Analysis of an IncR plasmid carried by carbapenem-resistant Klebsiella pneumoniae: A survey of swine Klebsiella pneumoniae in Jilin Province.

OBJECTIVES: This study was conducted in Jilin Province to investigate the mechanism involved in the antibiotic resistance and pathogenicity of Klebsiella pneumoniae. METHODS: Lung samples were collected from large-scale pig farms in Jilin Province. Antimicrobial susceptibility and mouse lethality assays were carried out. K. pneumoniae isolate JP20, with high virulence and antibiotic resistance, was chosen for whole-genome sequencing. The complete sequence of its genome was annotated, and the virulence and antibiotic resistance mechanism were analysed. RESULTS: A total of 32 K. pneumoniae strains were isolated and tested for antibiotic resistance and pathogenicity. Among them, the JP20 strain showed high levels of resistance to all tested antimicrobial agents and strong pathogenicity in mice (lethal dose of 1.35 × 1011 CFU/mL). Sequencing of the multidrug-resistant and highly virulent K. pneumoniae JP20 strain revealed that the antibiotic resistance genes were mainly carried by an IncR plasmid. We speculate that extended-spectrum ß-lactamases and loss of outer membrane porin OmpK36 play an important role in carbapenem antibiotic resistance. This plasmid contains a mosaic structure consisting of a large number of mobile elements. CONCLUSION: Through genome-wide analysis, we found that an lncR plasmid carried by the JP20 strain may have evolved in pig farms, possibly leading to multidrug resistance in the JP20 strain. It is speculated that the antibiotic resistance of K. pneumoniae in pig farms is mainly mediated by mobile elements (insertion sequences, transposons, and plasmids). These data provide a basis for monitoring the antibiotic resistance of K. pneumoniae and lay a foundation for an improved understanding of the genomic characteristics and antibiotic resistance mechanism of K. pneumoniae.

Impact of Sub-MIC Eugenol on Klebsiella pneumoniae Biofilm Formation via Upregulation of rcsB.

The Rcs phosphorelay system is present in many members of the Enterobacteriaceae. The aim of this study was to illustrate the possible mechanisms of eugenol on ultimate targets of Klebsiella pneumoniae (K. pneumoniae) Rcs phosphorelay, rcsB, and impact on biofilm formation. The minimum inhibitory concentration (MIC) of eugenol against K. pneumoniae KP1 and KP1 ΔrcsB strain was determined using the 2-fold micro-dilution method. Biofilm was measured by crystal violet staining. Transcriptome sequencing was performed to investigate sub-MIC eugenol on K. pneumoniae, and gene expression at mRNA level was analyzed by RT-qPCR. In vitro biofilm formation test and molecular docking were used to evaluate the effect of eugenol and to predict potential interactions with RcsB. MicroScale Thermophoresis (MST) was conducted for further validation. MIC of eugenol against K. pneumoniae KP1 and KP1 ΔrcsB strain was both 200 µg/ml. Transcriptome sequencing and RT-qPCR results indicated that rpmg, degP, rnpA, and dapD were downregulated, while rcsB, rcsD, rcsA, yiaG, and yiaD were upregulated in the eugenol-treated group. ΔrcsB exhibited a weakened biofilm formation capacity. Additional isopropyl-ß-d-thiogalactoside (IPTG) hinders biofilm formation, while sub-MIC eugenol could promote biofilm formation greatly. Docking analysis revealed that eugenol forms more hydrophobic bonds than hydrogen bonds. MST assay also showed a weak binding affinity between eugenol and RcsB. These results provide significant evidence that rcsB plays a key role in K. pneumoniae biofilm formation. Sub-MIC eugenol facilitates biofilm formation to a large extent instead of inhibiting it. Our findings reveal the potential risk of natural anti-biofilm ingredients at sub-MIC to treat drug-resistance bacteria.

Antibacterial activity and cytotoxicity of a novel bacteriocin isolated from Pseudomonas sp. strain 166.

Pseudomonas sp. strain 166 was isolated from soil samples from Changbai Mountains. A novel bacteriocin PA166 from Pseudomonas sp. 166 was purified using ammonium sulfate, dextran gel chromatography column and Q-Sepharose column chromatography successively. The molecular mass of bacteriocin PA166 was found to be 49.38 kDa by SDS-PAGE and liquid chromatography-mass spectrometry (MS)/MS. Bacteriocin PA166 showed stability at a wide range of pH (2-10), and thermal stability (40, 60, 80 and 100°C). The bacteriocin PA166 antimicrobial activity was slightly inhibited by Ca2+ , K+ and Mg2+ . The minimum bactericidal concentrations of bacteriocin PA166 against five Pasteurella multocida strains ranged from 2 to 8 µg ml-1 . Bacteriocin PA166 showed low cytotoxicity and a higher treatment index (TI = 82.51). Fluorescence spectroscopy indicated that bacteriocin PA166 destroyed the cell membrane to exert antimicrobial activity. In summary, bacteriocin PA166 had strong antibacterial activity, high TI and low toxicity, and hence could serve as a potential clinical therapeutic drug.

Genomic and Transcriptomic Analysis of Bovine Pasteurella multocida Serogroup A Strain Reveals Insights Into Virulence Attenuation.

Pasteurella multocida is one of the primary pathogens of bovine respiratory disease (BRD), and causes huge losses in the cattle industry. The Pm3 strain was a natural isolate, which is a strong form of pathogen and is sensitive to fluoroquinolones antibiotics. A high fluoroquinolone resistant strain, Pm64 (MIC = 64 µg/mL), was formed after continuous induction with subinhibitory concentration (1/2 MIC) of enrofloxacin, with the enhanced growth characteristics and large attenuation of pathogenicity in mice. This study reports the whole genome sequence and the transcription profile by RNA-Seq of strain Pm3/Pm64. The results showed an ineffective difference between the two strains at the genome level. However, 32 genes could be recognized in the gene islands (GIs) of Pm64, in which 24 genes were added and 8 genes were lost. Those genes are involved in DNA binding, trehalose metabolism, material transportation, capsule synthesis, prophage, amino acid metabolism, and other functions. In Pm3 strain, 558 up-regulated and 568 down-regulated genes were found compared to Pm64 strain, from which 20 virulence factor-related differentially expressed genes (DEGs) were screened. Mainly differentially transcribed genes were associated with capsular polysaccharide (CPS), lipopolysaccharide (LPS), lipooligosaccharide (LOS). Iron utilization, and biofilm composition. We speculated that the main mechanism of virulence attenuation after the formation of resistance of Pm64 comes from the change of the expression profile of these genes. This report elucidated the toxicity targets of P. multocida serogroup A which provide fundamental information toward the understanding of the pathogenic mechanism and to decreasing antimicrobial drugs resistance.