Rapid bactericidal and anti-virulence effects of Quercus infectoria gall extract against the pharyngitis causing pathogen Streptococcus pyogenes.

Introduction. In recent years, researchers have shown increasing interest in species of the Passiflora genus due to their potential biological and pharmacological properties. These species are an agronomically important crops and are used commercially in the fruit industry of South America. During of collection of fruits from cultivated plants, the leaves are removed. This plant material may be used for medicinal purposes. Our previous studies showed that crude extracts from leaves of P. alata, P. caerulea and P. incarnata contained various secondary metabolites such as phenolics, flavonoids, terpenoids. Moreover extract of P. alata showed the most effective activities against Acanthamoeba castellanii strain in vitro. Aim. The aim of our study was to evaluate and to compare the antibacterial and antifungal activities of the crude alcoholic extracts from leaf of P. alata, P. caerulea and P. incarnata. Material and methods. There was measurement of the minimal inhibitory concentration (MIC), the minimal bactericidal concentration (MBC), and the minimal fungicidal concentration (MFC) of the extracts by serial dilution method. Results. The results showed that the most active extracts against Enterococcus faecalis (ATCC 8040) were as follows from: P. incarnata = P. alata (MIC = 10.0 mg/ml, MBC >10.0 mg/ml) > P. caerulea (MIC = 10.0 mg/ml, MBC > 20.0 mg/ml); against Escherichia coli (PZH 026B6): P. incarnata (MIC = 10.0 mg/ml, MBC > 10.0 mg/ml) > P. caerulea (MIC = 10.0 mg/ml, MBC = 20.0 mg/ml) > P. alata (MIC = 10.0 mg/ml, MBC > 20.0 mg/ml); against Staphylococcus aureus (ATCC 6538P): P. incarnata (MIC = 2.5 mg/ml, MBC > 5.0) > P. caerulea (MIC = 5.0 mg/ml, MBC > 10.0) > P. alata (MIC = 10.0 mg/ml, MBC > 10.0); against Candida albicans (PCM 1409PZH): P. caerulea (MIC = 7.5 mg/ml, MBC = 15.0 mg/ml), P. incarnata (MIC = 10.0 mg/ml, MBC > 10.0 mg/ml), P. alata (MIC = 15.0 mg/ml, MBC > 20.0 mg/ml); against Microsporum gypseum K1: P. incarnata = P. caerulea = P. alata (MIC = 5.0 mg/ml, MBC = 5.0 mg/ml). Phytochemical study showed that the highest concentration of phenolic compounds was shown in extract of P. alata > P. caerulea > P. incarnata. Conclusions. Due to the fact that low antimicrobial activity has been demonstrated for raw extracts, there is a need for further studies of fractionated extracts and isolated compounds to assess their activity.

Background/Objectives: The increasing prevalence of antimicrobial-resistant bacteria highlights the need for improved methodologies to evaluate antimicrobial activity beyond conventional minimum inhibitory concentration testing. While resazurin-based assays are widely used for minimum inhibitory concentration determination due to their simplicity and sensitivity, minimum bactericidal concentration assessment still relies on labor-intensive colony-forming unit counting. The objective of this study was to develop and validate a resazurin-based microwell assay capable of determining both the minimum inhibitory concentration and the minimum bactericidal concentration without routine plate counting, thereby simplifying bactericidal evaluation. Methods: A two-step resazurin-based fluorescence assay was designed and performed in microplates. After determining the minimum inhibitory concentration using resazurin as a metabolic indicator, well-showing inhibited bacterial growths were subjected to a regrowth phase by transferring aliquots into fresh antimicrobial-free medium containing resazurin. This additional step allowed discrimination between reversible metabolic inhibition and irreversible bacterial death. The method was evaluated using ciprofloxacin and chloramphenicol against four bacterial species: Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, and Pseudomonas aeruginosa. Minimum bactericidal concentration values obtained using this assay were compared with those obtained through conventional colony counting on agar plates. Results: Minimum bactericidal concentration values obtained using the two-step fluorescence assay were fully concordant with the conventional colony-forming unit counting method for all tested antibiotics and bacterial species. Conclusions: The proposed two-step resazurin-based microwell assay represents a rapid, reliable, and less labor-intensive alternative for the determination of both the minimum inhibitory concentration and the minimum bactericidal concentration, with potential applications in clinical and industrial microbiology laboratories.

The aim of the preset study was to determine the efficacy of Annona Senegalensis stem and root extracts against Salmonella typhimurium Shigella flexneri and Escherichia coli through the evaluation of bacterial sensitivity and determination of the minimum inhibitory and bactericidal concentration of the extracts against the test isolates. Plant materials were collected and duly authenticated. The methanoic and aqueous extracts prepared from the powdered forms were tested on the bacterial after cultural and biochemical identification of the isolates. The antibiotic sensitivity test was carried out using the Kirby Bauer disk diffusion method while chloramphenicol was used as the standard control. The Minimum Inhibitory Concentration (MIC) of the plant extracts were determined by broth dilution method while the Minimum Bactericidal Concentration (MBC) was determined by a method described using standard protocols. The ratio of MBC:MIC was computed to determine the bactericidal or bacteriostatic effects of the extracts. Data were analyzed using the Minitab 16 statistical package. Descriptive statistics (mean and standard error) and analysis of variance tools were applied while mean separation was done Fischer’s method at 5% level of significance. Antibacterial sensitivity test showed that the control test (Chloramphenicol) had significantly higher antibacterial sensitivity ( P<0.05) than any of the plant extract. Minimum Inhibitory Concentration (MIC) of the plant extract ranged from 6.25 mg/ml to 25.0 mg/ml. The lowest MIC of 6.25mg/ml was observed in Salmonella typhimurium among all extract types. Root and stem had similar effects on the test organisms (P>0.05) but, methanoic root extract had the lowest MBC of 6.25mg/ml against S.typhimurium and S. flexneri. Based on the MBC/MIC ratio, all extract types had bactericidal effects on Salmonella typhimurium and Shigella flexneri except aqueous root extract that showed bacteriostatic effect. Only the methanoic root and stem extracts exhibited bactericidal effects on Escherichia coli. Annona Senegalensis root and stem could possibly be explored commercially as an antibacterial agent against species of Salmonella, Shigellia and Escherichia.

The objective of the study was to evaluate the antibacterial activities of N,N’[1,1’-dithiobis(phenylene)]bis(benzyldeneimine), referred to as L1 and o,o’-(N,N-dipicolinyldene) diazadiphenyldisulfide, referred to as L2, containing disulfide moieties against some ophthalmic pathogens (Klebsiella species, Escherichia coli, Streptococcus species, Proteus morganii, Pseudomonas species, Streptococcus pneumoniae, Acinetobacter species, Streptococcus pyogenes and Streptococcus viridans), urinary tract infectious pathogens (P. morganii, E. coli, Pseudomonas spp., Enterobacter species, and Klebsiella spp.) and antibiotic resistant pathogens (Staphylococcus species, Streptococcus, Pseudomonas spp., and Klebsiella spp.) for minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The MIC for the ophthalmic pathogens and antibiotic resistant pathogens were found to be 400 to 500 µg/ml, while for the urinary tract infectious pathogens a lower MIC value (200 µg/ml) was obtained. The MBC for the compounds against all the pathogens tested was 400 to 500 µg/ml. The synthesized Schiff L1 and L2 showed the MIC values for all the tested ophthalmic and antibiotic resistant bacterial pathogens more or less similar. Further studies are needed to prove the safe and efficacy needed for these compounds to develop as a drug after completing successful preclinical and clinical tests. Key words: Antibacterial activity, minimum bactericidal concentration (MBC), minimum inhibitory concentration (MIC), N, N’[1,1’-dithiobis(phenylene)]bis(benzyldeneimine), ophthalmic pathogens, Urinary tract infectious pathogens (UTI), antibiotic resistant pathogens.

Honey is one of the oldest traditional medicines that has been highly reputed and widely used for the treatment of several human diseases for thousands of years. The purpose of this study was to: (i)evaluate and compare the antibacterial and antibiofilm activities ofShawkiat and Rabiee honeys with those ofManuka honey against Escherichia coli and Staphylococcus aureus; (ii)assess the anti-virulence potential ofthese honeys, byexamining their impacts onthe expression offive selected genes inE.coli and seven genes inS.aureus that have been previously shown tobeinvolved inthe microcolony, biofilm and virulence inthe test organism. The antibacterial, antibiofilm and anti-virulence activities ofthese honeys against both bacteria were investigated byagar well diffusion, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), growth curve, time-kill curve, microtiter plate and reverse transcription-quantitative real time polymerase chain reaction (RT-qPCR). The susceptibility tests showed promising antibacterial activities ofShawkiat and Rabiee honeys compared with Manuka honey against E.coli and S.aureus. The results showed that Manuka honey possessed the lowest MIC value against both bacteria with 20% (w/v) MIC and MBC of25% (w/v). Slightly higher MIC values were shown byShawkiat and Rabiee honeys against both bacteria with 25% (w/v) MIC and 50% (w/v) MBC values compared with Manuka honey. Growth curves demonstrated nogrowth ofthe two bacteria after treatment with MIC ofall the tested honeys. Shawkiat and Rabiee honeys showed that both bacteria lost viability comparably with Manuka honey. The lowest concentration ofShawkiat and Rabiee honeys was able toinhibit and eradicate the biofilm ofboth bacteria compared with Manuka honey. The qPCR analysis showed that the expression ofall the selected genes inboth bacteria after treatment with all the tested honeys was downregulated and shared asimilar overall pattern ofgene expression, with atrend toward reduced expression ofthe virulence genes ofinterest. The results ofthis study indicate that Jordanian honeys possess antibacterial, antibiofilm and anti-virulence activities comparably with Manuka honey. This study revealed that Jordanian honey compared with Manuka honey inhibits E.coli and S.aureus planktonic cells and biofilm through the downregulation of genes required for growth, biofilm formation and motility.

The in vitro activity of tigemonam, a new oral monobactam, was studied with special attention to minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs). Against 250 clinical isolates, it inhibited 100% of Escherichia sp., Klebsiella sp., Serratia sp., Citrobacter sp., Providencia sp., Proteus sp., Morganetta sp., Aeromonas sp., Yersinia sp., Shigella sp., and Haemophilus sp. at 0.5 mg/L or less. With 1 mg/L, 75% of Enterobacter sp. were inhibited; however, three of the 20 strains tested needed more than 16 mg/L. Proteus sp., Morganella sp. and Providencia sp. were more susceptible, with MIC90S of 0.06 mg/L or less. The MBC was equal to or two times higher than the MIC. Increasing the inoculum size from 103 to 105 colony-forming units had little effect on MIC and MBC; with an inoculum of 107 or more, MIC and MBC increase three to eight times. MIC and MBC were a little lower in Mueller-Hinton base, and the presence of serum did not significantly change the MIC or the MBC. Tigemonam exhibits a rapid killing rate, but an increased antibiotic concentration was not accompanied by greater lethal effect, and the lethal rate at MBC was lower in trypticase soy broth (TSB) + 40% serum than in TSB alone.

Antibacterial activity of Achyrocline flaccida aqueous extract against Aeromonas hydrophila and its effects on biofilm formation

Background. Metal nanoparticles can have unique properties that differ from those of the solid metal from which they are obtained. This is what determines the increased interest in them on the part of researchers from all over the world. Objective of the study: quantitative assessment of the antibacterial effect of silver and zinc oxide nanoparticles on polyantibiotic-resistant strains of gram-positive and gram-negative microorganisms. Material and methods. The synthesis of silver and zinc oxide nanoparticles was performed by laser ablation in a liquid. The physical properties and sizes of these nanoparticles were studied on the basis of spectral characteristics and atomic force microscopy. The study of the antibacterial resistance of the studied microorganisms was carried out, as well as a quantitative assessment of the antibacterial effect of the obtained nanoparticles based on the determination of the minimum inhibitory and minimum bactericidal concentrations. Results. All microorganisms used in the study were clinical pathogenic strains with polyantibacterial resistance. In this case, the minimum inhibitory concentrations of silver nanoparticles ranged from 7.81 to 31.25 μg/ml, and the minimum bactericidal concentrations - from 31.25 to 62.50 μg/ml, while for zinc oxide nanoparticles the values of the minimum inhibitory concentrations were within the range from 125 to 500 μg/ml, and the values of the minimum bactericidal - from 250 to 1000 μg/ml. Conclusions. The laser ablation method allows the synthesis of chemically pure nanoparticles of silver, zinc oxide, and, if necessary, other metals. Silver and zinc oxide nanoparticles are effective antimicrobial agents against polyantibiotic-resistant pathogenic microbial flora. The obtained values of the minimum bactericidal and minimum inhibitory concentrations must be taken into account in the subsequent clinical implementation of medical devices based on the use of metal nanoparticles.

Information on the bactericidal activity of benzylpenycylin is important when planning antibiotic therapy for a number of diseases caused by Corynebacterium diphtheriae (endocarditis, bacteremia, septicemia, etc.). However, methods for determining the minimum bactericidal concentration (MBC) of antibiotics are complicated. The aim of the study was to develop a method for testing of diphtheria causative agent susceptibility to the bactericidal action of penicillin without MBC determination. The minimum inhibitory concentrations (MICs) and MBCs in 80 strains of C. diphtheriae were determined using the standard broth dilution method (macromethod). The MICs were registered after 24 and 48 years of growth. After the first day of growth the MIC of penicillin was in the range of 0.017 to 0.5 mg / L, after second day — in the range of 0.035 to 0.5 mg / liter. The increase of the MICs for the second day of growth was observed in 47.5±5.6% of strains. MIC50 and MIC90 both after 24 hours and after 48 hours of growth were 0.13 and 0.25 mg/L, respectively. MBCs of penicillin was in the range of 0.5 to 32.0 mg/L, MBC50 was 4.0 mg/l, MBC90 – 8.0 mg/l. In 35,0±5,3% of the studied strains, high MBCs (8.0 mg / l or higher) was detected. It has been established that in C.diphtheriae the relationship between the MIC and the MBC of penicillin is clearly expressed, taking into account the two values of the MIC – for the first and second day of growth. Investigated strains are divided into three conditional groups: 1) with MIC 0.13 mg/L and basically with low MBCs; 2) with MIC 0.25 mg/L, in most cases are not susceptible to bactericidal action, and 3) strains with MIC 0.5 mg/L and high MBCs. The most pronounced were differences in the prevalence of corynebacteria with high MBCs in two groups of strains: MICs which did not exceed 0.13 mg/L after 24 and 48 hours, and those in whom the MIC was 0.25 mg/L or higher already on the first day of incubation (Student's coefficient t=4.13, p<0.001). The obtained results can be used to improve the methods for determining of corynebacteria susceptibility to antimicrobials.

Chemical composition of Mentha pulegium and Rosmarinus officinalis essential oils and their antileishmanial, antibacterial and antioxidant activities

Background: Propolis is one of the useful bee colony products that have been used in traditional medicine for centuries. In this study, the physicochemical characters and their antibacterial effect of Iranian Propolis collected from Qazvin province was assessed.Methods: In this study, Thin Layer Chromatography and Vacuum Liquid Chromatography to detect different compounds of the extract have been used. In the initial evaluation of Propolis extract, it was found that the extract includes variable compounds with different polarity; so, the initial classification of extract with different polarity solvents was essential. Finally, 0.1 gr hydro alcoholic Propolis was injected to the HPLC by ultrasound. The antibacterial effect of Iranian ethanol extract Propolis was measured using a microdilution method against Pseudomonas aeruginosa: P. aeruginosa and Staphylococcus aureus: S.aureus standard strains and the minimum bactericidal and inhibitory concentration were defined.Results: Primary analysis of the ethanol extract by analytical Thin Layer Chromatography, demonstrated the presence of flavonoid and phenol in it. Minimum inhibitory concentration and Minimum Bactericidal Concentration for Staphylococcus aureus: S.aureus standard strain was 2.5mg/ml. The same procedure was done for Pseudomonas aeruginosa: P. aeruginosa standard strain and the Minimum inhibitory concentration and Minimum Bactericidal Concentration were 50mg/ml of Propolis extracts. Conclusion: According to the results, the alcoholic extract of propolis from Qazvin province of Iran provides significant antimicrobial activity. Its powerful activity may be due to high total phenolic and flavonoid contents.Keywords: Iranian propolis, Antibacterial activity, Phenolic compounds, Flavonoid compound

Enterococcus faecalis (E. faecalis) is implicated as the common etiological agent of root canal treatment failure. The study was aimed to compare the antibacterial efficacy and to assess the minimum inhibitory concentration (MIC) of 3 gutta-percha solvents-RC Solve, Endosolv-E, and xylene against E. faecalis. Agar well diffusion assay was performed to evaluate the antibacterial efficacy of the gutta-percha solvents against E. faecalis (ATCC 29212 and a clinical isolate). The standard drug vancomycin was used as the control. The MIC of the solvents was determined by broth microdilution assay. Two fold serial dilutions of the solvents were prepared and the lowest concentration of the solvent that inhibits visible bacterial growth was recorded as the MIC. Minimum bactericidal concentration (MBC) was determined by plating on Muller Hinton Agar plates and the lowest concentration of the solvent that resulted in 99% reduction of bacterial viability was scored as the MBC. The antibacterial activity of RC Solve against E. faecalis was found to be comparable to that of the standard drug-vancomycin. Xylene did not exhibit antibacterial activity while the antibacterial activity of Endosolv-E was lesser than RC Solve. RC Solve recorded lower MIC values against both E. faecalis ATCC29212 and clinical isolate (1:2 and 1:4 dilution) than Endosolv-E (undiluted). RC Solve was found to possess antibacterial activity against E. faecalis even at half the concentration that is routinely being used. Within the limitations of the study, we conclude that RC Solve can perform a dual role both as a gutta-percha solvent as well as a microbicide against E. faecalis.

The Fabaceae family is known for producing a diverse range of secondary metabolites having biological activity, including analgesic, anti-inflammatory, anticancer, antidiabetic, antirheumatic, antibacterial, and cytotoxic properties. Three Fabaceae species, Pterocarpus indicus, Adenanthera pavonina, and Falcataria falcata, possess pharmacological potential that has not been thoroughly investigated. The goals of this study were (1) to determine the antibacterial activity against Bacillus subtilis and Escherichia coli of leaves and twigs extract of these three Fabaceae species and (2) to identify the putative secondary metabolites in the leaves and twigs of the species exhibiting the lowest minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against B. subtilis and E. coli. The dried leaves and twigs of P. indicus, A. pavonina, and F. falcata were extracted using ultrasonic-assisted extraction, followed by an antibacterial bioassay utilizing broth dilution procedures. Qualitative and semiquantitative phytochemical screening was then carried out. Chemical substances were detected using liquid chromatography-mass spectrometry (LC-MS) analysis. F. falcata leaves methanol extract had MIC 1.30mg/mL and MBC 5.00mg/mL against B. subtilis and had trice higher antibacterial activity than P. indicus and A. pavonina. However, F. falcata leaves ethyl acetate extract had MIC 0.30mg/mL and MBC 1.0mg/mL against E. coil, twice higher than P. indicus and A. pavonina extract. This also positively correlated with the flavonoid contents obtained; F. falcata leaves ethyl acetate extract had higher, that is, 43.93±3.56mg QE/g extract. However, the highest total phenolic content was found in the methanol leaf extract of P. indicus, although the methanol leaf extract of F. falcata was still relatively large, namely, 172.79±1.51mg gallic acid equivalent (GAE)/g extract. LC-MS analysis revealed that F. falcata extract included phenolics, flavonoids, terpenoids, and carboxylic acids. Almost all extracts contained the active component kaempferol.

Streptococcus pyogenes one cause of respiratory tract infections including pharyngitis. Seventy-three percent of doctors prescribe antibiotics for pharyngitis. In developing countries, antibiotics are prescribed for 44–97% of hospitalized patients, sometimes with inappropriate doses. Improper antibiotic use can cause side effects such as allergies or diarrhea, increase health care costs, and increase the possibility of selection for antibiotic resistance. Research needs to be done on natural antibiotics found in natural ingredients. One of the natural ingredients empirically has many properties and is relatively safe is honey from bees. Honey has substances that are bactericidal and bacteriostatic like antibiotics. This study aimed Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) honey bee Trigona spp against Streptococcus pyogenes in vitro. In concentration of 2.5%, 5%, 7.5%, 10%, and 12, 5% and knowing the effect of honey bee Trigona spp with a concentration of 2.5%, 5%, 7.5%, 10%, and 12.5% against Streptococcus pyogenes in vitro. Type of research is the experiment with the Posttest-Only Control Group Design, the research material of honey from Trigona spp independent variable is the concentration of honeybees Trigona spp. The dependent variable is the level of turbidity produced in a tube to measure MIC and the number of colonies Streptococcus pyogenes on Blood Agar Plate media to measure MBC. The results of the MIC determination showed that all concentrations showed clear, MBC results the largest number of colonies occurred at a concentration of 2.5% of the total colony colonies and colonies growth the smallest occurred at a concentration of 10% of the number of colonies 0 colonies, Conclusions of MIC cone honey bee Trigona spp against bacteria Streptococcus pyogenes occurred at a concentration of 2.5%., MBC moth honey Trigona spp against bacteria Streptococcus pyogenes occurred at a concentration of 10%. There is an influence of the honey Trigona spp on the bacteria Streptococcus pyogenes, which is indicated by the value of p <0.05, which is 0, 000 using the Linear Regression test.

Group A Streptococci (GAS) or Streptococcus pyogenes is responsible for acute bacterial pharyngitis in children as well as adults. Streptococcal pharyngitis is initiated by successful attachment and colonization of the bacteria, followed by the establishment of the biofilm in various environments. In this study, we examined the antibacterial activities of in-house prepared aqueous and ethanolic extracts of 10 Atlantic Canada fruits in the context of minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), time-kill kinetics, and adhesion inhibition properties against S. pyogenes. Per our findings, MIC and MBC for all the tested extracts ranged from 0.25 to 8 mg/mL and from 4 to 64 mg/mL, respectively. Accordingly, at 1⁄2 × MBC, cranberry and sumac extracts also lowered the attachment of GAS to the uncoated and fibronectin-coated substratum. Particularly, cranberry and sumac aqueous extracts were more effective against the adhesion of S. pyogenes ATCC 19615 to the fibronectin-coated surface than a clinical strain. In conclusion, ethanolic and aqueous extracts of cranberry and sumac could potentially be incorporated into natural health products designed for the amelioration of strep throat, yet a detailed understanding of its mode of action (e.g., biofilm inhibition and eradication) could pave its path to the field of antibacterial natural health product discovery, design, and development.