Characterization and Pathogenicity of Mannheimia glucosida Isolated from Sheep

Abstract

Bacteria of the genus Mannheimia are major pathogens of respiratory diseases in ruminants and pose a significant threat to the global ruminant industry. However, the biological characteristics and pathogenic mechanisms of Mannheimia glucosida remain unclear. In this study, we isolated five strains of M. glucosida, which specifically hydrolyzed esculin, from sheep with respiratory disease in China. All five strains of M. glucosida were found to encode the adhesion-related gene adh and the anti-phagocytosis-related gene plpD, as determined by a virulence gene assay. Moreover, all M. glucosida isolates were resistant to streptomycin. Phylogenetic analysis based on 16S rRNA, infB, and sodA genes showed that the sodA gene could be a valuable indication for the analysis of bacterial genetic evolution in the genus Mannheimia. By mouse modeling, M. glucosida D251 was further found to cause multiorgan damage with an LD₅₀ of 1.35 × 10⁶ CFU. Meanwhile, by combining whole genome sequencing with bioinformatic analysis, we found that the D251 genome encodes a large number of virulence and drug resistance genes. Finally, we established a highly sensitive and specific PCR assay for M. glucosida. Collectively, these results indicate that M. glucosida may be an important pathogen in respiratory disease in sheep in China and provides a theoretical basis for the clinical diagnosis and treatment of this disease.

Keywords: Mannheimia glucosida; biochemical characterization; drug resistance; respiratory diseases; virulence.

Figure 1

Isolation and Identification of M. glucosida. (A) Growth status of M. glucosida isolates on blood agar plates. Amplification results of five strains of M. glucosida using (B) LKT, (C) LKT2, and (D) HP primers. M, Marker; 1–5, D251, G2, G3, G4, G5; N, Negative control. (E) Esculin hydrolysis assay.

Figure 2

Pathogenicity assays of M. glucosida in mice. (A) Results of the virulence gene test in M. glucosida. (B) HE staining observation of each organ tissue. The green arrow in the liver indicates hepatocellular steatosis. The yellow arrow in the spleen indicates splenic sinus stasis. Inflammatory cell infiltration is indicated by the blue arrow in the lung. Glomerular necrosis is indicated by the green arrow in the kidney. Scale bar, 50 μm. (C) Detection of M. glucosida in different tissue organs using LKT, LKT2, and HP primers. 1: heart; 2: liver; 3: spleen; 4: lung; 5: kidney; 6: brain; 7: rectum; 8: twelve Finger intestine; N: control.

Figure 3

Phylogenetic analysis of (A) lktA, (B) 16S rRNA, (C) infB, and (D) sodA genes in M. glucosida. Strain D251 is indicated by G1 in the figure.

Figure 4

Whole genome sequencing analysis of M. glucosida D251. (A) Genome map of strain D251. The outermost circle of the map identifies the genome size; the second and third circles show the CDS on the positive and negative strands, with different colors indicating the functional classification of the different COGs of the CDSs; the fourth circle shows the rRNAs and tRNAs; and the fifth circle shows the GC content. (B) Statistic of genomic protein COG function. (C) KEGG pathway annotation. A, Metabolism; B, Genetic Information Processing; C, Environmental Information Processing; D, Cellular Processes; E, Organismal Systems.

Figure 5

Establishment of a specific PCR detection method for M. glucosida. (A) Detection results of five isolates. 1–5: D251, G2, G3, G4, G5. N: control. (B) Optimization of PCR annealing temperature. 1–6: 60 °C, 59 °C, 57 °C, 55 °C, 53 °C, 51 °C. N: control. (C) PCR specificity analysis. 1: E. coli; 2: M. haemolytica; 3: M. ruminalis; 4: P. multocida; 5: M. ovipneumoniae; 6: M. glucosida; N: control. (D) Sensitivity analysis of genomic DNA for PCR detection. 1–7: 7.1 × 10¹~7.1 × 10⁻⁵ ng/μL. (E) Sensitivity analysis of CFU for PCR detection. 1–8: 5.6 × 10⁷~5.6 × 10⁰ CFU/mL. (F) PCR detection of M. glucosida in clinical samples. 1: control. 2–9: clinical samples.