicrobials to reduce economic losses from various bacterial diseases has led to the continuous emergence of resistant strains. In this study, the K-B method was used to detect the susceptibility of K. pneumoniae isolates to antimicrobial agents. The results indicate a concerning level of antibiotic resistance in camel-derived K. pneumoniae . All 14 isolated K. pneumoniae strains were MDR, showing 100% (14/14) resistance to β-lactams, aminoglycosides, macrolides, lincosamides, and glycopeptides, consistent with findings from pig farms in Xinjiang. The isolates were generally susceptible to tetracyclines, suggesting they could be considered for subsequent K. pneumoniae treatment, which differs somewhat from isolates from Xinjiang pig farms, where 21 K. pneumoniae isolates showed strong resistance to β-lactams, aminoglycosides, tetracyclines, sulfonamides, quinolones, and other antimicrobials, exhibiting multidrug resistance and sensitivity only to carbapenems (imipenem) and polymyxins (polymyxin B) (Wan et al., 2025b ), likely related to clinical drug use on the farms. Interestingly, the isolates were relatively sensitive (71.43%, 10/14) to the β-lactam ceftriaxone, the mechanism of which warrants further investigation. All isolated K. pneumoniae strains showed 100% intermediate susceptibility to polymyxin B, one of the "last line" antibiotics for clinical treatment. Bacterial resistance phenotypes are determined by resistance genotypes, but phenotype and genotype may not always align perfectly. PCR detected 9 resistance genes belonging to 4 classes (including bla TEM , bla CTX , bla NDM , bla VIM , aadA , sul2 , parC , gyrA , gyrB ). Among them, the detection rates for the β-lactam bla VIM and the quinolones gyrA and gyrB were 100%; for the β-lactams bla CTX , bla NDM , and the aminoglycoside aadA were 86%; for the β-lactam bla TEM was 43%; and for the quinolone parC and sulfonamide sul2 were relatively low at 14%. This discrepancy might be due to the presence of undetected or unknown relevant resistance genes. During farming, the extensive use of antibiotics, whether for treatment or growth promotion, can activate associated resistance genes (Jonas et al., 2021 ), thereby enhancing strain resistance and facilitating the transmission of these resistant strains to humans via the food chain or other transmission routes (Ovejero et al., 2017 ). In this study, K. pneumoniae isolates exhibited strong resistance to 5 classes out of the 20 antibiotics tested, while being relatively sensitive to only 2 antibiotics. It is recommended that farms strictly control antibiotic use to prevent the emergence of more resistant strains. Furthermore, new antimicrobial biological agents could be selected for prevention and control, enabling precise prevention, comprehensive management, and holistic treatment. Research (Shon et al., 2013 ) shows that the iucA can increase the virulence of hypervirulent K. pneumoniae (hvKP) and is a key virulence characteristic of hvKP. In this study, the iucA virulence gene was not detected in any of the 14 K. pneumoniae isolates, but the carriage rate for iroN was high (71.43%). The virulence genes iucA and iroN are associated with iron uptake systems, and the iro gene cluster primarily encodes salmochelin, the C-glucosylated form of enterobactin. Studies indicate that over 90% of K. pneumoniae strains causing liver abscesses can secrete salmochelin (Lam et al., 2018a ). In this study, the antimicrobial resistance and virulence gene profiles of isolates from workers' hands, soil, and camels were nearly identical, suggesting a risk of mutual transmission among humans, camels, and the environment. Additionally, the detection of ST495: KL121 strains in camels indicates that conventional ST495 may have acquired high virulence through plasmid uptake. Two ST495: KL121 isolates not only carried the rmpA gene but also exhibited high lethality in mouse pathogenicity assays, indicating strong virulence and potential “superbug” characteristics, Particular attention should be paid to the risk of transmission of such hv strains from animals to humans. Compared to the detection rates of iucA , iroN , and rmpA in porcine isolates from Xinjiang (57.14%, 88.89%, and 0%, respectively), our findings further highlight significant regional differences among K. pneumoniae clones. Multiple studies have elucidated the molecular mechanisms underlying the dissemination of virulence and resistance genes. For instance, the IncHI1B/repB -type hybrid virulence plasmid ( pHvKP51-Vir ) can promote the emergence of carbapenem-resistant hypervirulent K. pneumoniae (CR-hvKP) through mobile genetic element (MGE)-mediated recombination during conjugation (Huo et al., 2025 ). Non-conjugative virulence plasmids may also disseminate across strains via helper conjugative plasmids or fusion with multidrug-resistant plasmids (Li et al., 2020 ). The p15WZ-82_Vir plasmid represents one such product of integration between a hypervirulent plasmid and a multidrug-resistant plasmid (Yang et al., 2019 ). Moreover, hybrid conjugative virulence plasmids efficiently facilitate the transfer of virulence and resistance genes from ST11 CRKP to CR-hvKP (Gu et al., 2018 ; Liao et al., 2020 ). Additionally, some strains can integrate genetic elements, including virulence genes, into their chromosomes (Lam et al., 2018b ). Collectively, these studies demonstrate a close association among virulence genes, resistance genes, and their corresponding phenotypes in K. pneumoniae . Therefore, this study identified the presence of highly virulent and highly resistant superbugs in dairy camels. However, the molecular mechanisms underlying their formation require further investigation. 5. Conclusion This study elucidates the prevalent K. pneumoniae strains in Bactrian camel farms in Keping County, Xinjiang, China, revealing significant genetic diversity and multidrug-resistance profiles. Molecular typing identified four primary STs: ST495, ST297, ST42, and ST4089. Of particular concern is the ST495 lineage, which not only represents the predominant circulating strain in the region but was also isolated from camels, the environment, and dairy workers, suggesting a complex transmission cycle. Critically, this is the first identification of the ST495: KL121 strain in camels, characterized by concurrent hypervirulence and multidrug resistance, indicating a potential evolution towards a "superbug". Further analysis demonstrates that these camel-derived K. pneumoniae isolates commonly exhibit robust biofilm-forming capabilities and harbor a diverse array of antimicrobial resistance and virulence genes. This undoubtedly complicates clinical prevention and treatment strategies and poses a substantial threat to public health security. Abbreviations MDR multidrug-resistant hv hypervirulent K. pneumoniae Klebsiella pneumoniae MLST Multilocus sequence typing CR carbapenem resistance CPS capsular polysaccharide LPS lipopolysaccharide BHI Brain Heart Infusion ST sequence type HE hematoxylin-eosin hvKP virulence of hypervirulent K. pneumoniae CR-hvKP carbapenem-resistant hypervirulent K. pneumoniae MGE mobile genetic element khe K. pneumoniae hemagglutinin type 3 fimbrial gene 16S rRNA 16S ribosomal Ribonucleic Acid ropB RNA polymerase beta subunit tonB TonB protein phoE Phosphate limitation-inducible outer membrane porin E pgi Phosphoglucose isomerase mdh Malate dehydrogenase infB Translation initiation factor IF-2 gapA Glyceraldehyde-3-phosphate dehydrogenase A bla TEM Temoneira β-lactamase Bla CTX Cefotaxime-hydrolyzing β-lactamase bla NDM New Delhi metallo-β-lactamase bla OXA−48 Oxacillinase-48 bla IMP Active on imipenem β-lactamase bla VIM Verona integron-encoded metallo-β-lactamase bla DHA Verona integron-encoded metallo-β-lactamase Bla KPC Klebsiella pneumoniae carbapenemase aadA Aminoglycoside adenylyltransferase tet(B) Tetracycline resistance protein B cmlA Chloramphenicol resistance protein A floR Florfenicol/Chloramphenicol resistance protein sul2 Sulfonamide-resistant dihydropteroate synthase 2 parC ParC subunit of topoisomerase IV gyrA Gyrase A subunit gyrB Gyrase B subunit uge Uridine diphosphate galacturonate 4-epimerase wabG Wall polysaccharide biosynthesis protein G fimH Fimbrial adhesion subunit H mrkD Mrk fimbriae adhesion protein D entB Enterobactin synthetase component B iroN Iron-regulated outer membrane protein N kfu Klebsiella ferric uptake system icuA Iuc aerobactin synthetase A ureA Urease alpha subunit allS Allantoin metabolism regulatory protein S Declarations Ethics approval and consent to participate The mice and rabbits experiments were conducted in accordance with the protocol approved by the Scientific Ethics Committee of Tarim University (Permit No.PA20251024017). And the owner of the camels is aware of this and has given his consent for the collection of milk samples from the camels. Consent for publication Not applicable. Competing interests No potential conflict of interest was reported by the author(s). Funding This work was funded by the Tianchi Talents Introduction Program of Xinjiang Uygur Autonomous Region (BT-2025-TCYC-0041) and the Tarim University President’s Fund Project (TDZKBS202522, TDZKBS202521). Author Contribution QS, MN, LZ, JY and DT delineated the study conception and design. LZ, JY and DT supervised the study. QS, MN, MH, YH, JZ performed laboratory tests as well as analyzed the data, and MT, JG, YM, MN and QS participated in the animal experiments and analyzed the data. QS, MN and DT wrote the manuscript and approved the final version for publication. QS, MN, LZ, JY and DT participated in the manuscript discussion and revision. All authors have read and approved the final version of the manuscript. 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Wusiman M, Zuo J, Yu Y, Lv Z, Wang M, Nie L, Zhang X, Wu J, Wu Z, Jiang W, Pan Z, Zhang W, Yin H, Huang C, Chen Z, Miao J, Chen W, Han X. Molecular characterization of Klebsiella pneumoniae in clinical bovine mastitis in 14 provinces in China. Vet Res Commun. 2024;49:18. .https://doi.org/10.1007/s11259-024-10598-4 . Yang X, Wai-Chi Chan E, Zhang R, Chen S. A conjugative plasmid that augments virulence in Klebsiella pneumoniae. Nat Microbiol. 2019;4:2039–43. https://doi.org/10.1038/s41564-019-0566-7 . Ye H, Liu R, Shen J, Yang W, Hu T, Liu X, Wang K, Gong L, Xu H, Zhu J, Zheng Z, Zheng B. 2025. Unravelling tandem repeat-mediated mutagenesis drive rapid diversification of KPC enzymes: emergence of bla(KPC-263) and enhanced resistance to ceftazidime-avibactam. EBioMedicine 121, 105979.https://doi.org/10.1016/j.ebiom.2025.105979 Yin L, Lu L, He L, Lu G, Cao Y, Wang L, Zhai X, Wang C. Molecular characteristics of carbapenem-resistant gram-negative bacilli in pediatric patients in China. BMC Microbiol. 2023;23:136. .https://doi.org/10.1186/s12866-023-02875-0 . Zhang F, Liu X, Li Z, Li Z, Lei Z, Fan Y, Yang X, Liu Q, Ma Y, Lu B. Tracking international and regional dissemination of the KPC/NDM co-producing Klebsiella pneumoniae. Nat Commun. 2025;16:5574. https://doi.org/10.1038/s41467-025-60765-7 . Zhu J, Chen T, Ju Y, Dai J, Zhuge X. Transmission Dynamics and Novel Treatments of High Risk Carbapenem-Resistant Klebsiella pneumoniae: The Lens of One Health. Pharmaceuticals (Basel) 17. 2024. https://doi.org/10.3390/ph17091206 . Zhu J, Ju Y, Zhou X, Chen T, Zhuge X, Dai J. Epidemiological characteristics of SHV, cmlv, and FosA6-producing carbapenem-resistant Klebsiella pneumoniae based on whole genome sequences in Jiangsu, China. Front Microbiol. 2023;14:1219733. .https://doi.org/10.3389/fmicb.2023.1219733 . Tables Tables are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files supplementaryfile.docx Tables.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 08 Apr, 2026 Reviewers agreed at journal 29 Mar, 2026 Reviewers invited by journal 28 Mar, 2026 Editor assigned by journal 12 Feb, 2026 Submission checks completed at journal 11 Feb, 2026 First submitted to journal 11 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8650943","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":618053437,"identity":"018a96c9-c47a-467a-b474-9d1f092af55b","order_by":0,"name":"Meiting Niu","email":"","orcid":"","institution":"Tarim University","correspondingAuthor":false,"prefix":"","firstName":"Meiting","middleName":"","lastName":"Niu","suffix":""},{"id":618053438,"identity":"649476f0-075a-40ce-b0c2-b2afbe4d4d8d","order_by":1,"name":"Ling Zhao","email":"","orcid":"","institution":"Tarim University","correspondingAuthor":false,"prefix":"","firstName":"Ling","middleName":"","lastName":"Zhao","suffix":""},{"id":618053443,"identity":"4970a1f8-5c7d-457a-98e1-a13ac459f87e","order_by":2,"name":"Mengzhe Hou","email":"","orcid":"","institution":"Tarim University","correspondingAuthor":false,"prefix":"","firstName":"Mengzhe","middleName":"","lastName":"Hou","suffix":""},{"id":618053450,"identity":"93844f0a-7ff8-4594-8755-797eb5198bdf","order_by":3,"name":"Yu Han","email":"","orcid":"","institution":"Tarim University","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Han","suffix":""},{"id":618053452,"identity":"4d4f7d7c-c242-4200-aaa9-bdda99a4a09a","order_by":4,"name":"Jiabin Zhang","email":"","orcid":"","institution":"Tarim University","correspondingAuthor":false,"prefix":"","firstName":"Jiabin","middleName":"","lastName":"Zhang","suffix":""},{"id":618053455,"identity":"c9df4ba0-ff92-4f96-b8ac-ae92a6e9a724","order_by":5,"name":"Maimaiti Tuniyazi","email":"","orcid":"","institution":"Tarim University","correspondingAuthor":false,"prefix":"","firstName":"Maimaiti","middleName":"","lastName":"Tuniyazi","suffix":""},{"id":618053457,"identity":"bbb26371-39bd-4bd2-a121-2172702c83b4","order_by":6,"name":"Jindong Gao","email":"","orcid":"","institution":"Tarim University","correspondingAuthor":false,"prefix":"","firstName":"Jindong","middleName":"","lastName":"Gao","suffix":""},{"id":618053458,"identity":"65b8af4e-49a8-4bac-834a-8480a3d91c37","order_by":7,"name":"Yonghui Ma","email":"","orcid":"","institution":"Tarim University","correspondingAuthor":false,"prefix":"","firstName":"Yonghui","middleName":"","lastName":"Ma","suffix":""},{"id":618053459,"identity":"ea6001ba-2243-4779-b1c2-37feac085dff","order_by":8,"name":"Ruyue Zhang","email":"","orcid":"","institution":"Tarim University","correspondingAuthor":false,"prefix":"","firstName":"Ruyue","middleName":"","lastName":"Zhang","suffix":""},{"id":618053460,"identity":"d0de0a54-7a0f-4302-bb5b-9c98d638af7a","order_by":9,"name":"Qingshuo Ning","email":"","orcid":"","institution":"Tarim University","correspondingAuthor":false,"prefix":"","firstName":"Qingshuo","middleName":"","lastName":"Ning","suffix":""},{"id":618053461,"identity":"90bfa45c-af9d-4a21-a293-ff68853cbe2a","order_by":10,"name":"Jinhua Yin","email":"","orcid":"","institution":"Tarim University","correspondingAuthor":false,"prefix":"","firstName":"Jinhua","middleName":"","lastName":"Yin","suffix":""},{"id":618053462,"identity":"b3f6f65e-1259-419f-ad0c-fa7483f80b74","order_by":11,"name":"Dayong Tao","email":"","orcid":"","institution":"Tarim University","correspondingAuthor":false,"prefix":"","firstName":"Dayong","middleName":"","lastName":"Tao","suffix":""},{"id":618053463,"identity":"a02f2a43-18a1-4bbb-b8cd-fc4a2b7fa7cc","order_by":12,"name":"Qinghua Shang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6ElEQVRIie3PvWrDMBDA8SsCTWq1nmhwXuFCoFMeRsKlnQoZNQRq4SAPaciavIXHjDYGd1H3jAl9gm6FFvoxt9ju1kG/+f7cHUAU/UN83NTP73aWSOncUdtFf3KBPCURbqZq2+R0DG1/kqC4wvNlY8rq1qvTkg047PKBUGXsbJfV3pqMgyxWujsZtXOa7DmTzPmD2Y8Aw1PZnUBaahMEV19bDiZwILzrSzRVtUdBlfFz49mABK8nznnC7wSGJaJNGQRNalvnqEMren8ZF/njG9iP+40sTi+vdpHIYt2d/CD+Nh5FURT96hMiqE49zLEhmwAAAABJRU5ErkJggg==","orcid":"","institution":"Tarim University","correspondingAuthor":true,"prefix":"","firstName":"Qinghua","middleName":"","lastName":"Shang","suffix":""}],"badges":[],"createdAt":"2026-01-20 16:05:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8650943/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8650943/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106359105,"identity":"08272001-24c5-4671-ad75-101fd8eddf36","added_by":"auto","created_at":"2026-04-07 19:41:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":278946,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIsolation and identification of Klebsiella pneumoniae from mastitic camel milk samples. \u003c/strong\u003eA: Sampling location; B: Distribution of sample types (milkers, drinking water, camels, soil) and their respective positive detection and isolation rates; C: Electrophoresis of PCR detection from samples, positive samples show a band at 285 bp; D: Colony morphology of K. pneumoniae on BHI agar and microscopic image after Gram staining.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8650943/v1/c1a3244f6470c89909d5bc1c.png"},{"id":106404964,"identity":"d1f5e4bf-8136-4c4b-b1bc-c0854825aa24","added_by":"auto","created_at":"2026-04-08 09:18:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":789827,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSequence type analysis of the isolated Klebsiella pneumoniae strains. \u003c/strong\u003eA: Electrophoresis of PCR detection for the 7 housekeeping genes (1: ropB , 1075 bp; 2: infB , 462 bp; 3: pgi , 566 bp; 4: phoE , 602 bp; 5: gapA , 662 bp; 6: tonB , 539 bp; 7: mdh , 756 bp); B: Distribution of sequence types among the 14 K. pneumoniae strains; C: Minimum spanning tree based on MLST; D: Phylogenetic tree based on wzi alleles.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8650943/v1/5956760edf11ee326c913300.png"},{"id":106403891,"identity":"c2129b1a-e6bc-42d5-8bb0-087b9baadeac","added_by":"auto","created_at":"2026-04-08 09:15:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":345257,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntimicrobial resistance analysis of the isolated Klebsiella pneumoniae strains. \u003c/strong\u003eA: Disk diffusion antimicrobial susceptibility test results for the isolates; B: Electrophoresis of PCR detection for resistance genes in the isolates; C: a: Percentage of isolates sensitive in disk diffusion test; b: Percentage of isolates with intermediate susceptibility in disk diffusion test; c: Percentage of resistant isolates in disk diffusion test; d: Percentage of PCR-positive resistance genes; D: Number of strains with different resistance patterns.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8650943/v1/c298d0a79a256d72d4d0b47b.png"},{"id":106359110,"identity":"f0c005ba-dd2f-44d8-867f-ec1e4841f603","added_by":"auto","created_at":"2026-04-07 19:41:34","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":367759,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVirulence analysis of the isolated Klebsiella pneumoniae strains. \u003c/strong\u003eA: Electrophoresis of PCR detection for virulence genes in the isolates; B: Number of strains with different virulence gene combinations; C: String test of the isolated Klebsiella strains; D: Biofilm formation ability of the isolated strains; E: Distribution of resistance genes, virulence genes, and sequence types (STs) among the isolates.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8650943/v1/456ed111b83118465fdcf513.png"},{"id":106359111,"identity":"62a187fd-01ce-442d-8530-b3ad68f0096d","added_by":"auto","created_at":"2026-04-07 19:41:34","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":348386,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePathogenicity test results for selected isolates. \u003c/strong\u003eA: Survival curves of mice after intraperitoneal infection with selected isolates; B: Necropsy findings of a rabbit infected with KP386; C: Hematoxylin and eosin (H\u0026amp;E) staining results of tissues from the KP386-infected rabbit.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8650943/v1/00fe6dce85dd374b265006a3.png"},{"id":106406543,"identity":"b36b1c08-5b92-4948-a4c6-8a75bf2d2e2d","added_by":"auto","created_at":"2026-04-08 09:32:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3339201,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8650943/v1/e06d1056-7279-4af4-87e1-0b5e12c28dd6.pdf"},{"id":106403783,"identity":"e9497d72-f6ab-4a9d-9965-6af17ff9803a","added_by":"auto","created_at":"2026-04-08 09:14:58","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1803840,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryfile.docx","url":"https://assets-eu.researchsquare.com/files/rs-8650943/v1/cd3833445424a0192385a71d.docx"},{"id":106404358,"identity":"08b30ded-3c41-46f3-961e-198680a088af","added_by":"auto","created_at":"2026-04-08 09:15:52","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":30242,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-8650943/v1/269b3662694e4c3c646f9da0.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Molecular Characterization and Prevalence of Hypervirulent and Multidrug-Resistant Klebsiella pneumoniae in Mastitis of Bactrian Camels from Xinjiang, China","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e \u003cem\u003eK. pneumoniae\u003c/em\u003e is a flagellated, encapsulated bacterium ubiquitous in natural environments and recognized as an opportunistic pathogen. It primarily infects immunocompromised individuals, causing a range of serious human and animal diseases, including pneumonia, bacteremia, urinary tract infections, and liver abscesses(Wusiman et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) (Chen et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Dixon et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Raza et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Notably, the mortality rate for pneumonia caused by \u003cem\u003eK. pneumoniae\u003c/em\u003e is approximately 50% (Martin and Bachman, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In camels, it is also a causative agent of mastitis, a significant zoonotic disease that poses a serious threat to both public health and animal production (Chen et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Compared to bovine mastitis, research on the etiology of camel mastitis is relatively limited, with existing studies indicating bacterial infection as a primary cause (Aqib et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Environmental mastitis can be induced by various bacteria, among which \u003cem\u003eK. pneumoniae\u003c/em\u003e is one of the most clinically significant pathogens (Mao et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), widely present on animal mucosal surfaces and in the environment.\u003c/p\u003e \u003cp\u003eIn recent years, the convergence of carbapenem resistance (CR) and hypervirulence (hv) phenotypes has given rise to strains combining heightened pathogenicity with multidrug resistance, often termed \"highly virulent and highly resistant\" strains. The global dissemination of ST11 CR-HvKP, in particular, constitutes a \"superbug\" threat (Zhu et al., \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) (Zhang et al., \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) and represents one of the most severe challenges in current global public health.\u003c/p\u003e \u003cp\u003eClinically relevant lineages of \u003cem\u003eK. pneumoniae\u003c/em\u003e can be isolated from diverse animal and environmental sources, revealing their potential for cross-species transmission (Zhu et al., \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Whole-genome analyses have confirmed close phylogenetic relationships between clinical isolates and those from retail meat products (Abo-Shama et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), bovine mastitis cases (Holt et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), and environmental samples (Runcharoen et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Of particular concern is the situation in Southern Xinjiang, where camels are a distinctive economic livestock species, and products like camel milk are in increasing contact with humans. However, research on the virulence and drug resistance of \u003cem\u003eK. pneumoniae\u003c/em\u003e from camel hosts in this specific regional context remains scarce.\u003c/p\u003e \u003cp\u003eFurthermore, exacerbated by antibiotic misuse, \u003cem\u003eK. pneumoniae\u003c/em\u003e employs diverse antimicrobial resistance mechanisms. The increasing frequency of drug-resistant isolates complicates the treatment of \u003cem\u003eK. pneumoniae\u003c/em\u003e infections (Kot and Witeska, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The emergence of MDR strains has intensified this therapeutic challenge. Resistance in \u003cem\u003eK. pneumoniae\u003c/em\u003e is often associated with transferable plasmids carrying resistance and virulence genes, which can enhance bacterial survival in hostile environments (Da Silva and Mendon\u0026ccedil;a, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe diagnostic criteria and strain definition for hypervirulent \u003cem\u003eK. pneumoniae\u003c/em\u003e (HvKP) remain subjects of ongoing debate (Kochan et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Current detection methods primarily rely on screening for specific virulence gene markers. Genes such as \u003cem\u003eiroB\u003c/em\u003e, \u003cem\u003eiucA\u003c/em\u003e, \u003cem\u003epeg-344\u003c/em\u003e, \u003cem\u003ermpA\u003c/em\u003e, and \u003cem\u003ermpA2\u003c/em\u003e are considered reliable biomarkers for identifying HvKP (Russo et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Key virulence factors include capsular polysaccharide (CPS), lipopolysaccharide (LPS), allantoin metabolism, fimbriae, urease, and iron acquisition systems (Li et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Among the identified serotypes, K1 and K2 are regarded as the most virulent. The capsule, an extracellular polysaccharide matrix enveloping the bacterium, impedes phagocytosis, inhibits antimicrobial peptide activity, blocks complement components to prevent complement-mediated lysis and opsonization, and suppresses the acute activation of immune responses (Paczosa and Mecsas, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). LPS, composed of lipid A, core oligosaccharide, and O antigen, functions as both an immune activator and a virulence factor. Lipid A can dampen host inflammatory responses, while the diversity of the O antigen aids in immune evasion (Choby et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Clegg and Murphy, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Type I and Type III fimbriae are membrane-associated structures consisting of structural and adhesive subunits, encoded by specific gene clusters (\u003cem\u003efim\u003c/em\u003e and \u003cem\u003emrk\u003c/em\u003e, respectively) (Ara\u0026uacute;jo et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Huang et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Allantoin, a product of purine catabolism, can be degraded by \u003cem\u003eK. pneumoniae\u003c/em\u003e as a source of nitrogen and carbon. This metabolic pathway involves several enzymes, with the \u003cem\u003eallS\u003c/em\u003e gene playing a key role in its activation (Chou et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Among the siderophores secreted by \u003cem\u003eK. pneumoniae\u003c/em\u003e, enterobactin is encoded by the \u003cem\u003eentA, B, C, D, E\u003c/em\u003e, and \u003cem\u003eF\u003c/em\u003e gene cluster. The \u003cem\u003eentB\u003c/em\u003e gene is frequently used as a marker in various studies, as this siderophore is prevalent in both classical and hypervirulent strains (Bachman et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis study isolated 14 strains of \u003cem\u003eK. pneumoniae\u003c/em\u003e from camel milk, milker, and environmental samples collected from several farms in Xinjiang, China. The isolates were analyzed for antimicrobial resistance, pathogenicity, and the presence of resistance and virulence genes. These findings aim to inform precise treatment strategies for \u003cem\u003eK. pneumoniae\u003c/em\u003e infections in farmed camel populations and to highlight potential public health risks.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.1. Sample Collection\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFrom October 2024 to October 2025, a total of 510 samples were randomly collected from an intensive camel farm in Keping County, Aksu Prefecture, Xinjiang, China（Fig1. A）. These included 480 camel milk samples, 10 soil samples, 10 water samples, and 10 hand swab samples from milkers. Among the camel milk samples, 81 subclinical mastitis and 20 clinical mastitis samples, identified using the LMT diagnostic kit, were selected for this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.2. Isolation and Purification of K. pneumoniae\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUnder aseptic conditions, milk samples were inoculated into Brain Heart Infusion (BHI) broth (Qingdao Hope Bio-technology Co., Ltd., China) and incubated overnight at 37°C with shaking at 180 rpm. Genomic DNA was extracted from the milk samples using the boiling method and used as a template. Initial screening for \u003cem\u003eK. pneumoniae\u003c/em\u003e was performed via PCR amplification targeting the species-specific \u003cem\u003eK. pneumoniae hemagglutinin type 3 fimbrial gene\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/em\u003e (\u003cem\u003eKhe\u003c/em\u003e). The PCR protocol was as follows: initial denaturation at 95°C for 3 min; 30 cycles of denaturation at 94°C for 25 s, annealing at 61°C for 25 s (The primer sequences and related information are detailed in Table\u0026nbsp;1)(Wan et al., 2025b), and extension at 72°C for 1 min; followed by a final extension at 72°C for 10 min. Milk samples yielding positive PCR results were then streaked onto BHI agar plates and incubated at 37°C overnight. Single colonies were selected and repeatedly streaked onto fresh BHI plates for purification until uniform colony size and morphology were achieved. Colony characteristics, including size, morphology, and color, were recorded. Gram staining (Beijing Solarbio Science \u0026amp; Technology Co., Ltd., China) was performed for microscopic examination to confirm bacterial morphology and Gram reaction.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.3. Biochemical Identification\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBiochemical identification of the isolated strains was conducted using a biochemical test tube kit (Hangzhou Microbial Reagent Co., Ltd., China), following the manufacturer's instructions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.4. PCR Confirmation and 16S ribosomal Ribonucleic Acid Gene\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e(\u003cem\u003e16S rRNA\u003c/em\u003e)\u003cem\u003e\u0026nbsp;Sequencing\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGenomic DNA from bacterial isolates was extracted using the boiling method and served as the PCR template. The PCR protocol was: initial denaturation at 94°C for 5 min; 30 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s (The primer sequences and related information are detailed in Table\u0026nbsp;1), and extension at 72°C for 30 s; followed by a final extension at 72°C for 10 min. PCR products were verified by 1% agarose gel electrophoresis. The 16S rRNA gene of the isolates was amplified using universal bacterial primers. The purified PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The assembled full-length sequences were compared against the NCBI database using BLAST，and the sequence has been uploaded to GenBank (SUB15995834: X975972 - PX975985). All primers were synthesized by Youkang Biotechnology Co., Ltd. (Xinjiang, China).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.5. Capsular serotyping and MLST analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe wzi gene sequencing method was used to determine the capsular polysaccharide type of the isolates, while the MLST method was employed to identify the sequence type (ST) of the isolates as described in (Brisse et al., 2013), primers for the wzi gene of \u003cem\u003eK. pneumoniae and\u003c/em\u003e seven housekeeping genes (\u003cem\u003eGlyceraldehyde-3-phosphate dehydrogenase A\u003c/em\u003e: \u003cem\u003egapA\u003c/em\u003e, \u003cem\u003eTranslation initiation factor IF-2\u003c/em\u003e: \u003cem\u003einfB\u003c/em\u003e, \u003cem\u003eMalate dehydrogenase:\u003c/em\u003e \u003cem\u003emdh\u003c/em\u003e, \u003cem\u003ePhosphoglucose isomerase:\u003c/em\u003e \u003cem\u003epgi\u003c/em\u003e, \u003cem\u003ePhosphate limitation-inducible outer membrane porin E: phoE\u003c/em\u003e, \u003cem\u003eRNA polymerase beta subunit: ropB\u003c/em\u003e, and \u003cem\u003eTonB protein: tonB\u003c/em\u003e) were designed and subjected to PCR amplification. The primer sequences and related information are detailed in Table\u0026nbsp;2. PCR reaction program: 95\u0026nbsp;°C for 3\u0026nbsp;min for pre-denaturation; 94 °C for 25s for denaturation; 60\u0026nbsp;°C for 25s for annealing; 72\u0026nbsp;°C for 1\u0026nbsp;min for extension, set for 30 cycles; followed by a final extension at 72\u0026nbsp;°C for 5\u0026nbsp;min. Use 1% agarose gel electrophoresis to validate the PCR products. The PCR positive products were sequenced using the Sanger method, with the sequencing performed by General Biologicals (Shanghai, China). Submit the wzi gene sequence to the online (https://bigsdb.pasteur.fr/klebsiella/). The capsular serotypes of the isolates were obtained, and 7 housekeeping gene sequences were uploaded to MLST network database, and the ST types of the isolates were obtained. The wzi gene and housekeeping genes sequences have been uploaded to GenBank (PX991553 - PX991664).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.6. Quantitative biofilm production assay\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe ability of \u003cem\u003eK. pneumoniae\u003c/em\u003e to form biofilm was determined by crystal violet staining \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates were inoculated with LB solid medium by four-zone method and cultured at 37 °C for 12 h. A single colony was selected and inoculated in LB liquid medium, and cultured at 37 °C and 180 r/min until the concentration of the liquid reached 0.5 mL. 200 µL of the culture medium was transferred to the sterile 96-well plate and incubated at 37 °C for 48 h. After culture, the medium was discarded, washed 3 times with PBS buffer, and fixed with methanol solution (200 µL/Well) for 15 \u0026nbsp;min. PBS buffer was washed three times, 96-well cell plates were naturally dried and then stained with 0.1% crystal violet staining solution (200 µL/Well) for 10 min at room temperature, and a large amount of sterile distilled water was used for multiple washings until colorless, after decolorization with anhydrous ethanol (200 µL/well), the absorbance at 570 nm was measured by Enzyme-linked Immunosorbent Assay Reader. The critical ODc values (ODc=the mean of the blank control wells) were used to determine the results: OD ≤ ODc was considered to have no ability to form membrane, ODc \u0026lt; OD ≤ 2ODc was considered to have weak ability to form membrane, and 2ODc \u0026lt; OD ≤ 4ODc was considered to have moderate ability to form membrane, OD \u0026gt; 4ODc is the strong ability of film formation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.7. Antimicrobial Susceptibility Testing and Resistance Gene Detection\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe antimicrobial susceptibility of the isolates was determined using the disk diffusion method against the following antibiotics: β-lactams (Penicillin, Ampicillin, Piperacillin, Cefuroxime sodium, Ceftriaxone, Cephalexin, Cefoperazone, Ceftazidime, Cefazolin), aminoglycosides (Gentamicin, Kanamycin, Streptomycin, Amikacin), macrolides (Erythromycin), lincosamides (Lincomycin), glycopeptides (Vancomycin), tetracyclines (Tetracycline, Minocycline, Doxycycline), and polypeptides (Polymyxin B)(CLSI, 2022). Additionally, 16 antimicrobial resistance genes from six classes were detected by PCR in the 14 \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates (Candan and Aksöz, 2015; Tabaran et al., 2017; Wu et al., 2022): β-lactams (\u003cem\u003eTemoneira β-lactamase:\u003c/em\u003e \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eTEM\u003c/sub\u003e, \u003cem\u003eCefotaxime-hydrolyzing β-lactamase: bla\u003c/em\u003e\u003csub\u003eCTX\u003c/sub\u003e, \u003cem\u003eNew Delhi metallo-β-lactamase\u003c/em\u003e: \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e, Oxacillinase-48: \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA-48\u003c/sub\u003e, \u003cem\u003eActive on imipenem β-lactamase\u003c/em\u003e: \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u003c/sub\u003e, \u003cem\u003eVerona integron-encoded metallo-β-lactamase\u003c/em\u003e: \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eVIM\u003c/sub\u003e, \u003cem\u003eVerona integron-encoded metallo-β-lactamase\u003c/em\u003e: \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eDHA\u003c/sub\u003e, \u003cem\u003eKlebsiella pneumoniae carbapenemase\u003c/em\u003e: \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u003c/sub\u003e), aminoglycosides (\u003cem\u003eAminoglycoside adenylyltransferase\u003c/em\u003e: \u003cem\u003eaadA\u003c/em\u003e), tetracyclines (\u003cem\u003eTetracycline resistance protein B\u003c/em\u003e: \u003cem\u003etetB\u003c/em\u003e), chloramphenicol (\u003cem\u003eChloramphenicol resistance protein A\u003c/em\u003e: \u003cem\u003ecmlA\u003c/em\u003e, \u003cem\u003eFlorfenicol/Chloramphenicol resistance protein\u003c/em\u003e: \u003cem\u003efloR\u003c/em\u003e), sulfonamides (\u003cem\u003eSulfonamide-resistant dihydropteroate synthase 2\u003c/em\u003e: \u003cem\u003esul2\u003c/em\u003e), and quinolones (\u003cem\u003eParC subunit of topoisomerase IV\u003c/em\u003e: \u003cem\u003eparC\u003c/em\u003e, \u003cem\u003eGyrase A subunit\u003c/em\u003e: \u003cem\u003egyrA\u003c/em\u003e, \u003cem\u003eGyrase B subunit\u003c/em\u003e: \u003cem\u003egyrB\u003c/em\u003e). The PCR conditions were the same as described in section 2.2, and the specific primer information is detailed in Table 3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.8. Virulence Gene Detection\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on previous reports (Alcántar-Curiel et al., 2013; Candan and Aksöz, 2015; Lan et al., 2019; Russo et al., 2018), 12 virulence genes were detected in the \u003cem\u003eK. pneumoniae\u003c/em\u003e strains via PCR. These included genes associated with lipopolysaccharide (\u003cem\u003eUridine diphosphate galacturonate 4-epimerase\u003c/em\u003e: \u003cem\u003euge\u003c/em\u003e, \u003cem\u003eWall polysaccharide biosynthesis protein G\u003c/em\u003e: \u003cem\u003ewabG\u003c/em\u003e), fimbriae (\u003cem\u003eFimbrial adhesion subunit H\u003c/em\u003e: \u003cem\u003efimH\u003c/em\u003e, \u003cem\u003eMrk fimbriae adhesion protein D\u003c/em\u003e: \u003cem\u003emrkD\u003c/em\u003e), iron transport (\u003cem\u003eEnterobactin synthetase component B\u003c/em\u003e: \u003cem\u003eentB\u003c/em\u003e, \u003cem\u003eKlebsiella ferric uptake system\u003c/em\u003e: \u003cem\u003ekfu\u003c/em\u003e, \u003cem\u003eIron-regulated outer membrane protein N\u003c/em\u003e: \u003cem\u003eiron\u003c/em\u003e, \u003cem\u003eIuc aerobactin synthetase A\u003c/em\u003e: \u003cem\u003eicuA\u003c/em\u003e), urease (\u003cem\u003eUrease alpha subunit\u003c/em\u003e: \u003cem\u003eureA\u003c/em\u003e), and allantoin metabolism (\u003cem\u003eAllantoin metabolism regulatory protein S\u003c/em\u003e: \u003cem\u003eallS\u003c/em\u003e). The PCR reaction system and operational procedures followed the standard protocol outlined in section 2.2. Specific primer sequences and related information are provided in Table 4.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.9 String test\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eK. pneumoniae\u003c/em\u003e isolates were streaked onto MacConkey agar plates and incubated at 37 °C for 12 h. Using an inoculation loop, a single colony was gently touched and slowly lifted to observe the formation of a string-like growth. The length of the slime string was measured and recorded. Isolates were classified as mucoid if they produced a string and as Hv if the string length exceeded 5 mm (Kumabe and Kenzaka, 2014).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.10. Pathogenicity Assays in Mice and Rabbits\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThirty-two non-specific pathogen-free Kunming mice and six (6W) New Zealand white rabbits were all purchased from the Animal Experiment Center of Xinjiang Medical University (animal ethics committee approval number: No. PA20251024017).\u003c/p\u003e\n\u003cp\u003eThe virulence of the strains was assessed using the \"string test,\" PCR detection of virulence genes, a mouse infection model, and a New Zealand White rabbit infection model. Strains exhibiting strong adhesion in the string test and concurrently carrying a strong virulence gene profile via PCR were selected for the mouse intraperitoneal infection experiment (200 µL, 10^6 CFU/mouse). Strains demonstrating high pathogenicity in the mouse infection assay were subsequently used for the New Zealand White rabbit intratracheal challenge (500 µL, 10^7 CFU/rabbit). Following the challenge, animals were monitored daily. Any animal exhibiting a body weight loss of 30% or more was humanely euthanized. All remaining surviving animals were euthanized at the end of the study on day 7. Lung and liver tissues from the experimental rabbits were harvested, processed, and subjected to hematoxylin and eosin (H\u0026amp;E) staining. The euthanasia procedure involved an overdose of pentobarbital sodium administered after deep anesthesia. For mice, anesthesia was induced by intraperitoneal injection of ketamine (90 mg/kg) and sevoflurane (6 mg/kg), followed by euthanasia with pentobarbital sodium (150 mg/kg, intraperitoneal). For rabbits, anesthesia was induced by intramuscular injection of ketamine (40 mg/kg) and sevoflurane (6 mg/kg), and euthanasia was subsequently performed by intravenous injection of pentobarbital sodium (100 mg/kg) via the marginal ear vein. The absence of vital signs was verified in all animals to ensure death.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.11. Statistical Analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analyses were performed using GraphPad Prism 8.0. Comparisons between groups were conducted via Two-way ANOVA or Student's t-tests, as appropriate. Data are presented as mean ± standard deviation (SD). Statistical significance was defined as P \u0026lt; 0.05, ** P \u0026lt; 0.01, and *** P \u0026lt; 0.001.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Isolation Rate and Identification of Klebsiella pneumoniae\u003c/h2\u003e \u003cp\u003ePCR analysis revealed that \u003cem\u003eK. pneumoniae\u003c/em\u003e was detected in 21.78% of the 101 mastitic camel milk samples collected, with an isolation rate of 11.88%. Among 10 hand swab samples from dairy workers, the detection rate was 20% (2/10) and the isolation rate 10% (1/10). In soil samples, both detection and isolation rates were 10% (1/10), while no \u003cem\u003eK. pneumoniae\u003c/em\u003e was detected in any of the 10 water samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Agarose gel electrophoresis of PCR-positive samples showed a clear band at 286 bp (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). On BHI agar, the isolates formed large, moist, mucoid, raised, and grayish-white colonies. Gram staining revealed short, plump, pink-colored rods, occurring singly, in pairs, or in short chains (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Biochemical profiling indicated that all isolates were positive for glucose fermentation with gas production, lysine decarboxylase, citrate utilization, lactose fermentation, and Voges\u0026ndash;Proskauer test, but negative for hydrogen sulfide production, ornithine decarboxylase, indole production, dulcitol fermentation, phenylalanine deaminase, and urease. These biochemical characteristics are consistent with those of \u003cem\u003eK. pneumoniae\u003c/em\u003e as described in Bergey\u0026rsquo;s Manual of Determinative Bacteriology (data not shown). Furthermore, 16S rRNA gene sequencing confirmed the species identity, with sequence homologies ranging from 99.57% to 100% compared to reference \u003cem\u003eK. pneumoniae\u003c/em\u003e sequences in the GenBank database.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Sequence Typing of Klebsiella pneumoniae\u003c/h2\u003e \u003cp\u003eThe STs identified in this study included ST42, ST4089, ST297, and ST495. Specifically, there were two isolates of ST42, two of ST4089, two of ST297, and eight of ST495 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B). ST495 was isolated not only from camels but also from the hands of dairy workers (KP60) and soil samples (KP289). ST4069 clusters within the same clade as ST11, a hypervirulent sequence type commonly prevalent in China, whereas ST495, the most frequently isolated sequence type in this study, exhibits close phylogenetic relatedness to ST23. In contrast, ST297 was more distantly related (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Based on wzi gene sequencing, the 14 \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates were classified into four distinct wzi allele types (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Ten isolates of ST4069 and ST495 all exhibited the wzi122 allele, corresponding to serotype KL121. The two ST42 isolates carried the wzi464 allele, and the two ST297 isolates carried the wzi475 allele. Phylogenetic analysis indicated substantial genetic divergence among these three wzi allele types.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Antimicrobial Resistance Results\u003c/h2\u003e \u003cp\u003eAll 14 isolates (100%, 14/14) exhibited resistance to β-lactams (penicillin, ampicillin), aminoglycosides (kanamycin), macrolides (erythromycin), lincosamides (lincomycin), and glycopeptides (vancomycin). In contrast, susceptibility to tetracyclines was generally observed: susceptibility to tetracycline was 71.43% (10/14), intermediate susceptibility 14.29% (2/14), and resistance 14.29% (2/14); for minocycline, susceptibility was 28.57% (4/14) and intermediate susceptibility 71.43% (10/14); for doxycycline, susceptibility was 57.14% (8/14), intermediate susceptibility 28.57% (4/14), and resistance 14.29% (2/14). Susceptibility to the β-lactam agent ceftriaxone was 71.43% (10/14). All isolates (100%) displayed intermediate susceptibility to polymyxin B (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePCR-based screening identified nine distinct antimicrobial resistance genes, which were categorized into four classes: β-lactams (\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eTEM\u003c/sub\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX\u003c/sub\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eVIM\u003c/sub\u003e), aminoglycosides (\u003cem\u003eaadA\u003c/em\u003e), quinolones (\u003cem\u003eparC\u003c/em\u003e, \u003cem\u003egyrA\u003c/em\u003e, \u003cem\u003egyrB\u003c/em\u003e), and sulfonamides (\u003cem\u003esul2\u003c/em\u003e). The detection rates for \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eVIM\u003c/sub\u003e (β-lactams), \u003cem\u003egyrA\u003c/em\u003e, and \u003cem\u003egyrB\u003c/em\u003e (quinolones) were 100%; \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX\u003c/sub\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e (β-lactams), and \u003cem\u003eaadA\u003c/em\u003e (aminoglycosides) were detected in 86% of isolates; \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eTEM\u003c/sub\u003e (β-lactams) was present in 43% of isolates; while \u003cem\u003eparC\u003c/em\u003e (quinolones) and \u003cem\u003esul2\u003c/em\u003e (sulfonamides) showed relatively low detection rates, each at 14%. The resistance gene combination \u003cem\u003egyrA\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003egyrB\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eVIM\u003c/sub\u003e was the most prevalent, occurring in 100% of the isolates. In contrast, the combinations \u003cem\u003egyrA\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003egyrB\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eVIM\u003c/sub\u003e + \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX\u003c/sub\u003e + \u003cem\u003eaadA\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e + \u003cem\u003eparC\u003c/em\u003e, \u003cem\u003egyrA\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003egyrB\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eVIM\u003c/sub\u003e + \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX\u003c/sub\u003e + \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e, and \u003cem\u003egyrA\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003egyrB\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eVIM\u003c/sub\u003e + \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX\u003c/sub\u003e + \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e + \u003cem\u003eaadA\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003esul2\u003c/em\u003e were the least prevalent, each detected in only 14% of the isolates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Virulence Testing Results\u003c/h2\u003e \u003cp\u003eThrough polymerase chain reaction (PCR) analysis, a total of 10 virulence genes were identified, which were categorized into five functional groups: fimbria-associated genes (\u003cem\u003emrkD\u003c/em\u003e, \u003cem\u003efimH\u003c/em\u003e), urease gene (\u003cem\u003eureA\u003c/em\u003e), siderophore genes (\u003cem\u003ekfu\u003c/em\u003e, \u003cem\u003eentB\u003c/em\u003e, \u003cem\u003eiroN\u003c/em\u003e), lipopolysaccharide synthesis-related genes (\u003cem\u003ewabG\u003c/em\u003e, \u003cem\u003euge\u003c/em\u003e), and capsule-associated genes (\u003cem\u003emagA\u003c/em\u003e, \u003cem\u003ermpA\u003c/em\u003e). The detection rates of \u003cem\u003emrkD\u003c/em\u003e, \u003cem\u003eureA\u003c/em\u003e, \u003cem\u003ekfu\u003c/em\u003e, \u003cem\u003eentB\u003c/em\u003e, and \u003cem\u003ewabG\u003c/em\u003e were the highest, each reaching 100%. The capsule gene \u003cem\u003emagA\u003c/em\u003e was present in 86% of the isolates, while the siderophore gene \u003cem\u003eiroN\u003c/em\u003e was detected in 71%. The fimbrial gene \u003cem\u003efimH\u003c/em\u003e and the lipopolysaccharide gene \u003cem\u003euge\u003c/em\u003e showed relatively lower detection rates, both at 43%. In contrast, the \u003cem\u003ermpA\u003c/em\u003e gene had the lowest prevalence, detected in only 14% of isolates (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B, E). A conserved combination of virulence genes\u0026mdash;\u003cem\u003ewabG\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003ekfu\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eureA\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eentB\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003emrkD\u003c/em\u003e \u0026mdash;was universally present across all isolates, with 100% coverage (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). In the string test, 12 out of 14 isolates exhibited a positive phenotype, consistent with biofilm-forming capability (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Further integration of virulence gene profiles, antimicrobial resistance genes, and STs revealed that ST495 strains KP25 and KP386 both carried the \u003cem\u003ermpA\u003c/em\u003e virulence gene. Notably, KP386 also harbored the most diverse array of carbapenem resistance genes, suggesting a potentially greater public health risk.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn a murine intraperitoneal infection model, all tested isolates exhibited lethal effects. Strains KP99 and KP386 resulted in 100% mortality (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA); both belonged to ST495 and carried the \u003cem\u003ermpA\u003c/em\u003e virulence gene. Furthermore, in a rabbit intratracheal challenge assay, strain KP386 demonstrated high pathogenicity, causing pulmonary infection and liver abscesses (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Compared with the blank control, hematoxylin-eosin (HE) staining revealed significant pathological lesions in the lungs and liver of KP386-infected mice, as indicated by arrows (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). In summary, \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates obtained from camels in Xinjiang frequently harbor multiple virulence genes and include strains with high pathogenic potential. To evaluate the risk of cross-species transmission between animals and humans, further epidemiological surveillance in this region is warranted.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn 1882, \u003cem\u003eK. pneumoniae\u003c/em\u003e was first isolated from the lungs of a patient who died of pneumonia. Initially regarded as an opportunistic pathogen in the natural environment, it did not receive widespread societal attention (Bengoechea and Sa Pessoa, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). However, this bacterium has since spread extensively among humans, the environment, and animals, posing a potential threat to public health and the livestock industry (Giri et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In this study, the incidence of mastitis in Bactrian dairy camels was detected at 21% (101/480), slightly lower than the 25% incidence of clinical mastitis reported in organized camel herds in Bikaner, Rajasthan, India (Ranjan et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). This difference might be attributed to the early developmental stage of the camel dairy industry in Keping County, Xinjiang, where environmental pathogen loads may be lower. The detection rate of \u003cem\u003eK. pneumoniae\u003c/em\u003e in mastitic camel milk samples in this study was 21.78%, similar to the rate found in Libyan camel milk (1/5; 20%) (Azwai et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) and slightly lower than the proportion in Kuwaiti camel milk (12/33; 36%) (Rahmeh et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The isolation rate of \u003cem\u003eK. pneumoniae\u003c/em\u003e varies from 4.55% to 36.59% in pigs from certain regions of China (C\u0026oacute;rdova-Espinoza et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Hou et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2024b\u003c/span\u003e; Rui et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Wan et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2025a\u003c/span\u003e). Nobrega et al. detected \u003cem\u003eK. pneumoniae\u003c/em\u003e in most milk samples from 25 lactating dairy cows (Nobrega et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Furthermore, we also detected \u003cem\u003eK. pneumoniae\u003c/em\u003e in samples from workers' hands and soil, consistent with previous reports from dairy farms indicating that sources of \u003cem\u003eK. pneumoniae\u003c/em\u003e include organic bedding materials like wood by-products, cow manure, and dairy products (Cheng et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). This further highlights the severe challenge posed by \u003cem\u003eK. pneumoniae\u003c/em\u003e to global public health.\u003c/p\u003e \u003cp\u003eAs of now, the Pasteur MLST database lists over 7,000 STs of \u003cem\u003eK. pneumoniae\u003c/em\u003e globally. ST495 has been isolated from human blood, sputum, and the environment; ST297 from cattle (rumen, milk), seawater, and humans (blood, urine); ST42 from humans (blood, urine, sputum, feces); and ST4089 from human urine. Relevant literature reports that ST495 was isolated from a Greek hospital in 2011 (Giakkoupi et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Additionally, ST42 has been detected in samples from pig and dairy farms in Xinjiang (Hou et al., 2024a), in rivers receiving wastewater treatment plant effluent in 2024 (Swain et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), in the feces of a patient with a liver abscess in 2019 (Kim et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), in a tertiary care hospital in North India in 2021 (Nirwan et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and in a high-complexity hospital in Ecuador (Reyes et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Reports on ST297 and ST4089 are relatively scarce. In this experiment, 14 strains were isolated, all exhibiting multidrug resistance. ST495 was the predominant type in the camel farm. The study indicates that the four \u003cem\u003eK. pneumoniae\u003c/em\u003e types ST495, ST297, ST42, and ST4089 all pose a risk of transmission among humans, the environment, and camels, constituting a serious threat to public health. However, several recent reports from Xinjiang identified ST161 from bedding in a Urumqi dairy farm, and ST5387 from cloacal swabs of healthy laying hens and environmental fecal samples from a chicken farm. ST37 and ST967 \u003cem\u003eK. pneumoniae\u003c/em\u003e strains were isolated from pig farms (Wan et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2025b\u003c/span\u003e), suggesting significant differences in prevalent STs across different regions of Xinjiang. Ten isolates of ST4089 and ST495 all exhibited the wzi122 allele, corresponding to serotype KL121. In this study, the CC17 clonal complex (ST495) was identified among CRKP strains, and the expression of \u003cem\u003eNDM-1\u003c/em\u003e was observed in camel-derived isolates, aligning with previous reports of CC17-associated \u003cem\u003eNDM-1\u003c/em\u003e expression in neonatal infections(Yin et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Notably, these ST495 strains exhibited the KL121 capsular serotype, suggesting potential acquisition of virulence plasmids. Plasmids play a pivotal role in the dissemination of both antimicrobial resistance and hypervirulence in \u003cem\u003eK. pneumoniae\u003c/em\u003e (Dunn et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBiofilm formation is a crucial mechanism of drug resistance in \u003cem\u003eK. pneumoniae\u003c/em\u003e. The most important surface structures during \u003cem\u003eK. pneumoniae\u003c/em\u003e biofilm formation are type 3 fimbriae and CPS. Fimbriae mediate stable adhesion, while CPS ultimately influences biofilm structure and intercellular communication (Clegg and Murphy, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In this study, among the 14 isolated \u003cem\u003eK. pneumoniae\u003c/em\u003e strains, the detection rate for the \u003cem\u003efimH\u003c/em\u003e gene was 42.86% (6/14), primarily as ST495 strains generally did not carry the \u003cem\u003efimH\u003c/em\u003e gene. The detection rate for the \u003cem\u003emrkD\u003c/em\u003e gene was 100% (14/14), for the capsular gene \u003cem\u003emagA\u003c/em\u003e was 86% (12/14), and for \u003cem\u003ermpA\u003c/em\u003e was 14% (2/14). This differs from another study on \u003cem\u003eK. pneumoniae\u003c/em\u003e in pigs from Xinjiang, where among 21 isolated strains, the detection rates for \u003cem\u003efimH\u003c/em\u003e and \u003cem\u003emrkD\u003c/em\u003e were 100%, but no capsular genes (\u003cem\u003ermpA\u003c/em\u003e and \u003cem\u003emagA\u003c/em\u003e ) were detected (Wan et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2025b\u003c/span\u003e). Among these 14 strains, 6 exhibited strong biofilm-forming ability, and 6 had moderate ability, aligning with the multidrug resistance observed in susceptibility tests. Concurrently, the high detection rate of multiple resistance genes indicates that treating infections caused by these bacteria would be challenging. In this study, resistance genes for β-lactams, aminoglycosides, sulfonamides, and quinolones were detected in the \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates, with varying degrees of resistance, likely related to drug usage practices within the farm. The resistance mechanisms of K. pneumoniae are complex and diverse; the most common involves acquiring plasmids carrying resistance genes, while another involves the accumulation of gene mutations (Cheng et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Notably, the dissemination of these resistance genes heavily relies on mobile genetic elements (Koudoum et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). These plasmids and transposons can not only spread among human pathogens but also cross species barriers, widely disseminating among animal and environmental microbes, ultimately leading to a surge and persistent prevalence of multidrug-resistant strains (Pitout et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Tandem repeat-mediated mutagenesis is a significant, previously underestimated genetic mechanism driving the rapid diversification and adaptive evolution of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u003c/sub\u003e enzymes under antibiotic selection pressure (Ye et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). A limitation of our study is the lack of testing for resistance genes in the environment, which will be addressed in future work.\u003c/p\u003e \u003cp\u003eWith the intensification of animal farming, the extensive use of antimicrobials to reduce economic losses from various bacterial diseases has led to the continuous emergence of resistant strains. In this study, the K-B method was used to detect the susceptibility of K. pneumoniae isolates to antimicrobial agents. The results indicate a concerning level of antibiotic resistance in camel-derived \u003cem\u003eK. pneumoniae\u003c/em\u003e. All 14 isolated \u003cem\u003eK. pneumoniae\u003c/em\u003e strains were MDR, showing 100% (14/14) resistance to β-lactams, aminoglycosides, macrolides, lincosamides, and glycopeptides, consistent with findings from pig farms in Xinjiang. The isolates were generally susceptible to tetracyclines, suggesting they could be considered for subsequent \u003cem\u003eK. pneumoniae\u003c/em\u003e treatment, which differs somewhat from isolates from Xinjiang pig farms, where 21 K. pneumoniae isolates showed strong resistance to β-lactams, aminoglycosides, tetracyclines, sulfonamides, quinolones, and other antimicrobials, exhibiting multidrug resistance and sensitivity only to carbapenems (imipenem) and polymyxins (polymyxin B) (Wan et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2025b\u003c/span\u003e), likely related to clinical drug use on the farms. Interestingly, the isolates were relatively sensitive (71.43%, 10/14) to the β-lactam ceftriaxone, the mechanism of which warrants further investigation. All isolated \u003cem\u003eK. pneumoniae\u003c/em\u003e strains showed 100% intermediate susceptibility to polymyxin B, one of the \"last line\" antibiotics for clinical treatment. Bacterial resistance phenotypes are determined by resistance genotypes, but phenotype and genotype may not always align perfectly. PCR detected 9 resistance genes belonging to 4 classes (including \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eTEM\u003c/sub\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX\u003c/sub\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eVIM\u003c/sub\u003e, \u003cem\u003eaadA\u003c/em\u003e, \u003cem\u003esul2\u003c/em\u003e, \u003cem\u003eparC\u003c/em\u003e, \u003cem\u003egyrA\u003c/em\u003e, \u003cem\u003egyrB\u003c/em\u003e). Among them, the detection rates for the β-lactam \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eVIM\u003c/sub\u003e and the quinolones \u003cem\u003egyrA\u003c/em\u003e and \u003cem\u003egyrB\u003c/em\u003e were 100%; for the β-lactams \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX\u003c/sub\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e, and the aminoglycoside \u003cem\u003eaadA\u003c/em\u003e were 86%; for the β-lactam \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eTEM\u003c/sub\u003e was 43%; and for the quinolone \u003cem\u003eparC\u003c/em\u003e and sulfonamide \u003cem\u003esul2\u003c/em\u003e were relatively low at 14%. This discrepancy might be due to the presence of undetected or unknown relevant resistance genes. During farming, the extensive use of antibiotics, whether for treatment or growth promotion, can activate associated resistance genes (Jonas et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), thereby enhancing strain resistance and facilitating the transmission of these resistant strains to humans via the food chain or other transmission routes (Ovejero et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In this study, \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates exhibited strong resistance to 5 classes out of the 20 antibiotics tested, while being relatively sensitive to only 2 antibiotics. It is recommended that farms strictly control antibiotic use to prevent the emergence of more resistant strains. Furthermore, new antimicrobial biological agents could be selected for prevention and control, enabling precise prevention, comprehensive management, and holistic treatment.\u003c/p\u003e \u003cp\u003eResearch (Shon et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) shows that the \u003cem\u003eiucA\u003c/em\u003e can increase the virulence of hypervirulent \u003cem\u003eK. pneumoniae\u003c/em\u003e (hvKP) and is a key virulence characteristic of hvKP. In this study, the \u003cem\u003eiucA\u003c/em\u003e virulence gene was not detected in any of the 14 \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates, but the carriage rate for \u003cem\u003eiroN\u003c/em\u003e was high (71.43%). The virulence genes \u003cem\u003eiucA\u003c/em\u003e and \u003cem\u003eiroN\u003c/em\u003e are associated with iron uptake systems, and the iro gene cluster primarily encodes salmochelin, the C-glucosylated form of enterobactin. Studies indicate that over 90% of \u003cem\u003eK. pneumoniae\u003c/em\u003e strains causing liver abscesses can secrete salmochelin (Lam et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2018a\u003c/span\u003e). In this study, the antimicrobial resistance and virulence gene profiles of isolates from workers' hands, soil, and camels were nearly identical, suggesting a risk of mutual transmission among humans, camels, and the environment. Additionally, the detection of ST495: KL121 strains in camels indicates that conventional ST495 may have acquired high virulence through plasmid uptake. Two ST495: KL121 isolates not only carried the \u003cem\u003ermpA\u003c/em\u003e gene but also exhibited high lethality in mouse pathogenicity assays, indicating strong virulence and potential \u0026ldquo;superbug\u0026rdquo; characteristics, Particular attention should be paid to the risk of transmission of such hv strains from animals to humans. Compared to the detection rates of \u003cem\u003eiucA\u003c/em\u003e, \u003cem\u003eiroN\u003c/em\u003e, and \u003cem\u003ermpA\u003c/em\u003e in porcine isolates from Xinjiang (57.14%, 88.89%, and 0%, respectively), our findings further highlight significant regional differences among \u003cem\u003eK. pneumoniae\u003c/em\u003e clones. Multiple studies have elucidated the molecular mechanisms underlying the dissemination of virulence and resistance genes. For instance, the \u003cem\u003eIncHI1B/repB\u003c/em\u003e-type hybrid virulence plasmid (\u003cem\u003epHvKP51-Vir\u003c/em\u003e) can promote the emergence of carbapenem-resistant hypervirulent K. pneumoniae (CR-hvKP) through mobile genetic element (MGE)-mediated recombination during conjugation (Huo et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Non-conjugative virulence plasmids may also disseminate across strains via helper conjugative plasmids or fusion with multidrug-resistant plasmids (Li et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The \u003cem\u003ep15WZ-82_Vir\u003c/em\u003e plasmid represents one such product of integration between a hypervirulent plasmid and a multidrug-resistant plasmid (Yang et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Moreover, hybrid conjugative virulence plasmids efficiently facilitate the transfer of virulence and resistance genes from ST11 CRKP to CR-hvKP (Gu et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Liao et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Additionally, some strains can integrate genetic elements, including virulence genes, into their chromosomes (Lam et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2018b\u003c/span\u003e). Collectively, these studies demonstrate a close association among virulence genes, resistance genes, and their corresponding phenotypes in \u003cem\u003eK. pneumoniae\u003c/em\u003e. Therefore, this study identified the presence of highly virulent and highly resistant superbugs in dairy camels. However, the molecular mechanisms underlying their formation require further investigation.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis study elucidates the prevalent \u003cem\u003eK. pneumoniae\u003c/em\u003e strains in Bactrian camel farms in Keping County, Xinjiang, China, revealing significant genetic diversity and multidrug-resistance profiles. Molecular typing identified four primary STs: ST495, ST297, ST42, and ST4089. Of particular concern is the ST495 lineage, which not only represents the predominant circulating strain in the region but was also isolated from camels, the environment, and dairy workers, suggesting a complex transmission cycle. Critically, this is the first identification of the ST495: KL121 strain in camels, characterized by concurrent hypervirulence and multidrug resistance, indicating a potential evolution towards a \"superbug\". Further analysis demonstrates that these camel-derived \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates commonly exhibit robust biofilm-forming capabilities and harbor a diverse array of antimicrobial resistance and virulence genes. This undoubtedly complicates clinical prevention and treatment strategies and poses a substantial threat to public health security.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMDR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emultidrug-resistant\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ehv\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehypervirulent\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eK. pneumoniae\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eKlebsiella pneumoniae\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMLST\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMultilocus sequence typing\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecarbapenem resistance\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCPS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecapsular polysaccharide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLPS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003elipopolysaccharide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBHI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBrain Heart Infusion\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eST\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esequence type\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehematoxylin-eosin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ehvKP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003evirulence of hypervirulent K. pneumoniae\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCR-hvKP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecarbapenem-resistant hypervirulent K. pneumoniae\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMGE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emobile genetic element\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003ekhe\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eK. pneumoniae hemagglutinin type 3 fimbrial gene\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e16S rRNA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e16S ribosomal Ribonucleic Acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eropB\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRNA polymerase beta subunit\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003etonB\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTonB protein\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003ephoE\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePhosphate limitation-inducible outer membrane porin E\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003epgi\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePhosphoglucose isomerase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003emdh\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMalate dehydrogenase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003einfB\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTranslation initiation factor IF-2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003egapA\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGlyceraldehyde-3-phosphate dehydrogenase A\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eTEM\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTemoneira β-lactamase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eBla\u003c/em\u003e\u003csub\u003eCTX\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCefotaxime-hydrolyzing β-lactamase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNew Delhi metallo-β-lactamase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u0026minus;48\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eOxacillinase-48\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eActive on imipenem β-lactamase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eVIM\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eVerona integron-encoded metallo-β-lactamase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eDHA\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eVerona integron-encoded metallo-β-lactamase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eBla\u003c/em\u003e\u003csub\u003eKPC\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eKlebsiella pneumoniae carbapenemase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eaadA\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAminoglycoside adenylyltransferase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003etet(B)\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTetracycline resistance protein B\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003ecmlA\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eChloramphenicol resistance protein A\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003efloR\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFlorfenicol/Chloramphenicol resistance protein\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003esul2\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSulfonamide-resistant dihydropteroate synthase 2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eparC\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eParC subunit of topoisomerase IV\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003egyrA\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGyrase A subunit\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003egyrB\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGyrase B subunit\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003euge\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eUridine diphosphate galacturonate 4-epimerase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003ewabG\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eWall polysaccharide biosynthesis protein G\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003efimH\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFimbrial adhesion subunit H\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003emrkD\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMrk fimbriae adhesion protein D\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eentB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEnterobactin synthetase component B\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eiroN\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eIron-regulated outer membrane protein N\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ekfu\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eKlebsiella ferric uptake system\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eicuA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eIuc aerobactin synthetase A\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eureA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eUrease alpha subunit\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eallS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAllantoin metabolism regulatory protein S\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003e The mice and rabbits experiments were conducted in accordance with the protocol approved by the Scientific Ethics Committee of Tarim University (Permit No.PA20251024017). And the owner of the camels is aware of this and has given his consent for the collection of milk samples from the camels.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eNo potential conflict of interest was reported by the author(s).\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was funded by the Tianchi Talents Introduction Program of Xinjiang Uygur Autonomous Region (BT-2025-TCYC-0041) and the Tarim University President\u0026rsquo;s Fund Project (TDZKBS202522, TDZKBS202521).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eQS, MN, LZ, JY and DT delineated the study conception and design. LZ, JY and DT supervised the study. QS, MN, MH, YH, JZ performed laboratory tests as well as analyzed the data, and MT, JG, YM, MN and QS participated in the animal experiments and analyzed the data. QS, MN and DT wrote the manuscript and approved the final version for publication. QS, MN, LZ, JY and DT participated in the manuscript discussion and revision. All authors have read and approved the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003e We are grateful to the reviewers for their significant assistance in working on the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data generated and/or analyzed in this study are available in the [GenBank] repository under accession numbers [SUB15995834: PX975972\u0026ndash;PX975985; PX991553\u0026ndash;PX991664]. The data that support the findings of this study are openly available in this manuscript and are available from the corresponding author upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbo-Shama UH, Abo-Amer AES, Ahmed EAE, Alsaadawy RM, Sayed HH. Prevalence of multi-drug resistant and extended-spectrum β-lactamase producing Escherichia coli and Klebsiella pneumoniae among meat products sold at Sohag Governorate, Egypt. BMC Microbiol. 2025;25:636. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e.https://doi.org/10.1186/s12866-025-04392-8\u003c/span\u003e\u003cspan address=\".10.1186/s12866-025-04392-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlc\u0026aacute;ntar-Curiel MD, Blackburn D, Salda\u0026ntilde;a Z, Gayosso-V\u0026aacute;zquez C, Iovine NM, De la Cruz MA, Gir\u0026oacute;n JA. 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Front Microbiol. 2023;14:1219733. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e.https://doi.org/10.3389/fmicb.2023.1219733\u003c/span\u003e\u003cspan address=\".10.3389/fmicb.2023.1219733\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Klebsiella pneumoniae, camel mastitis, virulence, antimicrobial resistance, molecular characterization, public health risk","lastPublishedDoi":"10.21203/rs.3.rs-8650943/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8650943/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eAims\u003c/h2\u003e \u003cp\u003eThe escalating prevalence of multidrug-resistant (MDR) and hypervirulent (hv) \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e (\u003cem\u003eK. pneumoniae\u003c/em\u003e) strains represents a significant threat to both animal and human health globally. In Xinjiang, China, where Bactrian camels are an economically important livestock and human interaction with camel products is increasing, this study aimed to characterize the virulence, antibiotic resistance, and potential public health implications of \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates associated with camel mastitis.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eA total of 14 \u003cem\u003eK. pneumoniae\u003c/em\u003e strains were isolated from camel milk, milker hand swabs, and environmental samples. Antimicrobial susceptibility testing revealed widespread MDR, with 100% of isolates resistant to β-lactams, aminoglycosides, macrolides, lincosamides, and glycopeptides. PCR analysis identified multiple acquired resistance genes. Multilocus sequence typing (MLST) revealed diverse sequence types, including ST42, ST4089, ST297, and ST495, with ST495 being the dominant type. Virulence gene profiling confirmed the presence of various virulence-associated genes. Pathogenicity assays in mice and rabbits demonstrated high virulence in ST495:KL121 strains. Notably, ST495 was isolated from camels, dairy workers, and environmental samples, indicating a potential transmission cycle between these reservoirs.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThis study underscores the presence of highly MDR and potentially virulent \u003cem\u003eK. pneumoniae\u003c/em\u003e strains linked to camel mastitis in Xinjiang. The identification of ST495:KL121 strains, exhibiting both hypervirulence and multidrug resistance, suggests the emergence of \"superbug\" characteristics and poses a substantial public health risk. These findings emphasize the critical need for intensified surveillance and robust control measures targeting \u003cem\u003eK. pneumoniae\u003c/em\u003e in camel farming environments within the region to effectively mitigate the impact on both animal and human health.\u003c/p\u003e","manuscriptTitle":"Molecular Characterization and Prevalence of Hypervirulent and Multidrug-Resistant Klebsiella pneumoniae in Mastitis of Bactrian Camels from Xinjiang, China","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-07 19:41:26","doi":"10.21203/rs.3.rs-8650943/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-04-09T03:08:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"173859814273989637213089244304139598610","date":"2026-03-29T10:15:16+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-28T17:52:25+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-12T11:01:09+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-12T04:28:17+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Microbiology","date":"2026-02-12T04:23:41+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8a3ef029-a5fc-48bb-9e38-d162ce78cdcc","owner":[],"postedDate":"April 7th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-07T19:41:26+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-07 19:41:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8650943","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8650943","identity":"rs-8650943","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}