Modulation of virulence factors in biofilm-forming Klebsiella pneumoniae via selected South African medicinal plants and phytochemical compounds

Abstract

The emergence of multidrug-resistant (MDR) Klebsiella pneumoniae has become a growing public health concern across the globe, due to its significant role as a causative agent of severe nosocomial and community-acquired infections. Carbapenem-resistant (CBR) and extended spectrum beta lactamase (ESBL) producing K. pneumoniae strains, classified as MDR have been identified as the major causes of severe infections in humans. These strains often exhibit hypervirulent characteristics and pose a threat to treatment options due to their resistance to almost all classes of antibiotics, including the last line resorts. The identification of plant-based treatment options to target virulence factors of this pathogen is therefore a priority. Hence, this research was undertaken to explore the antibacterial and antivirulence properties, molecular modelling, virulence gene expression and safety use of bioactive South African medicinal plants and their metabolites against CBR and ESBL producing K. pneumoniae strains. Three South African medicinal plants namely Carpobrotus dimidiatus, Helichrysum populifolium and Lippia javanica were selected based on their therapeutic use for various K. pneumoniae associated infections. The plant extracts (ethyl acetate, dichloromethane, methanol, and water) were validated for their inhibitory activities against bacterial growth and virulence factors such as biofilm formation, exopolysaccharide (EPS) production, curli expression and hypermucoviscosity. The potent extract on K. pneumoniae biofilm was observed with a scanning electron microscope (SEM) while exopolysaccharide topography and surface parameters were observed using atomic force microscopy (AFM). Chemical profiling of the potent extract in-vitro was analysed using liquid chromatography-mass spectrometry (LC-MS). Virtual screening of selected compounds from the medicinal plants was carried out to interrupt the QS-associated SdiA transcriptional regulator protein in K. pneumoniae and attenuate its virulence. The crystal structure of SdiA, previously reported in E. coli as PDB ID: 4LFU was used as a template to model the structure of SdiA, serving as a prototype to search for compounds that could inhibit intercommunication and associated virulence activities. ProCheck, Verify3D, Ramachandran plot scores, and ProSA-Web all attested to the model’s good quality. Since SdiA protein in K. pneumoniae leads to the expression of virulence, 31 selected prospective bioactive compounds were docked against the SdiA modelled protein for antagonistic potential. The stability of the protein-ligand complex, atomic motions and inter-atomic interactions were further investigated through molecular dynamics simulations (MDS) at 100 ns production runs. The binding free energy was estimated using the molecular mechanics/poisson-boltzmann surface area (MM/PB-SA). The drug-likeness properties of the studied compounds were also validated. The promising phytochemical compounds (alpha-terpinene, camphene, fisetin, glycitein and phytol) were further evaluated in-vitro for their antibacterial and anti-biofilm associated virulence factors. Furthermore, the expression level of K. pneumoniae virulence genes after subjection to treatment with phytochemical compounds was determined using the Quantitative Real-Time PCR (qPCR). Cell viability and cytotoxicity activity of the studied compounds were assessed using African monkey kidney Vero cells. The antibacterial activity results revealed a noteworthy minimum inhibitory concentration (MIC) value for the C. dimidiatus dichloromethane extract at 0.78 mg/mL on CBR-K. pneumoniae. With regards to the virulence factors, L. javanica (ethyl acetate) showed the highest inhibition (67.25%) of the first biofilm stage-initial cell attachment for CBR-K. pneumoniae. Observations from the scanning electron microscope used for the in-situ visualization of the biofilms correlated the in-vitro findings, evidenced by a significant alteration of the biofilm architecture. Results of the EPS reduction assay, another virulence factor targeted in K. pneumoniae revealed the highest reduction of 34.18% for L. javanica (ethyl acetate), which was correlated by noticeable changes in the EPS surface topology as observed using atomic force microscope. Hypermucoviscosity, a phenotype which often characterizes hypervirulent K. pneumoniae was also reduced by L. javanica (ethyl acetate) to the least length mucoid string (1mm - 2mm) at 1mg/mL on both strains. Furthermore, curli expression often implicated with cell aggregation was inhibited by C. dimidiatus (aqueous) at 0.5 mg/mL in both K. pneumoniae strains. Chemical profiling of L. javanica (ethyl acetate), C. dimidiatus (aqueous) and H. populifolium (aqueous) identified diterpene (10.29%), hydroxy-dimethoxyflavone (10.24%) and 4,5-Dicaffeoylquinic acid (13.41%) respectively as dominant compounds. Molecular docking studies showed that phytol and glycitein possess the highest binding affinity of -9.205 kcal/mol and -9.752 kcal/mol for terpenes and flavonoids, respectively. The MDS of the protein in complex with the best-docked compounds revealed phytol with the highest binding energy of -44.2625 kcal/mol, a low root-mean-square deviation (RMSD) value of 1.54 Å and a root-mean-square fluctuation (RMSF) score of 1.78 Å. Analysis of the drug-likeness properties prediction and bioavailability of these compounds revealed their conformed activity to Lipinski’s rules with bioavailability scores of 0.55 F. In-vitro assessment of the compounds revealed fisetin as the best anti-CBR-K. pneumoniae, demonstrating MIC value of 0.0625 mg/mL. Phytol, glycitein and α-terpinene showed MIC values of 0.125 mg/mL for both strains. The assessment of the compounds for anti-virulence activity (exopolysaccharide reduction) revealed up to 65.91% reduction in phytol and camphene while phytol also showed the highest antiadhesion activity against CBR and ESBL- K. pneumoniae (54.71% and 50.05%), respectively. The expression levels of mrkA, rcsA and luxS genes in treated K. pneumoniae strains revealed fold expression ratio ranging between 0.607 (60.7%) and 1.00 (100%). MrkA gene targeted with phytol and rcsA gene targeted with camphene in CBR-K. pneumoniae strain revealed reduced fold expression values of 0.662 (66.2%) and 0.722 (72.2%), respectively compared to the untreated strain (16S rRNA as control) which revealed the value of 1.00 (100%). Phytol was shown to have no cytotoxic effect on the Vero cells whilst maintaining approximately 100% (p < 0.05) of the cell’s viability even at the highest tested concentration (0.25 mg/mL). The measured lactate dehydrogenase (LDH) activity showed that most of the tested compounds did not cause cell death in a dose-dependent manner. Overall, findings from this study revealed that the studied medicinal plants are not only rich sources of bioactive compounds but also possess antibacterial and antivirulence activities, with L. javanica (ethyl acetate) displaying the most remarkable activity. Secondary metabolites of the studied plants belonging to the terpene and flavonoids classes were also shown to reveal good binding affinities and high binding energies when bound to the transcriptional SdiA receptor which modulates biofilm formation and other virulence factors in K. pneumoniae. In-vitro antivirulence activities of the compounds revealed phytol to be the most potent antivirulence antibiofilm agent and was observed alongside camphene to downregulate the expression of mrkA and rcsA genes respectively. Phytol further revealed no cytotoxic effect on Vero cells while camphene revealed significant cell viability. The entire findings from this study presents the potent medicinal plants and compounds as promising leads for the development of novel drugs in the management of hypervirulent K. pneumoniae infections.