Plant-derived Antimicrobial Agents Research Articles

IN- VITRO ANTIBACTERIAL ACTIVITY OF SPILANTHES ACMELLA (AKARKARA) EXTRACT ON PORPHYROMONAS GINGIVALIS AND AGGREGATIBACTOR ACTINOMYCETEMCOMITANS

Periodontal diseases are caused by certain bacteria found in the bacterial plaque. Usage of plant-derived antimicrobial agents could serve as an effective alternative treatment against periodontal infections due to continuous rise seen in antibiotic resistance. Spilanthes acmella (S. acmella), a vital medicinal plant has been used for its various properties such as anti- inflammatory, antibacterial, antifungal, antinociceptive, anti-cancerous and hastening wound healing. This study was conducted to assess its antibacterial efficacy against common periodontal pathogens.Objective: The present study was conducted to assess the antibacterial activity of S. acmella plant extract against Porphyromonas gingivalis (Pg), and Aggregatibactor actinomycetemcomitans (Aa) and determine the presence of various phytochemicals in it.Materials and Methodology: An extract was prepared using dried S. acmella plant powder and mixed with methanol in 1:1 (w/v) ratio. Determination of minimal inhibitory concentration (MIC) was done by using tube dilution technique and time- kill assay was performed against Pg and Aa. Presence of phytochemicals was checked by thin layer chromatography (TLC) method.Results: MIC of S. acmella was found to be 40 μg/ml for Pg and 20 μg/ml for Aa within 2 h interval. Various phytochemicals were found in S. acmella extract which may be responsible for its anti- bacterial property.Conclusion: S. acmella extract shows a significant antibacterial effect against the major periodontal pathogens and hence may be a potential natural alternative for controlling the growth of these bacteria.

Citral and trans-cinnamaldehyde, two plant-derived antimicrobial agents can induce Staphylococcus aureus into VBNC state with different characteristics.

Viable but nonculturable (VBNC) state bacteria are difficult to detect in the food industry due to their nonculturable nature and their recovery characteristics pose a potential threat to human health. The results of this study indicated that S. aureus was found to enter the VBNC state completely after induced by citral (1 and 2mg/mL) for 2h, and after induced by trans-cinnamaldehyde (0.5 and 1mg/mL) for 1h and 3h, respectively. Except for VBNC state cells induced by 2mg/mL citral, the VBNC state cells induced by the other three conditions (1mg/mL citral, 0.5 and 1mg/mL trans-cinnamaldehyde) were able to be resuscitated in TSB media. In the VBNC state cells induced by citral and trans-cinnamaldehyde, the ATP concentration was reduced, the hemolysin-producing ability was significantly decreased, but the intracellular ROS level was elevated. The results of heat and simulated gastric fluid experiments showed different environment resistance on VBNC state cells induced by citral and trans-cinnamaldehyde. In addition, by observing the VBNC state cells showed that irregular folds on the surface, increased electron density inside and vacuoles in the nuclear region. What's more, S. aureus was found to enter the VBNC state completely after induced by meat-based broth containing citral (1 and 2mg/mL) for 7h and 5h, after induced by meat-based broth containing trans-cinnamaldehyde (0.5 and 1mg/mL) for 8h and 7h. In summary, citral and trans-cinnamaldehyde can induce S. aureus into VBNC state and food industry needs to comprehensively evaluate the antibacterial capacity of these two plant-derived antimicrobial agents.

TMT-Based Quantitative Proteomics Analyses Reveal the Antibacterial Mechanisms of Anthocyanins from Aronia melanocarpa against Escherichia coli O157:H7.

Aronia melanocarpa anthocyanins (AMAs), as natural plant extracts, can control pathogens and are attracting increasing attention. In this study, a tandem mass tag (TMT) quantitative proteomics method combined with multiple reaction monitoring (MRM) was used to explore the antibacterial mechanism of AMAs against Escherichia coli at the protein level. The results showed that 1739 proteins were identified in E. coli treated with AMAs, of which 628 were altered, including 262 downregulated proteins and 366 upregulated proteins. Bioinformatics analysis showed that these differentially expressed proteins have different molecular functions and participate in different molecular pathways. AMAs can affect E. coli protein biosynthesis, DNA replication and repair, oxidative stress response, peptidoglycan biosynthesis, and homeostasis. These pathways induce morphological changes and cell death. The results of this study help understand the molecular mechanism of the inhibitory effect of AMAs on food-borne pathogens and provide a reference for further development of plant-derived antimicrobial agents.

Proteomic analyses revealed the antibacterial mechanism of Aronia melanocarpa isolated anthocyanins against Escherichia coli O157: H7

Aronia melanocarpa anthocyanins (AMAs), as a natural plant extract, can control pathogens and are attracting increasing attention. However, at the proteomic level, the antibacterial mechanism of AMAs against Escherichia coli O157: H7 remains unclear. In this study, the tandem mass tag (TMT) quantitative proteomics was used to elucidate the potential antibacterial mechanisms of AMAs against E. coli O157: H7 cells. Ultrastructural observation and reactive oxygen species (ROS) levels detection showed that AMAs destroyed the integrity of E. coli O157: H7 cell membrane, led to the aggregation of contents and caused the increase of intracellular ROS level. TMT-based proteomic analysis suggested that AMAs can enter cells through mechanosensitive channels and inhibit the activity of heat shock proteins; disturb the metabolism of carbohydrates, amino acids and lipids in cells. The results of this study provide a reference for the further development of plant-derived antimicrobial agents.

Antibacterial prophylaxis and molecular docking studies of ketone and ester compounds isolated from Cyperus rotundus L. against Aeromonas veronii

Microbial infection outbreaks and the indiscriminate use of therapeutants, and other chemicals are causing an inevitable economic loss in the aquaculture industry. There is an ample research on plant-derived antimicrobial agents that can prevail over microbial resistance. The inflorescence of an invasive aquatic weed, Cyperus rotundus, was examined for its antibacterial properties against Aeromonas veronii in this study. The minimum inhibitory concentration (MIC) of lead compounds isolated from an antioxidant-rich purified methanol fraction was 31.25 µg/ml against the fish pathogen. Pre-feeding compound-based feed to experimentally challenged catfish, Pangasianodon hypophthalmus, resulted in a cumulative survival probability of 85% against A. veronii infection, compared to 10% in untreated challenged fish. Curative outcomes were also obtained from haematological and histopathological investigations. Compared to the conventional antibiotic, Oxytetracycline (−259.95 kcal/mol), the isolated ketone compound, 2-tert-Butyl-9, 10-anthraquinone (−267.14 kcal/mol), exhibited the most favourable docking with aerolysin. This study is the first to report on Cyperus inflorescence's prophylactic antibacterial activity against one of the most prevalent fish pathogens.

Evaluation of the Membrane Damage Mechanism of Chlorogenic Acid against Yersinia enterocolitica and Enterobacter sakazakii and Its Application in the Preservation of Raw Pork and Skim Milk.

Plant-derived antimicrobial agents have adequate antimicrobial effects on food-borne pathogens, which can be used as food preservatives. The purpose of this study was to evaluate the antibacterial mechanism of chlorogenic acid (CA) against Yersinia enterocolitica and Enterobacter sakazakii. The minimum inhibitory concentration (MIC) of CA was determined by employing the broth microdilution method. Then, the cell function and morphological changes of Y. enterocolitica and E. sakazakii treated with CA were characterized. Finally, the growth inhibition models of Y. enterocolitica in raw pork and E. sakazakii in skim milk were constructed through the response surface methodology. The results demonstrated that CA has a satisfactory inhibitory effect against Y. enterocolitica and E. sakazakii with a MIC of 2.5 mg/mL. In addition, CA inhibited the growth of Y. enterocolitica and E. sakazakii via cell membrane damage, such as depolarization of the cell membrane, reduction in intracellular adenosine triphosphate (ATP) and pH levels, and destruction of cell morphology. Moreover, CA reduced two log cycles of Y. enterocolitica in raw pork and E. sakazakii in skim milk at a certain temperature. According to the corresponding findings, CA has the potential to be developed as an effective preservative to control Y. enterocolitica and E. sakazakii-associated foodborne diseases.

Molecular docking and simulation studies of flavonoid compounds against PBP-2a of methicillin‐resistant Staphylococcus aureus

Methicillin-Resistant Staphylococcus aureus (MRSA), a pathogenic bacterium that causes life-threatening outbreaks such as community-onset and nosocomial infections as emerging ‘superbug’. Time and motion study of its virulent property developed resistance against most of the antibiotics such as Vancomycin. Thereby, to curb this problem entails the development of new therapeutic agents. Plant-derived antimicrobial agents have recently piqued people's interest, so in this research, 186 flavonoids compound selected to unmask the best candidates that can act as potent inhibitors against the Penicillin Binding Protein-2a (PBP-2a) of MRSA. Molecular docking performed using PyRx and GOLD suite to determine the binding affinities and interactions between the phytochemicals and the PBP-2a. The selected candidates strongly interact with the different amino acid residues. The 30 ns molecular dynamics (MD) simulations with five top-ranked compounds such as Naringin, Hesperidin, Neohesperidin, Didymin and Icariin validated the docking interactions. These findings are also strongly supported by root-mean-square deviation, root-mean-square fluctuation and the radius of gyration. ADME/T analysis demonstrates that these candidates appear to be safer inhibitors. Our findings point to natural flavonoids as a promising and readily available source of adjuvant antimicrobial therapy against resistant strains in the future. Communicated by Ramaswamy H. Sarma

Flavonoids as Potential anti-MRSA Agents through Modulation of PBP2a: A Computational and Experimental Study.

Recently, the interest in plant-derived antimicrobial agents has increased. However, there are no sufficient studies dealing with their modes of action. Herein, we investigate an in-house library of common plant-based phenolic compounds for their potential antibacterial effects against the methicillin-resistant Staphylococcus aureus (MRSA), a widespread life-threatening superbug. Flavonoids, which are considered major constituents in the plant kingdom, were found to be a promising class of compounds against MRSA, particularly the non-glycosylated ones. On the other hand, the glycosylated derivatives, along with the flavonolignan silibinin A, were able to restore the inhibitory activity of ampicillin against MRSA. To explore the mode of action of this class, they were subjected to an extensive inverse virtual screening (IVS), which suggested penicillin-binding protein 2a (PBP2a) as a possible target that mediates both the antibacterial and the antibiotic-synergistic effects of this class of compounds. Further molecular docking and molecular dynamic simulation experiments were conducted to support the primary IVS and the in vitro results and to study their binding modes with PBP2a. Our findings shed a light on plant-derived natural products, notably flavonoids, as a promising and readily available source for future adjuvant antimicrobial therapy against resistant strains.

Inactivation of Listeria monocytogenes, Escherichia coli O157:H7, and Pre-existing Bacteria on Spinach by Combined Treatment of Cudrania tricuspidata Leaf Extract Washing and Ultraviolet-C Irradiation

Sodium hypochlorite has been used in the food industry to reduce microbial contamination in food; however, the use of sodium hypochlorite is insufficient considering the human health hazards and environmental pollution. To overcome this issue, plant-derived antimicrobial agents have been developed. In our previous study, Cudrania tricuspidata leaf extract (0.4%) was used to wash fresh vegetables for 3 min; however, Cudrania tricuspidata leaf extract was not effective in inhibiting gram-negative pathogens. Therefore, to enhance microbial decontamination by Cudrania tricuspidata leaf extract, ultraviolet-C irradiation (1.8 kJ/m2) was used in combination on spinach as a model system. After the combined treatment, the microbial count of Listeria monocytogenes, Escherichia coli O157:H7, and pre-existing bacteria on spinach were reduced by 1.94, 1.93, and 2.10 log CFU/g, respectively. The log reduction levels of the combined treatment against Listeria monocytogenes, Escherichia coli O157:H7, and pre-existing bacteria on spinach were higher than those of 200 ppm sodium hypochlorite washing (1.46, 1.26, and 1.44 log reduction). Moreover, an increased amount of cellular constituents was released from the pathogens due to the combined treatment compared with the single treatment of Cudrania tricuspidata leaf extract washing or ultraviolet-C irradiation. The distorted and ruptured pathogens after the combined treatment were observed via scanning electron microscopy. Furthermore, combined treatment did not affect the color, texture, and bioactive compounds in spinach. These results suggest that combination of Cudrania tricuspidata leaf extract washing and ultraviolet-C irradiation used in the present study can be an effective method to replace sodium hypochlorite washing.

"Smart" Antimicrobial Nanocomplexes with Potential to Decrease Surgical Site Infections (SSI).

The emergence of resistant pathogens is a burden on mankind and threatens the existence of our species. Natural and plant-derived antimicrobial agents need to be developed in the race against antibiotic resistance. Nanotechnology is a promising approach with a variety of products. Biosynthesized silver nanoparticles (AgNP) have good antimicrobial activity. We prepared AgNPs with trans-cinnamic acid (TCA) and povidone–iodine (PI) with increased antimicrobial activity. We synthesized also AgNPs with natural cinnamon bark extract (Cinn) in combination with PI and coated biodegradable Polyglycolic Acid (PGA) sutures with the new materials separately. These compounds (TCA-AgNP, TCA-AgNP-PI, Cinn-AgNP, and Cinn-AgNP-PI) and their dip-coated PGA sutures were tested against 10 reference strains of microorganisms and five antibiotics by zone inhibition with disc- and agar-well-diffusion methods. The new compounds TCA-AgNP-PI and Cinn-AgNP-PI are broad spectrum microbicidal agents and therefore potential coating materials for sutures to prevent Surgical Site Infections (SSI). TCA-AgNP-PI inhibits the studied pathogens stronger than Cinn-AgNP-PI in-vitro and on coated sutures. Dynamic light scattering (DLS), ultraviolet-visible spectroscopy (UV-Vis), Fourier Transform infrared spectroscopy (FT-IR), Raman, X-ray diffraction (XRD), microstructural analysis by scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) confirmed the composition of TCA-AgNP-PI and Cinn-AgNP-PI. Smart solutions involving hybrid materials based on synergistic antimicrobial action have promising future perspectives to combat resistant microorganisms.

Plant-Derived Products as Antibacterial and Antifungal Agents in Human Health Care.

A number of papers reporting antimicrobial properties of extracts, essential oils, resins and various classes of compounds isolated from higher plants have been published in recent years; however, a comprehensive analysis of plant-derived antimicrobial agents currently applied in practice for the improvement of human health is still lacking. This review summarizes data on clinical efficacy, antimicrobial effects and the chemistry of commercially available antibacterial and antifungal agents of plant origin currently used in the prevention and treatment of gastrointestinal, oral, respiratory, skin, and urinary infections. As a result of an analysis of the literature, more than 40 plant-derived over-the-counter pharmaceuticals, dietary supplements, cosmetics, herbal medicines, and functional foods containing complex mixtures (e.g. Glycyrrhiza glabra extract, Melaleuca alternifolia essential oil, and Pistacia lentiscus resin), pure compounds (e.g. benzoic acid, berberine, eucalyptol, salicylic acid and thymol) as well as their derivatives and complexes (e.g. bismuth subsalicylate and zinc pyrithione) have been identified. The effectiveness of many of these products is illustrated by results of clinical trials and supported by data on there in vitro antimicrobial activity. A broad spectrum of various commercial products currently available on the market and their welldocumented clinical efficacy suggests that plants are prospective sources for the identification of new types of antimicrobial agents in future. Innovative approaches and methodologies for effective proof-of-concept research and the development of new types of plant-derived products effective against recently emerging problems related to human microbial diseases (e.g. antimicrobial resistance) are also proposed in this review.

Mediterranean herb extracts inhibit microbial growth of representative oral microorganisms and biofilm formation of Streptococcus mutans

In light of the growing antibiotic resistance, the usage of plant-derived antimicrobial agents could serve as an effective alternative treatment against oral infections. The aim of this study was to investigate the antimicrobial and antibiofilm activity of Mediterranean herb extracts against representative oral microorganisms. The extraction procedures and the analysis of the obtained extracts were performed under established experimental conditions. The minimum inhibitory (MIC) and bactericidal (MBC) concentrations of the methanol extracts of Cistus creticus ssp. creticus, Cistus monspeliensis, Origanum vulgare, Rosmarinus officinalis, Salvia sclarea and Thymus longicaulis against eight typical oral bacteria and the fungus Candida albicans were determined. The antibiofilm activity against Streptococcus mutans was also quantified using the microtiter plate test. Overall, all tested extracts inhibited effectively the screened obligate anaerobic microorganisms and in concentrations ≥0.3 mg ml-1 had moderate to high antibiofilm activity comparable to that of chlorhexidine (CHX) against S. mutans. In particular, R. officinalis (MIC: 0.08–5.00 mg ml-1) and S. sclarea (MIC: 0.08–2.50 mg ml-1) showed the highest antibacterial activity, while Cistus spp., R. officinalis and S. sclarea significantly inhibited S. mutans biofilm formation at 0.60, 1.25 and 2.50 mg ml-1, respectively. Porphyromonas gingivalis and Parvimonas micra were high susceptible to O. vulgare (MIC = 0.30 mg ml-1), whereas T. longicaulis eradicated all oral bacteria (MBC: 0.15–2.50 mg ml-1). Nevertheless, C. albicans showed no sensitivity to the tested extracts. In conclusion, the tested plant extracts could serve as alternative natural antibacterial and antibiofilm components against oral infections.

Essential oils from Origanum vulgare and Salvia officinalis exhibit antibacterial and anti-biofilm activities against Streptococcus pyogenes

In the present study, essential oils (EOs) extracted from oregano, sage, cloves, and ginger were evaluated for the phytochemical profile, antibacterial, and anti-biofilm activities against Streptococcus pyogenes. The broth microdilution method was used to determine the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of EOs. The minimum biofilm inhibitory concentrations (MBICs) were determined using MTT assay and fixed biofilms were observed through scan electron microscopy. The oregano and sage EOs showed the lowest MIC as well as MBC of 0.25–0.5 mg/mL. Time kill assay results showed that oregano and sage EOs exhibited bactericidal effects within 5 min and 4 h, respectively. Both oregano and sage extracts acts as a potent anti-biofilm agent with dual actions, preventing and eradicating the biofilm. The microscopic visualization of biofilms treated with EOs have shown morphological and density changes compared to the untreated control. Oregano EO was constituted predominantly carvacrol (91.6%) and in sage EO, higher levels of α-thujone (28.5%) and camphor (16.6%) were revealed. EOs of oregano and sage inhibit the growth and biofilm formation of S. pyogenes. Effective concentrations of oregano and sage EOs and their phytochemicals can be used in developing potential plant-derived antimicrobial agents in the management of streptococcal pharyngitis.

An in vitro study of antibacterial and antifungal activity of Cynodon dactylon

Background: Plant-derived antimicrobial agents remain an arena for research to overcome the issues pertaining to microbial resistance and adverse effects associated with synthetic drugs. Aims and Objectives: The present study is being performed to evaluate the antibacterial and antifungal activity of Cynodon dactylon. Materials and Methods: The methanol and n-butanol extracts of C. dactylon were prepared. The inoculum was prepared from stock cultures containing nutrient broth (antibacterial activity) and Sabouraud dextrose broth (antifungal activity) and incubated at 37°C (24 h) and at room temperature (48 h) respectively. Antibacterial and antifungal activity of n-butanol and methanol extracts of C. dactylon were screened with the aid of agar disc diffusion method on Muller-Hinton agar medium and Sabouraud dextrose agar medium respectively at 1000 μg/ml, 750 μg/ml, and 500 μg/ml concentrations. The measurement of diameter of zone of inhibition was performed and compared with controls-ampicillin for antibacterial activity and amphotericin-B for antifungal activity. Results: Methanol extract of C. dactylon was observed to have good antibacterial activity with Salmonella, Staphylococcus sps. being susceptible, and good antifungal activity against Aspergillus, Penicillium and Trichoderma viride at 1000 μg/ml. The n-butanol extract also had good antibacterial activity against Escherichia coli, Pseudomonas sps., and good antifungal activity against Aspergillus, Penicillium and T. viridae at 1000 μg/ml. Conclusion: From this study, we found that C. dactylon has promising antibacterial activity against Salmonella, Staphylococcus, E. coli, Pseudomonas, and potential antifungal activity against Aspergillus, Penicillium, T. viridae, Candida sps..

Efficacy of Plant-Derived Antimicrobials in Controlling Enterohemorrhagic Escherichia coli Virulence In Vitro

Escherichia coli O157:H7 is a major foodborne pathogen that can cause serious human illness characterized by hemorrhagic diarrhea and kidney failure. The pathology of enterohemorrhagic E. coli O157:H7 (EHEC) infection is primarily mediated by verotoxins, which bind to the globotriaosylceramide receptor on host cells. Antibiotics are contraindicated for treating EHEC infection because they lead to increased verotoxin release, thereby increasing the risk of renal failure and death in patients. Thus, alternative strategies are needed for controlling EHEC infections in humans. This study investigated the effect of subinhibitory concentrations of five plant-derived antimicrobial agents (PDAs) that are generally considered as safe, i.e., trans-cinnamaldehyde, eugenol, carvacrol, thymol, and β-resorcylic acid, on EHEC motility, adhesion to human intestinal epithelial cells, verotoxin production, and virulence gene expression. All tested PDAs reduced EHEC motility and attachment to human intestinal epithelial cells (P < 0.05) and decreased verotoxin synthesis by EHEC. The reverse transcription real-time PCR data revealed that PDAs decreased the expression of critical virulence genes in EHEC (P < 0.05). The results collectively suggest that these PDAs could be used to reduce EHEC virulence, but follow-up studies in animal models are necessary to validate these findings.

Label-free quantitative proteomic analysis of the inhibitory activities of juglone against translation and energy metabolism in Escherichia coli

Plant-derived antimicrobial agents have received increasing attention owing to their potential to control pathogens and excellent efficacy despite the growing prevalence of antibiotic resistance. However, the antibacterial mechanism of juglone, a traditional medicine used to cure skin infections, is still unclear. Therefore, in this study, in order to elucidate the mechanisms underlying the antibacterial activity of juglone, label-free quantitative proteomic technology was applied for analysis of the 417 proteins that were differentially expressed in Escherichia coli after treatment with juglone at one-half of the minimum inhibitory concentration. Gene ontology enrichment analysis of differentially expressed proteins suggested that juglone effectively repressed the expression of dehydrogenase and cytochrome oxidase, indicating that energy generation was blocked. Additionally, juglone induced RNA formation and ribosome assembly, resulting in inhibition of translation. This is the first study to adopt a proteomic approach to investigate the antibacterial mechanism of action of juglone against E. coli.

High-level antimicrobial efficacy of representative Mediterranean natural plant extracts against oral microorganisms.

Nature is an unexplored reservoir of novel phytopharmaceuticals. Since biofilm-related oral diseases often correlate with antibiotic resistance, plant-derived antimicrobial agents could enhance existing treatment options. Therefore, the rationale of the present report was to examine the antimicrobial impact of Mediterranean natural extracts on oral microorganisms. Five different extracts from Olea europaea, mastic gum, and Inula viscosa were tested against ten bacteria and one Candida albicans strain. The extraction protocols were conducted according to established experimental procedures. Two antimicrobial assays—the minimum inhibitory concentration (MIC) assay and the minimum bactericidal concentration (MBC) assay—were applied. The screened extracts were found to be active against each of the tested microorganisms. O. europaea presented MIC and MBC ranges of 0.07–10.00 mg mL−1 and 0.60–10.00 mg mL−1, respectively. The mean MBC values for mastic gum and I. viscosa were 0.07–10.00 mg mL−1 and 0.15–10.00 mg mL−1, respectively. Extracts were less effective against C. albicans and exerted bactericidal effects at a concentration range of 0.07–5.00 mg mL−1 on strict anaerobic bacteria (Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, and Parvimonas micra). Ethyl acetate I. viscosa extract and total mastic extract showed considerable antimicrobial activity against oral microorganisms and could therefore be considered as alternative natural anti-infectious agents.