Biological Characteristics and Comparative Genomic Analysis of Corynebacterium hindlerae from Bovine Skin Abscess

TL;DR

This paper studies the biology and genome of Corynebacterium hindlerae from bovine skin abscesses to aid in diagnosis and control.

Contribution

The study provides new insights into the genomic diversity and evolutionary relationships of C. hindlerae strains.

Findings

Strain LSKT01 showed optimal growth at 37°C, pH 5.5, and 1–2% NaCl with strong biofilm formation.

Phylogenetic analysis grouped five C. hindlerae strains into two subgroups with high ANI similarity.

Comparative genomics revealed conserved gene families and structural variations among strains.

Abstract

This study aimed to investigate the biological characteristics of Corynebacterium hindlerae, a pathogen discovered in bovine hosts, analyze its genomic features, clarify genetic relationships and differences among strains, and provide a scientific basis for comprehensive clinical prevention and control. The environmental tolerance, biofilm formation capability, and motility of the isolated strain LSKT01 were analyzed. A total of 12 Corynebacterium strains were selected for phylogenetic analysis based on core genes, evaluation of Average Nucleotide Identity (ANI), and comparative genomic analysis covering gene families, synteny, SNPs, InDels, and structural variations (SVs). The isolate exhibited optimal growth at 37 °C, pH 5.5, and 1–2% NaCl concentration, demonstrated strong biofilm-forming ability, but showed weak or no motility. Phylogenetic analysis divided the five Corynebacterium hindlerae strains into two subgroups, with strain LSKT01 being most closely related to strain 1864 (ANI 95.80–98.70%). Comparative genomic analysis revealed highly conserved gene families among strains, while LSKT01 possessed 20 specific paralogs. Genome synteny analysis showed an average similarity >90%, and strains ISL_960a and MAL_1082b exhibited more complex structural variations. The successful isolation and purification of LSKT01 revealed its biological characteristics and genomic features, elucidating the genetic relationships and evolutionary divergence among C. hindlerae strains from diverse sources. This provides a theoretical basis for the rapid diagnosis and precise prevention and control of Corynebacterium hindlerae in Xinjiang.

Figures

The growth curve of isolated bacteria under different temperature, salt concentration and pH. (a) Growth rate of the isolated bacteria over time at different temperatures. (b) Growth rate of the isolated bacteria over time at different pH levels. (c) Growth rate of the isolated bacteria over time at different salt concentrations.

Full text

1. Introduction

Corynebacterium, as a significant member of the Actinobacteria phylum [1], exhibits large circular chromosomes and abundant secondary metabolic gene clusters in its genome, demonstrating considerable plasticity and adaptability in its genomic structure [2,3]. These bacteria are widely distributed in environments such as soil, animal surfaces, and human mucosal membranes [4]. Most Corynebacterium species grow well on blood agar, while lipophilic strains require exogenous lipids such as Tween [5].

Reports on the isolation, identification, and phenotypic characteristics of Corynebacterium hindlerae are scarce. Corynebacterium hindlerae is a Gram-positive, non-spore-forming coccobacillus widely distributed in nature and on the skin and mucous membranes of various livestock such as cattle and sheep. In recent years, this bacterium has been identified in Xinjiang, China, and is closely associated with chronic suppurative lesions in domestic animals. The lesions typically occur in areas such as the head, neck, submandibular region, and tongue, and may also affect soft tissues. Cases of this disease may occur throughout all seasons of the year but are more commonly observed in warm and humid conditions during spring, summer, and early autumn, with an epidemic pattern that is sporadic in nature. The disease has a prolonged incubation period of 3 to 18 months, primarily manifesting as chronic suppurative lesions and granulomas [6]. These lesions not only lead to reduced appetite and slowed growth in animals but may also affect their appearance and health, resulting in quality deterioration. Consequently, this poses persistent economic losses to the local livestock farming industry [7]. Research on Corynebacterium hindlerae as a bovine pathogen is severely lacking, especially in major livestock regions such as Xinjiang [8]. This has resulted in significant gaps in understanding its transmission routes, pathogenicity, and impact within cattle populations. Furthermore, there is a notable absence of comprehensive comparative genomic studies on C. hindlerae isolates from different sources. Consequently, our understanding of its intraspecies genetic diversity, evolutionary relationships, mechanisms of host adaptation, and transmission dynamics within cattle populations remains limited.

With the rapid advancement of genomic technologies, comparative genomics has emerged as a powerful tool for elucidating the genetic diversity, virulence factors, environmental adaptability, and evolutionary relationships of pathogenic bacteria. Comparative genomic analysis among identical or similar species enables deeper insights into the biological characteristics, genetic relationships, and evolutionary differences of pathogens [9,10]. This study aims to isolate and purify Corynebacterium hindlerae, analyze its biological characteristics including environmental tolerance, biofilm formation capability, and motility, and utilize comparative genomics to examine its genomic features in terms of average nucleotide identity (ANI), gene families, genomic collinearity, single-nucleotide polymorphisms (SNPs), insertions/deletions (InDels), and structural variations (SVs). This provides a theoretical basis for the rapid identification and comprehensive prevention and control of Corynebacterium hindlerae.

2. Materials and Methods

2.1. Isolation and Purification of Bacteria

The strain LSKT01 was isolated from a large-scale cattle farm in Kuitun, Xinjiang. Affected cattle exhibiting typical chronic suppurative lesions or granulomas on the head, neck, and submandibular regions were selected. After removing superficial necrotic tissue, the lesions were disinfected using 5% iodine tincture swabs and 75% alcohol swabs. The nodules were incised, and deep purulent material was collected from the lesions using cotton swabs. The material was placed into collection tubes. Following successful isolation, the strain was stored at −20 °C in this laboratory. Genomic information has been uploaded to NCBI (accession number: PRJNA1314966). The following media and reagents were used for bacterial culture and phenotypic assays: Gauze’s Synthetic Medium No. 1, LB Broth, Columbia Blood Agar Base, Brain Heart Infusion (BHI) Broth, and defibrinated sheep blood (Qingdao Haibo Biotechnology Co., Ltd., Qingdao, China). For biofilm staining, 1% (w/v) crystal violet (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) and anhydrous methanol (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) were employed.

This study strictly relied on molecular biological methods for the identification of isolated strains. First, PCR amplification and sequencing were performed using universal 16S rRNA primers. The resulting sequences were subjected to BLAST alignment against the NCBI database, revealing the highest homology (98.64–99.86%) with Corynebacterium hindlerae. The frozen stock culture was thawed and inoculated into BHI broth. It was then incubated at 37 °C with shaking at 160 rpm for 36 h. Subsequently, the culture was streaked onto Columbia blood agar containing 5% sheep blood and onto Gauze’s Synthetic Medium No. 1, respectively. Target colonies were cultured for 24 h and then inoculated into fresh BHI broth. This was followed by incubation at 37 °C with shaking at 160 rpm for 20–24 h. A smear was prepared from the purified bacterial suspension using an inoculation loop for microscopic examination.

2.2. The Growth Curve and Environmental Tolerance Test of the Strain

A bacterial culture in the logarithmic growth phase was centrifuged, and the pellet was resuspended in PBS buffer to a concentration of 1 × 10^6^ CFU/mL. We prepared aliquots (1 mL) of LB broth. The prepared bacterial suspension of Corynebacterium hindlerae was inoculated into the LB broth. Cultures were incubated at 4 °C, 25 °C, 37 °C, and 45 °C, with three biological replicates for each temperature. The tubes were placed in incubators set to the corresponding temperatures. For the main growth assessment, cultures were incubated at 37 °C with shaking at 180 rpm for 24 h. Optical density at 600 nm (OD_600_) was measured and recorded at 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 h.

For pH tolerance, bacteria from the logarithmic phase were harvested, centrifuged, and resuspended in PBS to 1 × 10^6^ CFU/mL. LB broth was adjusted to pH 4.0, 5.5, 7.0, 8.5, and 10.0. The bacterial suspension was inoculated into each pH-adjusted broth, followed by incubation at 37 °C with shaking at 180 rpm for 24 h (three replicates per group). OD_600_ was measured at the same time intervals as above.

For the salt tolerance, a logarithmic-phase bacterial suspension was prepared as described (1 × 10^6^ CFU/mL in PBS). LB broth was supplemented with NaCl to final concentrations of 1%, 2%, 4%, 6%, and 8%. The bacterial suspension was inoculated into the salt-adjusted broths and incubated at 37 °C with shaking at 180 rpm for 24 h (three replicates per group). OD_600_ measurements were taken at the specified time points.

2.3. Determination of Biofilm Formation and Motility of Strains

2.3.1. Determination of Biofilm Formation, Morphology, and Structure

The biofilm-forming capacity of the isolate was determined using the crystal violet staining method. The prepared bacterial suspension was aliquoted into a 24-well plate, using LB broth as the negative control. The plates were incubated at 37 °C for 8, 24, 48, and 72 h, with three replicates for each time point. Discard the bacterial suspension from the wells, wash the plate 3 times, and fix the biofilm with anhydrous methanol for 15 min. After discarding the methanol, air-dry the plate at room temperature for 30 min. Wash the plate 3 times again, and then stain with 1% crystal violet for 15 min. Rinse off the staining solution gently under running tap water. Invert the 96-well plate onto sterile filter paper and dry completely in a 37 °C incubator. Finally, add 95% ethanol to each well and destain for 25 min.

The optical density of the resulting solution was measured at 570 nm (OD_570_) using a microplate reader. The OD_570_ value represents the amount of biofilm formed, with the average absorbance of the negative control wells defined as ODc. The biofilm formation capacity was categorized as follows: no biofilm formation if OD_570_ < ODc; weak formation if ODc < OD_570_ < 2 × ODc; moderate formation if 2 × ODc < OD_570_ < 4 × ODc; and strong formation if OD_570_ > 4 × ODc [11]. The experimental data represent the mean values from three independent experiments, with error bars indicating the standard error of the mean. Intergroup comparisons were performed using t-tests and one-way analysis of variance (ANOVA). All data exhibited a normal distribution. Prism 6.02 software was used to conduct t-tests on the OD_570_ values of culture broths from each isolate and LB broth. A p-value < 0.05 was set as the criterion for bacterial biofilm formation. Graphs were generated, data analyzed, and statistical analyses performed using GraphPad Prism 9.0.

A bacterial suspension (1 × 10^6^ CFU/mL) was inoculated into a 96-well plate (200 µL per well) and incubated statically at 37 °C for 12, 24, 36, and 48 h. After incubation, the suspension was discarded, and each well was gently washed three times with ultrapure water. Biofilms were fixed with formaldehyde for 15 min, after which the fixative was removed. The plate was air-dried at room temperature for 30 min, washed three times with ultrapure water, and stained with 1% (w/v) crystal violet at room temperature for 15 min. Excess stain was carefully rinsed off under running tap water. The plate was then inverted on filter paper and dried in a 37 °C incubator. Biofilm morphology was examined under an optical microscope.

2.3.2. Detection of Motility

Purified bacteria were cultured to the logarithmic growth phase. BHI semi-solid medium containing 0.5% agar was prepared. Using an inoculation needle, 2 µL of the bacterial suspension was spot-inoculated onto the surface of the semi-solid medium. The plates were incubated at 37 °C for 24 h. Motile Bacillus subtilis and non-motile Staphylococcus aureus ATCC 25922 were used as positive and negative controls, respectively. Bacterial motility was assessed by measuring the diameter of the migration zone formed by the spreading bacteria.

2.4. Histopathologic Examination

Twelve healthy 8-week-old female Kunming mice were selected. An intraperitoneal infection model was established by injecting six mice with 500 µL of a bacterial suspension containing 1 × 10^8^ CFU/mL. The remaining six mice in the control group received an equal volume of sterile physiological saline. At 24 h post-infection, all mice were euthanized by cervical dislocation. Necropsy was performed immediately. Tissue samples from the liver, spleen, lungs, kidneys, and proximal jejunum were collected for pathological analysis. The specimens were fixed in 10% paraformaldehyde solution, processed into hematoxylin and eosin (H&E)-stained sections, and examined for histological alterations under a light microscope. Images were captured for subsequent analysis [12].

2.5. Genomic DNA Extraction, Sequencing, Quality Control Analysis, and Assembly of the Bacterial Isolate

For genomic sequencing, the E.Z.N.A.^®^ Bacterial DNA Kit (Omega Bio-Tek, Norcross, GA, USA) was used to extract genomic DNA. Sequencing libraries were prepared using the NEBNext^®^ Ultra™ II DNA Library Prep Kit (New England Biolabs, Ipswich, MA, USA). Library quantification was performed with the TBS380 Picogreen assay (Invitrogen, Carlsbad, CA, USA). Paired-end sequencing was conducted on an Illumina platform using the TruSeq SBS Kit (300 cycles) (Illumina, San Diego, CA, USA). Genomic DNA was extracted from the cell pellet of a logarithmic-phase bacterial culture after centrifugation at 12,000 rpm for 5 min. Sequencing libraries were constructed using the Illumina TruSeq™ Nano DNA Sample Prep Kit (Illumina, San Diego, CA, USA). Raw sequencing reads were quality-controlled using Trimmomatic [13]. De novo assembly was performed with ABySS using multiple K-mer parameters to obtain the optimal assembly [14]. This assembly was further refined by local gap filling and base error correction using GapCloser [15].

2.6. Comparative Genomic Analysis

By comparing whole-genome sequences across different species, one can analyze the composition of their core genomes and variable genomes, as well as genomic structural variations [16]. Based on strain LSKT01 data, this study selected four additional Corynebacterium hindlerae strains and seven other representative Corynebacterium species for comparative genomic analysis [17,18], as shown in Table 1.

2.6.1. Phylogenetic Analysis

Based on core genes, a phylogenetic tree was constructed. For single-copy core genes identified in analyses of shared and unique genes, the MUSCLE software (version 3.8.31) was used to align the protein multiple sequences of these single-copy core genes. The results were then converted into CDS (codon-encoded sequence) data for phylogenetic tree construction. The maximum likelihood phylogenetic tree was constructed using PhyML (v3.0) [19,20]. The default replacement model is the JTT model (Jones–Taylor–Thornton model). Node support is calculated using the Bootstrap sampling method (self-expansion method) with 100 bootstraps.

2.6.2. ANI Analysis

ANI is considered an indicator of the genetic relationship between two genomes at the nucleotide level. It serves as a genomic alternative to traditional DNA-DNA hybridization for defining microbial species. Characterized by high interspecies differentiation, A DNA-DNA hybridization (DDH) value of 70% corresponds to approximately 95% average nucleotide identity (ANI). Subsequently, 95% ANI has been widely adopted as the threshold criterion for defining prokaryotic species using genomic data [21]. Analysis in this experiment was performed using the fast ANI (v 1.32) software [22]. The analysis defaults to whole-genome alignment using the BLASTN algorithm (ANIb), with a fragment size set to 1020 bp. All other parameters use the software’s default values.

2.6.3. Gene Family Analysis

Protein alignment across the multiple target genomes was performed using BLAST software (v2.13.0). Unreliable results were filtered out, and the Solar tool was employed to eliminate redundancy. Subsequently, the proteins were clustered to obtain the gene family clustering results. To screen for reliable homology relationships, an E-value threshold ≤ 1 × 10^−5^ is set, and query coverage must be >50%.

2.6.4. Common Genes and Specific Genes

The cd-hit software (v4.8.1) was used to cluster the protein sequences of multiple samples to be analyzed, and the screening parameters of Identity and alignment length were set [23]. The clustering of all protein sequences was obtained according to the analysis results of the software. Set the sequence consistency (Identity) threshold to ≥50%, and ensure that the alignment length covers ≥ 50% of the longer sequence.

2.6.5. Genome Collinearity Analysis

MUMmer software (v4.0.1) was used to compare the target genome and the reference genome to determine the large-scale collinearity between the genomes [24]. The maximum interval is set to 500, and mincluster is set to 100 to anchor large-scale collinearity blocks. LASTZ is used to compare regions, confirm local positional alignments, and find Translocation/Trans, Inversion/Inv, and Trans + Inv regions.

2.6.6. SNP/InDel Statistics

SNPs represent DNA sequence polymorphisms arising from single-nucleotide variations, primarily involving transitions or transversions. To identify SNPs, each sample genome was globally aligned against the reference sequence using MUMmer software. Potential SNP sites were initially screened based on sequence discrepancies between the sample and reference. The sequences surrounding these sites were extracted and validated by realigning them against the assembled genome using BLAT software (v2.13.0). Following filtering, high-confidence SNPs were retained for further analysis. Extract 100 bp upstream and downstream sequences for each potential SNP site on the reference sequence. Align this sequence against the sample’s assembly results. If the best alignment length is <101 bp, the SNP is deemed unreliable and excluded. If the sequence aligns to multiple genomic locations, the SNP is considered located in a repetitive region and excluded.

InDels refer to the insertion or deletion of short nucleotide fragments at specific genomic positions relative to a reference sequence. For InDel detection, sample genomes were aligned against the reference using LASTZ software (v1.04.03) to identify optimal alignments. The resulting alignments were subsequently validated and filtered using the bwa (v0.7.17) and samtools software (v1.15.1) suites to ensure accuracy. Extract a 150 bp region (±75 bp) upstream and downstream of each preliminarily identified InDel site on the reference sequence as the validation target region.

2.6.7. SV Statistics

SV refers to alterations in DNA sequences longer than 50 bp within the genome. Compared to SNPs and InDels, SVs involve a broader sequence range. The software SyRI (v1.6.3) was used to detect genomic structural variations in five strains of Corynebacterium hindlerae [25], including deletions, insertions, duplications, inversions, and translocations of DNA segments within the genome. By default, sequence changes ≥50 bp in length are defined as structural variants.

3. Results

3.1. Isolation, Purification and Identification of Bacteria

As shown in Figure 1, the isolate formed smooth, moist, circular, raised, white to cream-colored colonies on Columbia blood agar supplemented with 5% sheep blood, demonstrating good growth under both aerobic and 5% CO_2_ conditions. On Gauze’s Synthetic Medium No. 1, growth was slow; no colonies were visible at 12 h. After 30 h of incubation, small, opaque, white colonies developed, exhibiting slightly dry surfaces with slightly raised centers and well-defined edges. No pigment diffusion was observed around the colonies, but a slight yellowish pigment deposition was noted at the interface with the medium. Microscopic examination revealed Gram-positive staining, although the staining was uneven and tended to fade in older cultures, as shown in Figure 1b. Individual cells exhibited slender, irregular short rod or coccobacillary morphology, predominantly occurring singly, in V-shaped arrangements, or in parallel palisade-like formations, as shown in Figure 1c. In BHI liquid medium, the bacterial suspension exhibits a viscous consistency. When stationary, distinct filamentous or flocculent aggregates are visible suspended within the medium. Upon gentle shaking, the suspension flows slowly. When stationary, distinct filamentous or flocculent aggregates are visibly suspended within the medium. Upon gentle shaking, the suspension flows slowly, as shown in Figure 1d.

3.2. Environmental Tolerance Test of Isolates

3.2.1. Temperature Tolerance Test

The bacterial suspension was inoculated into LB broth and incubated statically at 4 °C, 25 °C, 37 °C, and 45 °C, with three biological replicates per temperature group. Samples were taken from 1 to 32 h to measure the optical density at 600 nm (OD_600_). The isolate exhibited robust growth at 37 °C, with the fastest growth rate observed between 18 and 20 h. Growth was reduced at 45 °C, although a relatively high rate was maintained after 20 h, reaching a peak OD_600_ of 0.61 ± 0.015 at 28 h. At 25 °C, growth was significantly slower, with an OD_600_ of 0.108 ± 0.0027 at 24 h. At 4 °C, growth was slow or nearly stagnant, showing no significant increase over 32 h, as shown in Figure 2a.

3.2.2. pH Tolerance Test

Bacterial suspensions were inoculated into LB broth adjusted to pH 4.0, 5.5, 7.0, 8.5, and 10.0, followed by incubation at 37 °C with shaking at 180 rpm for 24 h. OD_600_ was measured from 1 to 32 h. The isolate grew fastest overall at pH 5.5, entering the exponential phase at 4 h, peaking at 20 h, and stabilizing by 22 h. At pH 7.0, growth was also relatively rapid, with the exponential phase beginning at 6 h and peaking at 20 h. At pH 8.5, the peak growth rate occurred between 18 and 20 h, reaching a maximum OD_600_ of 0.61 ± 0.016 at 20 h. Growth was significantly slower at pH 4.0, with OD_600_ declining after reaching 0.16 at 20 h. No significant growth was observed throughout the incubation period at pH 10.0, as shown in Figure 2b.

3.2.3. Salt Concentration Tolerance Test

LB broth was prepared with NaCl added to final concentrations of 1%, 2%, 4%, 6%, and 8%. Isolates cultured to the logarithmic growth phase were inoculated into the respective media and incubated at 37 °C with shaking at 180 rpm for 24 h, with three biological replicates per concentration. The optical density at 600 nm (OD_600_) was measured at intervals from 1 to 32 h. The isolates grew robustly at 1%, 2%, and 4% NaCl. The OD_600_ values increased gradually over time, peaking at 20 h with no significant differences observed among these three concentrations before entering a stationary phase. Growth was slightly slower at 4% NaCl compared to 1% and 2%. At 6% NaCl, the growth rate decreased markedly, reaching a reduced peak OD_600_ of 0.46 ± 0.039 at 20 h. Although the isolate remained capable of proliferation at 8% NaCl, growth was severely inhibited, increasing slowly until 24 h before declining, as shown in Figure 2c.

The growth curve of isolated bacteria under different temperature, salt concentration and pH. (a) Growth rate of the isolated bacteria over time at different temperatures. (b) Growth rate of the isolated bacteria over time at different pH levels. (c) Growth rate of the isolated bacteria over time at different salt concentrations.

3.3. Determination of Biofilm Formation and Motility of Isolates

3.3.1. Determination of Biofilm Amount by Crystal Violet Staining Method

As shown in Figure 3, bacterial suspensions were inoculated into a 96-well plate alongside negative controls, with three biological replicates per group. After static incubation at 37 °C for 8, 24, 48 and 72 h, biofilm formation was assessed using the crystal violet staining method. In this experiment, the ODc (cut-off value of the negative control) was 0.21. At 8 h, the OD_570_ of the isolate was 0.21 ± 0.026, indicating weak biofilm-forming ability, corresponding to the initial attachment phase. At 24 h, the OD_570_ increased to 0.4 ± 0.017, still classified as weak but showing a statistically significant difference compared with both the negative-control group and the 8-h group (p < 0.05). By 48 h, the OD_570_ reached 0.63 ± 0.039, reflecting moderate biofilm-forming capacity. At 72 h, the OD_570_ peaked at 0.79 ± 0.025, also within the moderate range; this value differed significantly from those of all other time points (p < 0.05).

3.3.2. Observation of Biofilm Morphology and Structure

As shown in Figure 4, at 12 h, bacterial cells had extensively adhered to the microplate surface, forming scattered microcolonies. Localized areas displayed sparse grid-like structures with cells connected into chains, indicating the initial stage of biofilm formation, as shown in Figure 4a. By 24 h (Figure 4b), the grid-like structures had expanded, forming a more continuous biofilm architecture. Cell density increased significantly. At 36 h (Figure 4c), the biofilm matured into a dense and distinct irregular grid structure. By 48 h (Figure 4d), most grid structures had further condensed, reducing the number of visible water channels. Some areas exhibited discontinuous punctate patterns, with evidence of multilayered vertical accumulation. These observations indicate that the period of 12–24 h represented the adhesion phase, while 24–48 h constituted the maturation phase of biofilm development.

3.3.3. Motility Assay Results of the Isolates

As shown in Figure 5, after 24 h of incubation, the average migration zone diameter for the isolated strain was 0.8 cm. This value was not significantly different from that of the non-motile Staphylococcus aureus ATCC 25922, indicating that strain LSKT01 exhibits weak or no motility.

3.4. Histopathological Examination

All mice in the blank control group survived without abnormal symptoms. In contrast, infected mice exhibited loss of appetite, hunched posture, ruffled fur, and significantly reduced activity. Their eyes were red, swollen, and difficult to open, with purulent discharge present. Defecation was impaired, characterized by dry, hardened feces adhering tightly to the perianal region. Necropsy revealed markedly pale livers in deceased mice, with pinpoint necrotic foci visible on the surface. The lungs appeared whitish, showing diffuse pallor on both the surface and cross-sections of the lobes. Among the submitted tissue samples (liver, spleen, lung, kidney, and intestine), the control group showed no significant pathological alterations. The majority of organs in the experimental group exhibited varying degrees of pathological changes, including edema, inflammatory cell infiltration, congestion, and fatty degeneration. Hepatic tissue showed extensive hepatic cell fatty degeneration (yellow arrows), with small round vacuoles appearing in the cytoplasm; significant vascular congestion (orange arrows) was observed, accompanied by leukocyte infiltration within the vessels (brown arrows), as depicted in Figure 6a,b. Spleen tissue demonstrated extensive connective tissue proliferation in the capsule, accompanied by abundant infiltration of lymphocytes, granulocytes, and macrophages (yellow arrows). The red pulp and marginal zones exhibited significant granulocyte infiltration (green arrows), with frequent extramedullary focal aggregates (orange arrows), as shown in Figure 6c–e. The lung tissue shows moderate granulocyte infiltration (orange arrows), occasional foamy cells within alveoli (green arrows), and focal alveolar hemorrhage (green polygons). Leukocytes and eosinophilic material are occasionally visible within bronchiolar lumens (brown arrows). The interstitium, including pulmonary connective tissue and vessels, shows marked congestion (yellow arrows), as seen in Figure 6f,g. Renal tissue demonstrates mild dilation of some tubules (green polygons), with eosinophilic material frequently present within tubular lumens (orange arrows), as seen in Figure 6h,i. In intestinal tissue, mild edema of mucosal epithelial cells (blue arrow) was observed with loosely stained cytoplasm. Separation between mucosal epithelium and lamina propria was frequently noted (orange arrow), while lymphocytic and granulocytic infiltration was present in the submucosa (green arrow), as shown in Figure 6g,k.

3.5. Results of Comparative Genomic Analysis

3.5.1. Phylogenetic Analysis Results

A core-genome-based phylogenetic tree revealed clustering patterns of different Corynebacterium species, as shown in Figure 7. The core genome corresponding to Corynebacterium hindlerae comprises 513 genes. The five C. hindlerae strains were divided into two distinct subgroups, corresponding to two separate biotypes. Strain LSKT01 clustered on the same branch as strain 1864, indicating the closest evolutionary relationship between them. Furthermore, the genomes of strains LSKT01, NML-93-0612, 1864, ISL_960a, and MAL_1082b formed a tightly clustered clade, demonstrating high conservation in their genomic architecture. Phylogenetic analysis suggested that Corynebacterium species cluster according to pathogenicity, with pathogenic and non-pathogenic strains occupying separate branches on the evolutionary tree [26]. Corynebacterium kalinowskii 1959 and Corynebacterium epidermidicanis DSM 45586 showed a closer relationship to C. hindlerae, residing within the same major clade and indicating pathogenic potential, whereas they were distantly related to the industrial strain Corynebacterium glutamicum ATCC 13032.

3.5.2. ANI Analysis Results

Average Nucleotide Identity (ANI) is a key metric in comparative genomics for assessing inter-genomic identity. The results of ANI analysis are presented in Figure 8, where color intensity represents the percentage identity of aligned regions between genome pairs. Tools for whole-genome species comparison and identification, such as ANI, DDH, and rpoB gene sequencing, have become essential standards for identifying Corynebacterium species, effectively distinguishing the genomes of most closely related species [27,28]. Typically, 16S rRNA gene sequence identity is below 98.7%, dDDH values are less than 70%, intraspecific ANI values exceed 95%, and interspecific ANI values remain below 83% [29]. Strain LSKT01 shared ANI values ranging from 95.80% to 98.70% with the other four C. hindlerae strains, all exceeding the 95% intraspecific threshold. The highest ANI was observed with strain 1864, while identity with MAL_1082b was relatively lower. These results are consistent with the branching pattern in the phylogenetic tree, reflecting genetic diversity within the species. ANI values among different biotypes of C. hindlerae strains ranged from 96.20% to 98.70%. In contrast, interspecific ANI values between strain LSKT01 and other Corynebacterium species were all below 80.00%.

3.5.3. Gene Family Analysis Results

A gene family is a group of genes sharing a common ancestor across multiple genomes. Nucleotide and amino acid sequences within the family exhibit significant similarity, typically retaining related molecular functions or domains. As shown in Table 2 and Figure 9, this serves as a basis for inferring the functions of unknown genes and provides clues about gene evolutionary history. The five Corynebacterium hindlerae strains exhibit minimal variation in the number of single-copy homologous genes, indicating highly conserved gene family structures and stable genomic architecture within this species. Most genes among the five strains are conserved, with fewer genes present in specific gene families. Strain LSKT01 possesses a total of 2478 genes, comprising 2380 genes across all gene families and 20 specific paralogs. Among the five Corynebacterium hindlerae strains, strain LSKT01 contained the highest number of polygenic homologs, species-specific paralogs, and unique gene families, reflecting its adaptive evolution to specific environmental conditions across different species. Among 8 species of 12 Corynebacterium strains, strain ATCC-13032 contained significantly higher numbers of multicopy homologous genes, specific paralogs, and unique gene families compared to other Corynebacterium species, indicating specific expansion of gene families in Corynebacterium glutamicum.

3.5.4. Common Genes and Unique Genes

To gain a more comprehensive understanding of the genetic composition of LSKT01 within the core-pan genome, protein sequences from multiple samples requiring analysis were clustered using the CD-HIT software. As shown in Figure 10a, among 8 species and 12 strains of Corynebacterium, the core genome identified 513 direct homologs, while the number of non-shared dispensable genes identified was 8824. Strain ATCC-13032 (Corynebacterium glutamicum) harbored the highest number of dispensable genes at 1334, while strain ISL-960a possessed the fewest at 28. Among the Corynebacterium species, Corynebacterium hindlerae contained the lowest number of dispensable genes, with strain LSKT01 possessing 156 dispensable genes. Based on dispensable gene distribution across samples, a heatmap of sample clustering was generated to visualize groupings. Analysis results (Figure 10b) show distinct clusters for different Corynebacterium species. Strain 1864 exhibits the closest phylogenetic relationship to LSKT01, followed by strains NML 93-0612, ISL_960a, and MAL_1082b. This result is consistent with the phylogenetic tree and ANI analysis findings.

3.5.5. Genome Synteny Analysis

Synteny analysis is a bioinformatics method used to determine the conserved order of gene sequences by comparing two or more genomes. By performing synteny analysis between two genomes, one can observe sequence insertions, deletions, and other structural changes, thereby gaining macro-scale insights into the evolutionary processes between strains and the overall stability of genome architecture. Synteny analysis was conducted on the genomes of five Corynebacterium hindlerae strains, as shown in Table 3 and Figure 11. Comparisons between strain LSKT01 and the other four C. hindlerae strains (NML-93-0612, 1864, ISL_960a, and MAL_1082b) revealed an average similarity >90%. Among all genome alignments, the highest degree of collinearity was observed between strains LSKT01 and MAL_1082b (Figure 11d), whereas the highest similarity was found between LSKT01 and 1864 (Figure 11b)—consistent with the phylogenetic tree and ANI results. The corresponding genomic regions between these two strains showed lower homology but were dominated by translocations, indicating significant structural similarity and an evolutionary relationship between their genomes.

3.5.6. SNP/InDel Statistics Results

SNP (Single-Nucleotide Polymorphism) primarily refers to DNA sequence polymorphism at the genomic level caused by single nucleotide variation, typically involving transitions or transversions. As genetic markers, SNPs exhibit characteristics such as high abundance, widespread distribution, and genetic stability, making them applicable in research fields including population genetics and individual identification. As shown in Table 4, using strain LSKT01 as the reference genome, the number of SNPs in four Corynebacterium hindlerae strains, from highest to lowest, is as follows: ISL_960a, MAL_1082b, NML 93-0612, and 1864. InDel (Insertion/Deletion) refers to the insertion or deletion of short nucleotide sequences at specific positions in the genome relative to the reference sequence within an individual or population. InDel results indicate that strain MAL_1082b exhibits the highest total InDel count and the highest CDS_InDel count.

3.5.7. SV Statistics Results

Genomic structural variation (SV) typically refers to DNA segment deletions, insertions, duplications, inversions, and translocations within the genome. Strain LSKT01 and MAL_1082b exhibited the highest number of deletion and insertion sites (Figure 12d), along with the most translocation and translocation-inversion regions. Strain LSKT01 and strain 1864 showed the fewest deletion and insertion sites (Figure 12b), fewer inversion and translocation regions, and predominantly translocation sites. The total number of SVs between LSKT01 and ISL_960a (Figure 12c) and between LSKT01 and MAL_1082b was significantly higher than that in the other two Corynebacterium hindlerae strains, reaching 231 and 225, respectively. This suggests that environmental differences across regions may have driven more complex genomic rearrangements in these strains.

4. Discussion

The isolated strain grew well under both aerobic and 5% CO_2_ conditions, and was capable of growth on both blood agar and Gauze’s Synthetic Medium No. 1. on Columbia blood agar supplemented with 5% sheep blood, it formed smooth, moist, circular, raised, white to cream-colored colonies. This result aligns with that reported by Bernard et al., who also observed black colony formation accompanied by a black halo on modified Tinsdale medium [30]. Microscopic examination revealed Gram-positive cells exhibiting a slender, irregular short rod or coccobacillary morphology. Environmental tolerance assays indicated that strong acids and strong bases significantly inhibited strain growth. The cell wall structure of Corynebacterium species plays a crucial role in resisting environmental stress. Beyond this structural barrier, our comparative genomic analysis revealed that the LSKT01 genome contains a series of genes associated with osmotic protection. This may be one of the key reasons why Corynebacterium hindlerae can still grow under varying salinity and pH conditions [31,32]. Biofilms are key virulence factors for bacterial pathogenicity, exhibiting drug resistance, phagocytosis resistance, and strong adhesion properties that enable them to withstand host immune defenses. The biofilm formation capacity of the isolated strains indicates their robust biofilm-forming ability. Most members of the Corynebacterium genus possess biofilm-forming capabilities, a trait associated with their persistent colonization within hosts or environmental settings [31]. It is noteworthy that for pathogenic species such as Corynebacterium diphtheriae and Corynebacterium pseudotuberculosis, biofilms are primarily considered key virulence determinants. The isolates exhibited weak or no motility, consistent with the characteristics of most Corynebacterium species, suggesting their dispersal likely relies more on host contact or external mechanical transmission [33].

Comparative genomic analysis elucidated the genetic diversity and evolutionary relationships of Corynebacterium hindlerae. Both core-genome phylogenetic reconstruction and ANI analysis consistently demonstrated that LSKT01 exhibits high genomic homology with other C. hindlerae strains, with ANI values exceeding 95% and highly conserved genomic architecture. Strain LSKT01 shows the closest phylogenetic relationship to the Australian human-derived strain 1864, suggesting it may possess broader cross-host transmission capability and a wider host range. The Kenyan bovine isolates ISL_960a and MAL_1082b exhibited more complex genomic structural variations in SNP/InDel and SV analyses, with significantly higher numbers of structural variants than other strains. This indicates that their genomic architecture has undergone more extensive rearrangement, likely to confer stronger environmental adaptability—a finding consistent with previous reports [2].

Gene family analysis revealed that Corynebacterium hindlerae possesses the fewest unique genes compared to other Corynebacterium strains, with strain LSKT01 harboring 156 unique genes. The LSKT01 genome exhibits overall conservation but shows evidence of gene family expansion and horizontal gene transfer. Horizontal gene transfer is a key mechanism for acquiring novel virulence factors in Corynebacterium species. The presence of 20 strain-specific paralogs in LSKT01 may indicate recent gene family expansion or horizontal acquisition, potentially linked to niche adaptation. For example, the Shiga-like toxin gene in Enterobacter ulcerans exhibits a significantly lower GC content compared to the genomic average, indicating that this gene was acquired through horizontal transfer [34,35]. Synteny analysis revealed numerous translocation and inversion regions between LSKT01 and other strains. While extensive homologous genes exist across genomes, significant structural recombination is present. Conserved regions indicate functional similarities among these strains.

Research indicates that multiple pathogenic species within the genus Corynebacterium (such as Corynebacterium ulcerans and Corynebacterium pseudotuberculosis) share a conserved virulence factor system, including phospholipase D, neuraminidase, and fimbriae structures [36]. Previous virulence gene analysis indicates that LSKT01 shares virulence factors associated with adhesion and iron uptake, which are prevalent among many Corynebacterium species and likely serve primarily to adapt to specific ecological niches. Meanwhile, multiple immune evasion genes—including capsular (rmlB), tyrosine phosphatase (ptpA), and catalase (katA)—demonstrate the strain’s capacity for persistent infection. Pal et al. [26] revealed through comparative genomics that core genes in pathogenic Corynebacterium species are more enriched in pathways related to metabolism and host–pathogen interactions, while LSKT01 exhibited the highest proportion of genes related to the cell periphery and biofilms in cellular composition. This indicates that the LSKT01 strain is extensively involved in cell membrane structure and transmembrane transport functions, demonstrating heightened activity in exchanging substances with the external environment and metabolizing intracellular materials. It can switch metabolic states when the microenvironment changes. These functional adaptations in LSKT01 may contribute to its pathogenicity and environmental persistence.

It should be noted that this study has certain limitations. The number of strains analyzed is relatively limited, and the biological characteristics are characterized based on only a single isolate. Therefore, whether the described genomic features and phenotypes—such as environmental tolerance and strong biofilm formation—are common among other C. hindlerae strains, especially those from different hosts or geographical origins, requires further validation with an expanded sample size in the future. The findings regarding the pathogenicity, cross-host transmission, and epidemiological significance of LSKT01 presented in this paper should be regarded as preliminary observations and hypotheses concerning this strain. Nevertheless, these preliminary results provide key reference information for subsequent research on this emerging pathogen.

5. Conclusions

This study successfully isolated and purified Corynebacterium hindlerae, which exhibited optimal growth at 37 °C, pH 5.5, and 1–2% salt concentration. The strain demonstrated strong biofilm-forming ability, with biofilms reaching maturity within 24–48 h and forming mature structures within 36 h. Motility was weak or absent. Comparative genomics analysis revealed high similarity between LSKT01 and four other Corynebacterium hindlerae strains (particularly strain 1864), with ANI values ranging from 95.80% to 98.70%. Significant genomic rearrangements were observed among the strains, accompanied by specific gene and gene family expansions within their genomes to adapt to specific environmental conditions. Conserved core genes and specific paralogs identified through genomic analysis can aid in developing rapid, accurate subunit vaccines or molecular diagnostic tools. Characterization of environmental tolerance provides a basis for formulating more effective disinfection protocols.