Extracts of Selected South African Medicinal Plants Mitigate Virulence Factors in Multidrug-Resistant Strains of Klebsiella pneumoniae.

The emergence of multidrug-resistant (MDR) Klebsiella pneumoniae remains a global health threat due to its alarming rates of becoming resistant to antibiotics. Therefore, identifying plant-based treatment options to target this pathogen's virulence factors is a priority. This study examined the antivirulence activities of twelve plant extracts obtained from three South African medicinal plants (Lippia javanica, Carpobrotus dimidiatus, and Helichrysum populifolium) against carbapenem-resistant (CBR) and extended-spectrum beta-lactamase (ESBL) positive K. pneumoniae strains. 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). Results revealed a noteworthy minimum inhibitory concentration (MIC) value for the C. dimidiatus dichloromethane extract at 0.78 mg/mL on CBR- K. pneumoniae. L. javanica (ethyl acetate) showed the highest cell attachment inhibition (67.25%) for CBR- K. pneumoniae. SEM correlated the in-vitro findings, evidenced by a significant alteration of the biofilm architecture. The highest EPS reduction of 34.18% was also noted for L. javanica (ethyl acetate) and correlated by noticeable changes observed using AFM. L. javanica (ethyl acetate) further reduced hypermucoviscosity to the least length mucoid string (1 mm-2 mm) at 1.00 mg/mL on both strains. C. dimidiatus (aqueous) showed biofilm inhibition of 45.91% for the ESBL-positive K. pneumoniae and inhibited curli expression at 0.50 mg/mL in both K. pneumoniae strains as observed for H. populifolium (aqueous) extract. 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. Overall, the ethyl acetate extract of L. javanica revealed potent antivirulence properties against the studied MDR K. pneumoniae strains. Hence, it is a promising medicinal plant that can be investigated further to develop alternative therapy for managing K. pneumoniae-associated infections.

Correction: Adeosun et al. Anti-Biofilm and Associated Anti-Virulence Activities of Selected Phytochemical Compounds against Klebsiella pneumoniae. Plants 2022, 11, 1429.

In the original publication [...].

Antioxidant and Antimicrobial Evaluations of Moringa oleifera Lam Leaves Extract and Isolated Compounds.

Moringa oleifera, native to India, grows in tropical and subtropical regions around the world and has valuable pharmacological properties such as anti-asthmatic, anti-diabetic, anti-inflammatory, anti-infertility, anti-cancer, anti-microbial, antioxidant, and many more. The purpose of this study was to assess the free radical scavenging ability of two extracts and two pure compounds of M. oleifera Lam (hexane, ethanol, compound E3, and compound Ra) against reactive oxygen species, as well as their reducing power and antimicrobial activities. Bioautography antioxidant assay, 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydrogen peroxide (H2O2) free radical scavenging, and iron (iii) (Fe3+ to Fe2+) chloride reducing power assays were used to assess the extracts' qualitative and quantitative free radical scavenging activities. Furthermore, the extract and the compounds were tested against both Gram-positive and Gram-negative bacterial strains suspended in Mueller-Hinton Broth. The extracts and pure compounds showed noteworthy antioxidant potential, with positive compound bands in the Rf range of 0.05-0.89. DPPH), H2O2, and Fe3+ to Fe2+ reduction assays revealed that ethanol extract has a high antioxidant potential, followed by compound E3, compound Ra, and finally hexane extract. Using regression analysis, the half maximal inhibitory concentration (IC50) values for test and control samples were calculated. Compound Ra and ethanol exhibited high antioxidant activity at concentrations as low as ≈0.28 mg/mL in comparison with n-hexane extract, compound E3, ascorbic acid, and butylated hydroxytoluene standards. The radical scavenging activity of almost all M. oleifera plant extracts against DPPH was observed at 0.28 mg/mL; however, the highest activity was observed at the same concentration for ascorbic acid and butylated hydroxytoluene (BHT) with a low IC50 value of 0.08 mg/mL and compound Ra and ethanol with a low IC50 of 0.4 mg/mL, respectively. The extracts and pure compounds of M. oleifera have little to no antibacterial potential. M. oleifera extracts contain antioxidant agents efficient to alleviate degenerative conditions such as cancer and cardiovascular disease but have little activity against infectious diseases.

Molecular modelling of SdiA protein by selected flavonoid and terpenes compounds to attenuate virulence in Klebsiella pneumoniae.

Klebsiella pneumoniae is one of the perturbing multidrug resistant (MDR) and ESKAPE pathogens contributing to the mounting morbidity, mortality and extended rate of hospitalization. Its virulence, often regulated by quorum sensing (QS) reinforces the need to explore alternative and prospective antivirulence agents, relatively from plants secondary metabolites. Computer aided drug discovery using molecular modelling techniques offers advantage to investigate prospective drugs to combat MDR pathogens. Thus, this study employed virtual screening of selected terpenes and flavonoids from medicinal plants to interrupt the QS associated SdiA protein in K. pneumoniae to attenuate its virulence. 4LFU was used as a template to model the structure of SdiA. 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 prospective bioactive compounds were docked 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). Furthermore, the drug-likeness properties of the studied compounds were validated. Docking studies showed phytol possesses the highest binding affinity (-9.205 kcal/mol) while glycitein had -9.752 kcal/mol highest docking score. 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 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. The studied terpenes and flavonoids compounds effectively thwart SdiA protein, therefore regulate inter- or intra cellular communication and associated in virulence Enterobacteriaceae, serving as prospective antivirulence drugs.Communicated by Ramaswamy H. Sarma.

Anti-Biofilm and Associated Anti-Virulence Activities of Selected Phytochemical Compounds against Klebsiella pneumoniae.

The ability of Klebsiella pneumoniae to form biofilm renders the pathogen recalcitrant to various antibiotics. The difficulty in managing K. pneumoniae related chronic infections is due to its biofilm-forming ability and associated virulence factors, necessitating the development of efficient strategies to control virulence factors. This study aimed at evaluating the inhibitory potential of selected phytochemical compounds on biofilm-associated virulence factors in K. pneumoniae, as well as authenticating their antibiofilm activity. Five phytochemical compounds (alpha-terpinene, camphene, fisetin, glycitein and phytol) were evaluated for their antibacterial and anti-biofilm-associated virulence factors such as exopolysaccharides, curli fibers, and hypermucoviscosity against carbapenem-resistant and extended-spectrum beta-lactamase-positive K. pneumoniae strains. The antibiofilm potential of these compounds was evaluated at initial cell attachment, microcolony formation and mature biofilm formation, then validated by in situ visualization using scanning electron microscopy (SEM). Exopolysaccharide surface topography was characterized using atomic force microscopy (AFM). The antibacterial activity of the compounds confirmed fisetin as the best anti-carbapenem-resistant K. pneumoniae, demonstrating a minimum inhibitory concentration (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 an up to 65.91% reduction in phytol and camphene. Atomic force microscopy detected marked differences between the topographies of untreated and treated (camphene and phytol) exopolysaccharides. Curli expression was inhibited at both 0.5 and 1.0 mg/mL by phytol, glycitein, fisetin and quercetin. The hypermucoviscosity was reduced by phytol, glycitein, and fisetin to the shortest mucoid string (1 mm) at 1 mg/mL. Phytol showed the highest antiadhesion activity against carbapenem-resistant and extended-spectrum beta-lactamase-positive K. pneumoniae (54.71% and 50.05%), respectively. Scanning electron microscopy correlated the in vitro findings, with phytol significantly altering the biofilm architecture. Phytol has antibiofilm and antivirulence potential against the highly virulent K. pneumoniae strains, revealing it as a potential lead compound for the management of K. pneumoniae-associated infections.

Quorum sensing modulation and inhibition in biofilm forming foot ulcer pathogens by selected medicinal plants.

The crisis of antibiotic resistance necessitates the search of phytochemicals as potential antibacterial, anti-quorum sensing and antibiofilm forming agents. For the present study, fifteen (15) selected medicinal plants were evaluated to inhibit the biological activities of multi-drug resistant (MDR) pathogenic bacteria (Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis) associated with diabetic foot ulcer. Antibacterial activities revealed noteworthy minimum inhibitory concentration (MIC) values ≤1 mg/mL for thirteen (13) out of the sixty (60) plant extracts screened. The potent extracts included Euclea natalensis ethyl acetate (0.25 mg/mL), Aloe ferox methanol (0.5 mg/ml) and Warburgia salutaris aqueous (0.5 mg/mL) extracts. Chemical profiling of the active extracts using gas chromatography-mass spectrometry (GC-MS) identified neophytadiene, guanosine, squalene, cis megastigma-5,8-diene-4-one and sorbitol as prevalent compounds among the active extracts. Anti-quorum sensing activities of E. natalensis (ethyl acetate), A. ferox (methanol) and W. salutaris (aqueous) extracts ranged from 4.81 - 58.34% with E. natalensis (ethyl-acetate) showing the highest activity. Molecular docking against CviR protein showed selected compounds having high docking scores with sorbitol showing the highest score of -7.04 kcal/mol. Warburgia salutaris aqueous extract exhibited the highest biofilm inhibition (73%) against E. coli. Euclea natalensis, Aloe ferox and Warburgia salutaris compounds act as antagonist of N-acyl homoserine lactone (AHL) signaling, thus may serve as candidates in antipathogenic and antibiofilm phytomedicine development for MDR foot ulcer bacterial pathogens.

An Andrographolide from Helichrysum caespitium (DC.) Sond. Ex Harv., (Asteraceae) and Its Antimicrobial, Antiquorum Sensing, and Antibiofilm Potentials.

Helichrysum caespititium (DC.) Sond. Ex Harv., (Asteraceae) is a medicinal plant indigenous to South Africa. Its non-polar extracts exhibit significant antimicrobial and, in particular, antigonorrheal activity. This study aimed at isolating and purifying the active antigonorrheal compound from its chloroform extract and validating its inhibition potential on quorum sensing (QS) and biofilm formation of multi-drug resistant (MDR) pathogens. Phytochemical investigation of aerial parts of H. caespititium afforded a diterpene lactone (CF6). The effect of CF6 on violacein production and biofilm formation was studied using in vitro quantitative violacein inhibition (Chromobacterium violaceum) and biofilm formation (Streptococcus pyogenes, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Neisseria gonorrhoeae, and Pseudomonas aeruginosa). The structure of CF6 was characterized using FTIR, NMR, and UPLC-MS data accordingly, as 10-methyl-8-(propan-17-ylidene)naphthalen-9-yl)-11-vinyl-14-hydroxyfuran-16-one. The susceptibility testing of the pathogens against CF6 revealed Neisseria gonorrhoeae was noticeably susceptible with a MIC value of 60 µg/mL, while Streptococcus pyogenes and Staphylococcus aureus showed MIC of 125 µg/mL. All gram-negative pathogens, Escherichia coli, Klebsiella pneumonia and Pseudomonas aeruginosa were inhibited at 250 µg/mL. CF6 also inhibited the production of violacein by 51.88% at 250 µg/mL and prevented cell attachment by 40.76-81.18%, with N. gonorrhoeae being highly prohibited from forming biofilm. In conclusion, 10-methyl-8-(propan-17-ylidene)naphthalen-9-yl)-11-vinyl-14-hydroxyfuran-16-one is the first of its kind to be isolated from the non-polar (chloroform) extract of South African Helichrysum caespititium with antigonorrheal, antimicrobial, antiquorum sensing, and antibiofilm properties. The compound may serve as a drug candidate against MDR pathogens.

In Silico and In Vitro Screening of Antipathogenic Properties of Melianthus comosus (Vahl) against Pseudomonas aeruginosa.

Bacterial quorum sensing (QS) system regulates pathogenesis, virulence, and biofilm formation, and together they contribute to nosocomial infections. Opportunistic pathogens, such as Pseudomonas aeruginosa, rely on QS for regulating virulence factors. Therefore, blocking the QS system may aid management of various infectious diseases caused by human pathogens. Plant secondary metabolites can thwart bacterial colonization and virulence. As such, this study was undertaken to evaluate three extracts from the medicinal plant, Melianthus comosus, from which phytochemical compounds were identified with potential to inhibit QS-dependent virulence factors in P. aeruginosa. Chemical profiling of the three extracts identified 1,2-benzene dicarboxylic acid, diethyl ester, neophytadiene and hexadecanoic acid as the common compounds. Validation of antibacterial activity confirmed the same MIC values of 0.78 mg/mL for aqueous, methanol and dichloromethane extracts while selected guanosine showed MIC 0.031 mg/mL. Molecular docking analysis showed anti-quorum sensing (AQS) potential of guanosine binding to CviR' and 2UV0 proteins with varying docking scores of -5.969 and -8.376 kcal/mol, respectively. Guanosine inhibited biofilm cell attachment and biofilm development at 78.88% and 34.85%, respectively. Significant swimming and swarming motility restriction of P. aeruginosa were observed at the highest concentration of plant extracts and guanosine. Overall, guanosine revealed the best swarming motility restrictions. M. comosus extracts and guanosine have shown clear antibacterial effects and subsequent reduction of QS-dependent virulence activities against P.aeruginosa. Therefore, they could be ideal candidates in the search for antipathogenic drugs to combat P.aeruginosa infections.

Calpurnia aurea (Aiton) Benth Extracts Reduce Quorum Sensing Controlled Virulence Factors in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is the causative agent of several life-threatening human infections. Like many other pathogens, P. aeruginosa exhibits quorum sensing (QS) controlled virulence factors such as biofilm during disease progression, complicating treatment with conventional antibiotics. Thus, impeding the pathogen's QS circuit appears as a promising alternative strategy to overcome pseudomonas infections. In the present study, Calpurnia aurea were evaluated for their antibacterial (minimum inhibitory concentrations (MIC)), anti-quorum sensing/antivirulence (AQS), and antibiofilm potential against P. aeruginosa. AQS and antivirulence (biofilm formation, swimming, and swarming motility) activities of plant extracts were evaluated against Chromobacterium violaceum and P. aeruginosa, respectively. The in vitro AQS potential of the individual compounds were validated using in silico molecular docking. Acetone and ethanolic extracts of C. aurea showed MIC at 1.56 mg/mL. The quantitative violacein inhibition (AQS) assay showed ethyl acetate extracts as the most potent at a concentration of 1 mg/mL. GCMS analysis of C. aurea revealed 17 compounds; four (pentadecanol, dimethyl terephthalate, terephthalic acid, and methyl mannose) showed potential AQS through molecular docking against the CviR protein of C. violaceum. Biofilm of P. aeruginosa was significantly inhibited by ≥60% using 1-mg/mL extract of C. aurea. Confocal laser scanning microscopy correlated the findings of crystal violet assay with the extracts significantly altering the swimming motility. C. aurea extracts reduced the virulence of pseudomonas, albeit in a strain- and extract-specific manner, showing their suitability for the identification of lead compounds with QS inhibitory potential for the control of P. aeruginosa infections.

Exploring Common Culinary Herbs and Spices as Potential Anti-Quorum Sensing Agents.

Quorum sensing controls bacterial pathogenesis and virulence; hence, interrupting this system renders pathogenic bacteria non-virulent, and presents a novel treatment for various bacterial infections. In the search for novel anti-quorum sensing (AQS) compounds, 14 common culinary herbs and spices were screened for potential antipathogenicity activity against Chromobacterium violaceum ATCC 12472. Extracts of Glycyrrhiza glabra (liquorice), Apium graveolens (celery), Capsicum annuum (cayenne pepper) and Syzygium anisatum (aniseed) demonstrated good AQS potential, yielding opaque halo zones ranging from 12⁻19 mm diameter at sub-minimum inhibitory concentrations (0.350⁻4.00 mg/mL). For the same species, the percentage reduction in violacein production ranged from 56.4 to 97.3%. Zones with violacein inhibitory effects were evident in a celery extract analysed using high performance thin layer chromatography-bio-autography. The major active compound was isolated from celery using preparative-high performance liquid chromatography-mass spectrometry and identified using gas chromatography-mass spectrometry (GC-MS) as 3-n-butyl-4,5-dihydrophthalide (sedanenolide). Potent opaque zones of inhibition observed on the HPTLC-bio-autography plate seeded with C. violaceum confirmed that sedanenolide was probably largely responsible for the AQS activity of celery. The bacteriocidal properties of many herbs and spices are reported. This study, however, was focussed on AQS activity, and may serve as initial scientific validation for the anti-infective properties ascribed to several culinary herbs and spices.

Characterization and flocculation efficiency of a bioflocculant produced by a marine Halobacillus.

We reported earlier on the bioflocculant production potential of Halobacillus sp. Mvuyo, a marine bacteria isolated from Algoa Bay sediment samples. In this paper we report on the detailed characterization of the purified bioflocculant composed of polysaccharide and protein. The optimum dose of the purified bioflocculant for the clarification of 4 g l(-1) kaolin clay suspension was 0.2 mg ml(-1) at neutral pH. Scanning electron micrograph (SEM) revealed the bioflocculant to have an amorphous structure. The Fourier transform infrared (FTIR) spectrum exhibited the presence of hydroxyl, carboxyl and amino groups in its structure. The bioflocculant was thermostable with relative bioflocculant activity residue of 74.4% after heat treatment at 100 degrees C. Moreover thermogravimetric analysis (TGA) exhibited a degradation temperature (Td) of - 140 degrees C. The flocculation efficiency of the bioflocculant was 86.2% compared with 82.6%, 74.5% and 70.9% for polyethylimine, ferric chloride and alum, respectively. This bioflocculant has immense promise as a substitute to inorganic and synthetic flocculants in view of their hazard implications.

Thermostable bacterial bioflocculant produced by Cobetia spp. isolated from Algoa Bay (South Africa).

A novel bioflocculant-producing bacteria was isolated from sediment samples of Algoa Bay in the Eastern Cape Province of South Africa and the effect of culture conditions on the bioflocculant production was investigated. Analysis of the partial nucleotide sequence of the 16S rDNA of the bacteria revealed 99% similarity to Cobetia sp. L222 and the sequence was deposited in GenBank as Cobetia sp. OAUIFE (accession number JF799092). Cultivation condition studies revealed that bioflocculant production was optimal with an inoculum size of 2% (v/v), initial pH of 6.0, Mn(2+) as the metal ion, and glucose as the carbon source. Metal ions, including Na(+), K(+), Li(+), Ca(2+)and Mg(2+) stimulated bioflocculant production, resulting in flocculating activity of above 90%. This crude bioflocculant is thermally stable, with about 78% of its flocculating activity remaining after heating at 100 °C for 25 min. Analysis of the purified bioflocculant revealed it to be an acidic extracellular polysaccharide.

Production and characterization of bioflocculant produced by Halobacillus sp. Mvuyo isolated from bottom sediment of Algoa Bay.

A bioflocculant-producing bacteria isolated from marine sediment of Algoa Bay was assessed for its bioflocculant-producing potentials. Based on 16S recombinant deoxyribonucleic acid (rDNA) sequence analysis, the isolate was identified as Halobacillus sp. and deposited in the Genbank as Halobacillus sp. Mvuyo with accession number HQ537125. The bacteria produced bioflocculant optimally in the presence of glucose (76% flocculating activity) and ammonium chloride (93% flocculating activity) as sole sources of carbon and nitrogen, respectively. The flocculating capabilities of the flocculant were increased by the addition of Ca2+ (76% flocculating activity) and the highest flocculating activity was observed at neutral pH (7.0). The chemical analysis of the bioflocculant revealed that it contained mainly polysaccharide and protein.

Studies on bioflocculant production by Arthrobacter sp. Raats, a freshwater bacteria isolated from Tyume River, South Africa.

A bioflocculant-producing bacteria was isolated from Tyume River in the Eastern Cape Province, South Africa and identified by 16S rRNA gene nucleotide sequence to have 91% similarity to Arthrobacter sp. 5J12A, and the nucleotide sequence was deposited in GenBank as Arthrobacter sp. Raats (accession number HQ875723). The bacteria produced an extracellular bioflocculant when grown aerobically in a production medium containing glucose as sole carbon source and had an initial pH of 7.0. Influences of carbon, nitrogen and metal ions sources, as well as initial pH on flocculating activity were investigated. The bacteria optimally produced the bioflocullant when lactose and urea were used as sole sources of carbon and nitrogen respectively with flocculating activities of 75.4% and 83.4% respectively. Also, the bacteria produced the bioflocculant optimally when initial pH of the medium was 7.0 (flocculating activity 84%), and when Mg(2+) was used as cation (flocculating activity 77%). Composition analyses indicated the bioflocculant to be principally a glycoprotein made up of about 56% protein and 25% total carbohydrate.

Halomonas sp. OKOH--a marine bacterium isolated from the bottom sediment of Algoa Bay--produces a polysaccharide bioflocculant: partial characterization and biochemical analysis of its properties.

A bioflocculant-producing bacterium isolated from seawater was identified based on 16S rRNA gene nucleotide sequence to have 99% similarity to that of Halomonas sp. Au160H and the nucleotide sequence was deposited as Halomonas sp. OKOH (Genbank accession number is HQ875722). Influences of carbon source, nitrogen source, salt ions and pH on flocculating activity were investigated. The bioflocculant was optimally produced when glucose (87% flocculating activity) and urea (88% flocculating activity) were used as sources of carbon and nitrogen, respectively. Also, initial pH of 7.0 and Ca²âº supported optimal production of the bioflocculant with flocculating activities of 87% respectively. Chemical analyses revealed the bioflocculant to be a polysaccharide.

Bioflocculant production by Virgibacillus sp. Rob isolated from the bottom sediment of Algoa Bay in the Eastern Cape, South Africa.

A bioflocculant-producing marine bacterium previously isolated from marine sediment of Algoa Bay was screened for flocculant production. Comparative analysis of 16S rDNA sequence identified the isolate to have 99% similarity to Virgibacillus sp. XQ-1 and it was deposited in the GenBank as Virgibacillus sp. Rob with accession number HQ537127. The bacterium produced biflocculants optimally in glucose (70.4%) and peptone (70.4%) as sole sources of carbon and nitrogen, alkaline pH (12) (74%); and the presence of Fe2+ (74%). Chemical analysis of the bioflocculant revealed it to be a polysaccharide.