Virulence characteristics and antibiotic resistance analysis of Klebsiella pneumoniae isolated from pig farms in Xinjiang, China: revealing potential zoonotic risks

Abstract

Background

This study aimed to analyze the antimicrobial resistance and pathogenicity of Klebsiella pneumoniae(K. pneumoniae) isolates from pigs, evaluate their potential threat to pig farming and public health, and provide a theoretical basis for controlling K. pneumoniae infections in pig farms.

Methods

Nasal swabs collected from pigs were subjected to bacterial isolation, biochemical identification, species-specific PCR, and 16S rRNA sequencing to identify K. pneumoniae. Serotyping and multilocus sequence typing (MLST) were conducted using the wzi and MLST methods, respectively. Biofilm formation was assessed using crystal violet staining. Antimicrobial susceptibility was evaluated via the Kirby-Bauer disk diffusion method, and resistance and virulence genes were identified using PCR. Pathogenicity was determined through string testing and mouse infection models.

Results

21 strains of K. pneumoniae were isolated and identified from 50 swabs of pig nasal cavities. The isolates were classified into serotypes wzi 19 and wzi 81 and sequence types ST37 and ST967. Ten isolates exhibited strong biofilm-forming ability, while 11 showed moderate biofilm production. Antimicrobial susceptibility testing revealed resistance to β-lactams, aminoglycosides, quinolones, tetracyclines, sulfonamides, aminoalcohols, and glycopeptides, with sensitivity restricted to imipenem and polymyxins. Ten resistance genes and eight virulence genes were detected. Pathogenicity testing in mice revealed a moderate virulence level, with a median lethal dose (LD₅₀) of 4.0 × 10⁶ CFU/mL. Infected mice exhibited significant lesions in the liver, lungs, and small intestine.

Conclusion

These findings highlight a potential risk to pig farming and public health, emphasizing the need for effective control measures against K. pneumoniae infections in pig farms.

Introduction

K. pneumoniae is a flagellate, encapsulated bacterium widely distributed in nature and is considered as an opportunistic pathogen that can cause diseases such as pneumonia, meningitis, septicemia, and bronchitis in humans and animals, especially when host immunity is compromised or after prolonged use of antimicrobial drugs. In pigs, K. pneumoniae infections commonly present as suppurative fibrinous pneumonia, mastitis, and septicemia [1, 2].

Several outbreaks of K. pneumoniae in pig farms have caused significant economic losses. Between 2011 and 2014, outbreaks in 13 UK pig farms led to septicemia and rapid mortality in piglets [3]. Similarly, in 2017, an outbreak in Victoria, Australia, resulted in a mortality rate of approximately 60% among piglets, severely affecting the pig farming industry [4]. Despite these high fatality rates, K. pneumoniae infections in animals remain underrecognized and under-researched.

The increasing scale of livestock farming, improper management practices, overuse of antibiotics, and prevalence of immunosuppressive diseases have contributed to a rise in antimicrobial-resistant K. pneumoniae. The emergence of multidrug-resistant (MDR) strains has exacerbated treatment challenges [5, 6]. Resistance in K. pneumoniae is often associated with transferable plasmids carrying both resistance and virulence genes, which enhance bacterial survival in adverse environments [7].

The pathogenicity of K. pneumoniae is primarily attributed to its four key virulence factors: capsular polysaccharides, pili, lipopolysaccharides (LPS), and siderophores. The polysaccharide capsule aids in evasion from host immune defenses, including phagocytosis, lysozyme, and complement-mediated damage. Of the 78 capsular serotypes identified, K1 and K2 are considered the most virulent [8]. Adhesins and type 1 (fim) and type 3 (mrk) pili facilitate bacterial adherence to epithelial and immune cells, promoting biofilm formation. LPS, composed of lipid A, core polysaccharide, and O antigen, functions as an immune activator and virulence factor. Lipid A, in particular, helps suppress host inflammatory responses, while the diversity of O antigens enables immune evasion [9, 10].

Siderophores, including enterobactin, salmochelin, yersiniabactin, and aerobactin, allow K. pneumoniae to thrive in iron-limited environments by outcompeting host iron-chelating proteins [11]. Additional virulence factors, such as hemolysin and enterotoxin, further contribute to its pathogenic potential [12].

In this study, 21 strains of K. pneumoniae were isolated from nasopharyngeal swabs collected at a large-scale pig farm 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 pig populations and highlight potential public health risks.

Materials and methods

Animal sampling and testing

Sampling

From May to August 2024, we choosed Wujiaqu, a city situated in central Xinjiang, renowned for its numerous pig farms, as the geographical area for our research, and collected 50 nasal swabs from the largest pig farm with 15,000 hybrid pig (Duroc × Landrace × Yorkshire). From 5 pig pens, we collected nasal swabs from 50 healthy hybrid piglets, aged 30 days and weighing (10 ± 0.5) kg. The nasal swabs were collected by inserting them into the animals’ nasal cavities and rotating them 2–3 times. The samples were immediately stored at 4 °C for subsequent analysis. The swine farm is located in the central part of Xinjiang, which has developed transportation (Fig. 1). At the same time, the management of the swine farm is strict, and it can sell a large number of piglets and pork products to the outside world.

Experimental animals

A cohort of 48 six-week-old Kunming strain white mice was procured from the Laboratory Animal Center of Xinjiang Medical University. The mice were used for pathogenicity assays under controlled laboratory conditions (Approval Letter for Ethics Review by Biology Ethics Committee of Shihezi University, Approval Number: A2024-412).

Isolation and purification of K. pneumoniae

Nasal swabs from pigs were inoculated into brain heart infusion (BHI) liquid medium (Haibo Biotech, Qingdao, China) under aseptic conditions and incubated overnight at 37 °C with shaking at 180 rpm. The enriched cultures were then streaked onto MacConkey agar plates (Haibo Biotech, Qingdao, China) and incubated at 37 °C for 24 h.

Single colonies were selected and repeatedly streaked onto fresh MacConkey agar plates for purification until uniform colony size and morphology were achieved. Colony characteristics, including size, morphology, and color, were recorded. Gram-staining (Solarbio Technology, Beijing, China) was performed for microscopic examination to confirm bacterial morphology and Gram reaction.

Identification of K. pneumoniae

Biochemical identification

The isolated strains were identified using a biochemical identification tube kit (Hangzhou Microbial Reagent Co., Hangzhou, China) according to the manufacturer’s instructions.

PCR identification and 16 S rRNA gene genetic evolution analysis

The genomic DNA of bacterial isolates was extracted by boiling methods as template. PCR detection was performed using specific primers designed for the Khe gene unique to K. pneumoniae, with the genomic DNA of bacterial isolates serving as the template. PCR program: 3 min pre-denaturation at 95 °C; 25s denaturation at 94 °C; 25s annealing at 61 °C (specific annealing temperature see Table 1); 1 min extension at 72 °C, for a total of 30 cycles; 10 min extension at 72 °C. Verify PCR products using 1% agarose gel electrophoresis. Using universal bacterial primers [13] to amplify the 16 S rRNA gene of the isolated strains, the purified PCR products were sent to General Biologicals Co., Ltd. (Anhui, China) for sequencing. The assembled complete sequences were subjected to BLAST comparison on NCBI, and a phylogenetic tree was constructed using MEGA 11.0. The primers were synthesized by Xinjiang Youkang Biotechnology Co., Ltd. (Urumqi, China), and the primer sequences are shown in Table 1.

Table 1 Primer sequences for identification of K. pneumoniae

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 [14], primers for the wzi gene of K. pneumoniae and seven housekeeping genes (gapA, infB, mdh, pgi, phoE, ropB, and 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 (Anhui, 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.

Table 2 K. pneumoniae typing identification primer sequence

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 < OD ≤ 4ODc was considered to have moderate ability to form membrane, OD > 4ODc is the strong ability of film formation.

Antimicrobial susceptibility testing

The paper disc diffusion method (Kirby-Bauer method) was used to assess the sensitivity of isolated strains to common antibiotics. A 200µL aliquot of the logarithmic growth-phase culture of K. pneumoniae was uniformly spread on a Mueller-Hinton agar (MHA) plate. Discs containing 17 different antibiotics(PEN, AMX, CFB, CTX, K, GM, ENR, CIP, DX, TMP, FON, PB, AMP, IPM, TCY, CRO, CA) were placed at appropriate positions on the plate, which was then incubated at 37 °C for 24 h. The diameters of the inhibition zones were measured and recorded, with the experiment repeated three times to obtain an average value. Concurrently, K. pneumoniae ATCC 700,603 and Escherichia coli ATCC 25,922 were used as quality control strains for parallel testing to ensure the validity of the results. The sensitivity of the bacteria to the antibiotics was determined based on the guidelines from the Clinical and Laboratory Standards Institute (CLSI) [15– 16] (Table 3).

Table 3 Criteria for determining the dosage and drug resistance of tablets

Antimicrobial resistance gene detection

Referring to relevant literature [17,18,19,20,21], Using PCR methods to detect six categories of 16 antibiotic resistance genes in isolates of K. pneumoniae, which include β-lactam (bla_TEM, bla_CTX, bla_NDM, bla_OXA−48, bla_IMP, bla_VI, bla_DHA, bla_KPC), aminoglycosides (aadA), tetracyclines (tet(B)), chloramphenicol (cmlA, floR), sulfonamides (sul2), and quinolones (parC, gyrA, gyrB). The PCR reaction conditions are the same as described in Sect. 2.3.2, with specific primer information detailed in Table 4.

Table 4 Primer sequences for resistance genes of K. pneumoniae

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 hypermucoviscous (HV) if the string length exceeded 5 mm [22].

Virulence gene detection

Referring to relevant literature [18, 23,24,25,26,27], the PCR method was employed to detect 12 virulence genes in K. pneumoniae strains: lipopolysaccharide-related genes (uge, wabG), fimbriae-related genes (fimH, mrkD), iron transport-related genes (entB, kfu, iroN, icuA), urease-related gene (ureA), and allantoin-related gene (allS). The PCR reaction system and procedure were consistent with Sect. 2.3.2, and the primer sequences and related information are provided in Table 5.

Table 5 Primer sequences for virulence genes of K. pneumoniae

Mouse pathogenicity test

Based on the detection of resistance and virulence genes, a multidrug-resistant strain, KP-20, belonging to ST 967 and carrying eight virulence genes and seven resistance genes, was selected as the challenge strain.

Experimental design

A total of 48 six-week-old Kunming strain mice were randomly assigned to eight groups (one control group and seven experimental groups), with six mice per group. Prior to the experiment, the mice were fasted but allowed access to water for 24 h.A fresh bacterial culture in the logarithmic growth phase was prepared and subjected to a 10-fold gradient dilution. The experimental group of mice was intraperitoneally injected with 0.4 mL of bacterial suspensions at various doses ranging from 1.6 × 10⁹ to 1.6 × 10³ CFU per mouse. The control group was injected with an equal volume of sterile saline.

Observation and data collection

Post-infection, the clinical symptoms and mortality of the mice were observed and recorded every four hours. The median lethal dose (LD₅₀) of KP-20 was calculated using the modified Karber method.

Necropsy and pathological examination

Mice that succumbed to the infection underwent necropsy to assess organ lesions. Diseased tissues were collected for bacterial isolation and culture. Isolates were identified as K. pneumoniae using specific primers. Additionally, affected tissues were fixed in 4% formaldehyde, embedded in paraffin, sectioned, and stained for histopathological examination under a microscope.

Statistical analysis

All statistical analyses were performed using GraphPad Prism 9.0,Graphs and charts were generated to visually represent the statistical findings, aiding in the interpretation of the results.

Results

Isolation and identification of K. pneumoniae

A total of 21 bacterial strains were isolated from the collected samples. The isolated strains grew as pink, mucoid, circular colonies on MacConkey agar (Fig. 2A). Gram-staining microscopy reveals the presence of single, paired, or chained, blunt-ended, pink short bacilli (see Fig. 2B). Biochemical characteristic analysis shows that the isolated strain tests positive for glucuronate, peptone water, citrate, gas production from glucose, lysine, gossypol, sorbitol, marigold alcohol, and xylose. Negative reactions are observed for hydrogen sulfide, indole, phenylalanine, semi-solid, and ornithine tests. The biochemical characteristics of the isolated bacterium are consistent with those of K. pneumoniae as described in the Bergy’s Manual of Determinative Bacteriology. PCR identification of the isolated strain was performed using Khe gene primers specific to K. pneumoniae, with the PCR amplification product showing a specific band at 285 bp, consistent with the expected fragment size (see Fig. 2C).

Through the homology analysis of the 16 S rRNA gene, it was found that the 16 S rRNA gene sequences of 21 isolated strains exhibited a similarity of 99.86–100% with the 16 S rRNA sequences of K. pneumoniae in the GenBank database, further confirming that these isolates are indeed K. pneumoniae. Phylogenetic analysis indicated that the isolates mainly clustered into two branches, with the closest genetic relationship to the pig-derived K. pneumoniae previously isolated from the Yili region of Xinjiang [26] (KP-wzi19 and KP-wzi33-64) (see Fig. 3). Based on the biological characteristics of the isolates, the PCR amplification results of the Khe gene, and the analysis of the 16 S rRNA sequences, all 21 isolates were identified as K. pneumoniae and were designated as KP-01, KP-02, KP-03, KP-21. Among the 50 nasal swab samples, the overall isolation rate of K. pneumoniae was 42% (21/50).

Fig. 1

Geographical distribution of sampling sites in Xinjiang

Fig. 2

Identification of isolated strains. Note A Colony morphology of isolated bacteria on MacConkey agar medium; B Gram-staining microscopy results of isolated bacteria (100x); C PCR amplification results of Khe gene of isolated bacteria (M: DL-700 Marker 1–22: Isolate Strain N: Negative Control)

Fig. 3

Phylogenetic tree based on 16 S rRNA gene sequence. Note The phylogenetic tree of 16 S rRNA gene sequence of isolated strains (the strains marked with circles are all K. pneumoniae isolated in this study, and the hollow circles represent type ST 967; The solid circle represents the ST 37

Capsular serotyping and multilocus sequence typing

Based on the wzi gene sequence, 21 isolates of K. pneumoniae can be classified into two different wzi allele types. Among these, the wzi81-KL81KL120-K81 type accounts for 42.86% (9/21), while the wzi19-KL19-K19 type has the highest representation at 57.14% (12/21). The evolutionary tree of the wzi gene is shown in Fig. 4. According to 7 butler gene sequences, 21 strains of K. pneumoniae can be divided into two STs types, mainly ST 37 and ST 967. Among them, ST 37 accounted for 42.86 (9/21), and ST 967 accounted for 57.14% (12/21). The MLST minimum spanning tree found that the isolates ST 37 and ST 967 were far apart from each other (as shown in Fig. 5). The K. pneumoniae isolates of wzi81 belong to ST 37, and the isolates of wzi19 belong to ST 967, indicating that the two types of typing results are highly consistent. The detailed typing of K. pneumoniae isolates is shown in Table 6.

Fig. 4

Identification of isolated strain serotypes

Fig. 5

MLST minimum spanning tree

Table 6 Classification of K. pneumoniae isolates is shown

Biofilm production

According to crystal violet staining method, OD_570nm of 21 isolates were all greater than 2 ODc (as shown in Fig. 6), among which 10 had strong film forming ability and 11 had medium film forming ability. There were 3 strains with strong film forming ability and 6 strains with medium film forming ability of type ST 37. There were 7 strains with strong film forming ability and 5 strains with medium film forming ability of type ST 967. It is suggested that these strains have strong ability to resist the external environment and the bactericidal effect of antibiotics.

Fig. 6

Isolate the biofilm formation ability of the strain

Antimicrobial susceptibility testing

The drug susceptibility test results of K. pneumoniae isolates were shown in Fig. 7. The 21 isolates showed strong resistance to β-lactam antibiotics and tetracycline antibiotics. Among them, penicillin, amoxicillin, cefazolin, ampicillin, cefotaxime, ceftriaxone for β-lactam antibiotics; Aminoglycoside gentamicin; Tetracycline doxycycline, flufenicol, tetracycline; Cotrimoxazole sulfanilamide; The drug resistance rate of 12 antibiotics such as glycopeptide vancomycin was up to 100%. The resistance rate of kanamycin to aminoglycoside was 71.43%. The resistance rates to Enrofloxacin and ciprofloxacin quinolones were 57.14% and 95.24%, respectively. These isolates were sensitive to carbapenem imipenem and polymyxin B, with resistance rates of 19.05% and 9.21% respectively. Each K. pneumoniae strain is resistant to at least 12 antibiotics, and according to the definition of bacterial multi-drug resistance (MDR), all isolates belong to the MDR strain.

Fig. 7

Overall durg resistance of 21 strains of K. pneumoniae to different types of antibiotics

Antimicrobial resistance genes

The K. pneumoniae isolates were tested for 16 kinds of drug resistance genes, and the results were shown in Fig. 8. A total of 10 drug resistance genes in 6 classes (bla_TEM, bla_DHA, aadA, cmlA, floR, sul2, tet(B), parC, gyrA, gyrB) were detected in 21 isolates. The detection rates of aminoglycoside resistance gene (aadA), chloramphenicol resistance gene (cmlA, floR), sulfa resistance gene (sul2) and quinolone resistance gene (gyrA) were all 100%. The detection rates of quinolone resistance genes parC and gyrB were 57.14% and 61.90%, respectively. The detection rates of β-lactam resistance genes bla_TEM and bla_DHA were 47.62%. The detection rate of tetracycline resistance gene tet(B) was relatively low, only 4.76%. The comparison between the two serotypes showed that the probability of ST 967 K. pneumoniae isolate carrying bla_TEM was 8.33% (1/12), while the probability of ST 37 K. pneumoniae isolate carrying bla_TEM was 100% (9/9). There was no significant difference in the carrying of other resistance genes between the two serotypes.

Fig. 8

Detection of virulence genes in 21 strains of K. pneumoniae

The analysis of drug resistance gene profiles of K. pneumoniae isolates showed that 12 kinds of drug resistance gene combinations were detected in 21 isolates. Among them, aadA + clmA + floR + sul2 + parC + gyrA + gyrB + bla_DHA+bla_TEM was the combination with the largest number of drug-resistance genes, with a carrying rate of 4.76%. aadA + clmA + floR + sul2 + gyrA + gyrB + bla_DHA+bla_TEM had the largest number of drug resistance gene combinations, with a carrying rate of 19.05% (Fig. 9). These results indicate that it is common for K. pneumoniae of pig origin to carry multiple drug resistance genes in Xinjiang.

As shown in Table 7, the drug-resistance phenotype and drug-resistance genotype of K. pneumoniae isolates were consistent. The detection rates of β-lactam, aminoglycoside and sulfonamide resistance genes were 100%. The coincidence rate of quinolone resistance was 95.24%. The compliance rate of tetracycline resistance was 4.76%.

Fig. 9

Antimicrobial Resistance gene combinations of different strains of K. pneumoniae

Table 7 The detection rate of resistant genes of K. pneumoniae was consistent with the resistant phenotype

Mucoid phenotyping

In the string test, none of the 21 strains of K. pneumoniae isolated showed a mucous thread length exceeding 5 mm, and all results were negative (Fig. 10), indicating that a high-mucus phenotype of K. pneumoniae was not detected.

Fig. 10

Drawing test result of some Klebsiella pneumoniae isolates

Virulence gene detection

The detection of virulence genes of 21 K. pneumoniae isolates was shown in Fig. 8. A total of 8 virulence genes (uge, wabG, entB, iroN, icuA, fimH, mrkD and ureA) were detected. Among them, the highest detection rate was LPS related genes (wabG, uge, entB), fimH, mrkD and ureA related genes (ureA), accounting for 100%. The detection rates of iucA and iroN were 57.14% and 42.86%, respectively. Capsular-associated genes (rmpA, magA), ferrifer-associated genes (kfu) and allantoin genes (allS) were not detected. The comparison of virulence genes carried by two different serotypes showed that ST 967 K. pneumoniae isolates specifically carried virulence gene icuA, while most ST 37 K. pneumoniae isolates carried virulence gene iroN. There was no significant difference between the two serotypes in carrying other virulence genes.

According to the statistics of multiple virulence gene carrier, this isolate carried at least 6 virulence genes, of which fimH + mrkD + uge + wabG + entB + iucA + ureA had the largest virulence gene, accounting for 52.38%. The proportion of fimH + mrkD + uge + wabG + entB + ureA virulence gene and fimH + mrkD + uge + wabG + entB + iucA + ureA + ironN was the smallest, accounting for only 4.76% (Fig. 11). These results indicated that multiple virulence genes carried by K. pneumoniae from pigs were common in Xinjiang.

Fig. 11

Virulence gene combinations of different strains of K. pneumoniae

Mouse pathogenicity test

A comprehensive analysis was conducted on the drug resistance phenotypes, resistance genes, and virulence genes of 21 isolated strains, leading to the selection of the isolate KP-20 for a pathogenicity experiment in mice. The results revealed clinical symptoms in the experimental mice, including lethargy, reduced food and water intake, rapid breathing, and ruffled fur, beginning 4 h after intraperitoneal injection of the bacterial suspension. After 6 hours of infection, mice in the experimental group given 1.6 × 10¹⁰ CFU began to die, exhibiting symptoms such as hind limb convulsions and respiratory distress before death. By 24 h post-infection, all mice in four experimental groups receiving 1.6 × 10¹⁰, 1.6 × 10⁹, 1.6 × 10⁸, and 1.6 × 10⁷ CFU were dead, while three mice in the group receiving 1.6 × 10⁶ CFU survived; the remaining mice in other experimental groups were all alive. After 72 h, the surviving mice recovered to a normal state, with no abnormalities observed in the control group. The survival status of mice in each experimental group at different time points is shown in Fig. 12. Necropsy of the deceased mice revealed diffuse pulmonary hemorrhage, varying degrees of enlargement and congestion in the liver and spleen, localized distension in the small intestine, thinning of intestinal mucosa, and bleeding (Fig. 13). Bacteria were isolated from the lungs, liver, and spleen tissues of the dead mice, and PCR identification confirmed that the isolated strain matched the pathogenic strain (Fig. 14). Based on the death rates of the mice post-infection, the median lethal dose (LD₅₀) of the isolate KP-20 was calculated to be 4.0 × 10⁶ CFU/mL. Pathological histological observations indicated disorganized liver cell arrangement, significant infiltration of red blood cells in the central vein, considerable hemorrhage and congestion in lung tissue, markedly reduced lymphocyte numbers in the white pulp of the spleen, and a significant increase in red blood cell numbers in the red pulp; the intestinal villi were found to be broken and sloughed off, with an influx of neutrophils in the submucosa, along with numerous villus fragments in the intestinal lumen (Fig. 15).

Fig. 12

Survival curves of mice

Fig. 13

Pathological examination of mice

Fig. 14

PCR amplification of Khe gene of bacteria isolated from various organs of infected mice. M: DL-700 Marker; 1:bacteria isolated from the lungs; 2: bacteria isolated from the livers N: negative control

Fig. 15

Histopathological observation of mice. Note A, B, C, D liver, lung, spleen and small intestine of mice in the control group; a, b, c, d liver, lung, spleen and small intestine of 4 × 10⁹ CFU/mL mice in the experimental group. Note a The arrow indicates a high concentration of red blood cells.; b The arrow indicates a high concentration of red blood cells; c The arrow points to an area with red blood cells and inflammatory exudate; d The arrow marks the site of small intestinal villi shedding

Discussion

In 1882, K. pneumoniae was first isolated from the lungs of a patient who had died from pneumonia. Initially regarded as an opportunistic pathogen present in the natural environment, it did not receive widespread attention from society [29]. Currently, pneumonia caused by animal-derived Klebsiella bacteria typically demonstrates multidrug resistance and strong pathogenicity. The bacterium can acquire and spread antibiotic resistance genes through horizontal gene transfer mechanisms [30], it spreads widely among humans, the environment, and animals, posing a potential threat to public health and livestock farming [31]. In 2020, Dong [32] isolated 45 strains of K. pneumoniae from 123 samples of diseased tissue collected from various pig farms in the Cangzhou region, resulting in an isolation rate of 36.59%.In 2021, Fu [33] isolated a strain of K. pneumoniae from the diseased materials of a pig farm in Shaanxi. In 2022, Li [2] isolated a strain of K. pneumoniae (type ST-35) from the lung tissue of sick pigs at a large-scale pig farm in Guizhou.In 2022, Zuo [34] isolated three strains of K. pneumoniae from stool swab samples of diarrhea-prone Tibetan pigs from the Tibet region, with an isolation rate of 5%. In 2023, Xu [35] isolated 58 strains of K. pneumoniae from fecal swab samples collected from pig farms of varying scales in Fujian, achieving an isolation rate of 17.1% (58/340). In 2023, Hou [28] and others isolated three strains of K. pneumoniae from large-scale pig farms in the Xinjiang region, with an isolation rate of 4.55% (3/66) (one strain belonging to ST-967 type, one to ST-42 type, and one of unknown type). This research indicates that K. pneumoniae is prevalent in pig populations in China and has certain pathogenicity towards pigs.The study isolated 21 strains of K. pneumoniae from nasal swab samples of healthy pigs at another large-scale farm in Xinjiang, with an isolation rate of 42% (21/50). Mouse infection trials confirmed that the isolated strains exhibited moderate virulence in mice, consistent with existing research findings.

As of now, official MLST data shows that there are a total of 7,622 ST types of K. pneumoniae worldwide. ST 37 is primarily found in Europe, Asia, and North America, with most strains being of human origin. In contrast, ST 967 is mainly distributed in Asia and Europe, with some strains being of human origin and others of porcine origin. The porcine K. pneumoniae strains of ST 37 and ST 967 isolated in this experiment align with the known distribution patterns of K. pneumoniae. In 2017, China reported for the first time the isolation of ST 37 type K. pneumoniae from a newborn [36]. In 2024, Jing [37] and others isolated 13 strains of ST 37 type K. pneumoniae from the pig farm.This indicates that the ST 37 strain of K. pneumoniae is distributed among both the human population and pig populations in our country. In August 2019, researchers isolated the ST 967 strain of K. pneumoniae from a rehabilitated patient in Armenia [38]. So far, there has been little research on the ST 967 type of K. pneumoniae. In this experiment, 12 strains of ST 967 were isolated, all of which are multidrug-resistant. The ST 967 type is the predominant strain in this pig farm. Studies have indicated that both ST 37 and ST 967 types of K. pneumoniae pose a risk of transmission between humans and pigs, presenting a serious threat to public health safety.

Since the outbreak of African Swine Fever in 2018, the biosafety work of each farm is extremely strict, and disinfection is the top priority. The formation and spread of K. pneumoniae isolates in each enclosure is closely related to its strong biofilm formation ability. Li et al. [39] found that biofilm is an important mechanism of drug resistance of K. pneumoniae. The most important surface structures in the biofilm formation process of K. pneumoniae are type 3 pili and capsular polysaccharide. Fimbril mediates stable adhesion, while CPS ultimately affect biofilm structure and intercellular communication [10]. In this experiment, 21 strains of K. pneumoniae were isolated, and the detection rate of fimH and mrkD genes was 100%, but no capsular genes (rmpA and magA) were detected. Among the 21 strains, 10 strains had strong biofilm forming ability and 11 strains had medium biofilm forming ability, which was consistent with the multiple drug resistance shown in the drug susceptibility test. Meanwhile, the high detection rate of multiple drug resistance genes also indicated that the treatment of the bacteria after infection would be difficult. β-lactam, aminoglycosides, sulfonamides and quinolone resistance genes were detected in the KP isolates in this study to varying degrees, which is related to the clinical use of drugs in farms. Studies [40] have shown that resistance genes can be transmitted across strains and even between Gram-negative and Gram-positive bacteria. There are more than 400 acquired AMR genes in the genome of K. pneumoniae, twice the number of E. coli, and K. pneumoniae can acquire AMR genes from the environment [41].

With the intensive farming of animals, in order to reduce the economic losses caused by various bacterial diseases, antibacterial drugs are used in large quantities, resulting in the continuous emergence of drug-resistant strains. In this study, K-B method was used to detect the sensitivity of K. pneumoniae isolates to antibiotics. The results showed that 21 isolates showed strong resistance to β-lactam, aminoglycoside, tetracycline, sulfonamides, quinolones and other antibacterial drugs, and showed multiple drug resistance, and were only sensitive to carbapenem imipenem and polymyxins polymyxins B. These two drugs may be considered for later treatment of K. pneumoniae infection. Guan Zhongbin et al. isolated K. pneumoniae from 51 large-scale pig farms in Southwest China and showed high drug resistance to amoxicillin, ampicillin, kanamycin, gentamicin and other drugs, which was similar to the test results in this paper [42]. K. pneumoniae can synthesize β-lactamase and aminoglycoside passivase [43] and act on various parts of antibiotics, causing drugs to lose their original functions and thus developing resistance to corresponding antibiotics [44]. The resistance rate of K. pneumoniae isolates to penicillin G, amoxicillin, cefazolin, gentamicin and other drugs was 100%, which was consistent with the previous conclusions. The drug-resistant phenotype of bacteria is determined by the drug-resistant genotype, but the drug-resistant phenotype and drug-resistant genotype may not be completely consistent. The comparison between the drug-resistance phenotype and drug-resistance genotype of K. pneumoniae isolates showed that the compatibility rate of β-lactam, aminoglycoside, sulfonamides and quinolone resistance genes and drug-resistance phenotype of K. pneumoniae isolates was more than 95%, and the compatibility rate of tetracycline resistance genes and drug-resistance phenotype was only 4.76%. It is speculated that the cause may be the presence of undetected or unknown related resistance genes. In this study, the coincidence rate between β-lactam, aminoglycoside, drug resistance phenotype and drug resistance genes was similar to the results of Pang et al. [45]. In the process of breeding, a large amount of antibiotics, whether used for treatment or growth promotion, will activate relative drug resistance genes [46], which will contribute to the enhancement of strain resistance and increase the spread of these resistant strains to humans through the food chain or other transmission routes [43]. In this study, K. pneumoniae isolates developed strong resistance to 7 classes of 15 antibiotics, and were relatively sensitive to only 2 antibiotics. It is suggested that farms strictly control the use of antibiotics to prevent the emergence of more drug-resistant strains, and new antibacterial biologics can also be selected for prevention and control, so as to achieve accurate prevention and control and comprehensive treatment.

HvKP strains usually contain virulence genes such as iucA, iroB, rmpA and rmpA2 [24]. Studies [47] have shown that iron uptake related genes (iucA) can increase the virulence of hvKP and is a key virulence characteristic of hvKP. In this study, the detection rate of iucA virulence gene in 21 K. pneumoniae isolates was 57.14%, and the positive strains were all ST 967. None of the 9 ST 37 isolates carried iucA, but a high probability of carrying iroN (88.89%). At the same time, the detection rate of iucA was higher than that of K. pneumoniae isolated by Min et al. [48] from various human samples from a hospital in Ningbo, China. Virulence genes iucA and iroN are related to the iron uptake system, while iro gene cluster mainly encodes salmonella, which is the C-glucosylated form of enterobacteriin. Studies have shown that more than 90% of K. pneumoniae that causes liver abscess can secrete salmonella [49]. It is speculated that the virulence of the two serotypes of K. pneumoniae isolates will be different, and further study will be conducted in the future. The expression of entB gene can produce more iron carriers, promote the development and growth of biofilm, and enhance the virulence of hvKP [50]. fimH and mrkD are adhesins encoding type I and type III fimilus, which can help K. pneumoniae break through the host defense system and are important virulence factors causing urinary tract infections. wabG is a Lipopolysaccharides (LPS) related gene encoding galactosyltransferase, which plays an important role in the synthesis of LPS, LPS can inhibit the phagocytosis of macrophages, thus leading to host susceptibility to infection [51]. K. pneumoniae toxic genes mrkD, fimH, wabG are conserved and can help bacteria grow and reproduce. In this study, the detection rates of mrkD, fimH and wabG of K. pneumoniae isolates were 100%, which was similar to the results of Zhu [52] and Zhang [53] et al.Studies have shown [54] that the virulence genes carried by K. pneumoniae are associated with resistance genes and antibiotic resistance phenotypes.Current research has found [55] that plasmids can transmit antibiotic resistance genes and virulence related genetic elements to K. pneumoniae. These plasmids can undergo frequent gene transcription, leading to plasmid fusion, resulting in an increasing number of highly virulent and carbapenem resistant K. pneumoniae strains.

In this study, the isolated strain KP-20 contained up to 8 virulence genes and 7 drug resistance genes, and had strong resistance to 13 antibiotics. The results of the mouse challenge test showed that the median lethal dose (LD50) of this strain to mice was 4.0 × 10⁶ CFU/mL, showing moderate pathogenicity and significantly stronger virulence than the pigeon derived K. pneumoniae isolated by Chen et al. [56] (LD50 = 4.68 × 10⁹ CFU/mL). After 8 h infection with high dose of KP-20, the mice died. The liver, lungs and spleen of the dead mice were bleeding and swollen to varying degrees, which fully demonstrated the pathogenicity of this strain. Although this strain was derived from healthy weaned piglets, it did not cause infection in the pig farm, which may be related to the strict implementation of various biosafety measures in the large-scale pig farm, but the test data showed that this strain has a certain potential threat to low-age piglets, pigs with low immunity and breeders.

Conclusions

The porcine-derived K. pneumoniae isolated in this study has a strong ability to form biofilms, exhibits a strong phenomenon of multiple drug resistance, carries various drug resistance and virulence genes, has a moderate virulence to mice, and poses a potential threat to pig farming and public health safety. The use of polymyxin and imipenem antibiotics has a certain clearance effect on K. pneumoniae, and the use of β- lactam antibiotics should be strictly controlled to prevent other bacteria from developing antibiotic resistance except for K. pneumoniae. This study can provide a reference basis for the prevention and control of K. pneumoniae in pig farms.