Modulation of Anti-Microbial Resistant <i>Salmonella heidelberg</i>Using Synbiotics (Probiotics and Prebiotics) in Two <i>In-Vitro</i>Assays (Cross-Streaking and Agar Wells Diffusion)

Salmonellosis is the most prevalent bacterial foodborne disease in many countries worldwide. Utilization of probiotics is one of the most accepted ways to reduce Salmonella, especially lactic acid bacteria, as it has proven to reduce the enteric pathogens in monogastric and ruminant livestock animals through different mechanisms such as antimicrobials production, competitive adhesion to the gastrointestinal tract, and immune stimulation. Prebiotics could be utilized solely for health benefits as an alternative to probiotics or in addition to probiotics for a synergistic effect known as synbiotics. The aim of this study was to compare effects of different probiotic strains (Lactobacillus acidophilus (La-14), Lactobacillus paracasei (Lpc-37), Streptococcus thermophiles (St-21), Bifidobacterium bifidum (Bb-06), and Aspergillus niger (ATCC®16888TM) and without prebiotics (Mannose;Xylose;Galactooligosaccharides GOS;Inulin;and Dandelion extract) on lowering Salmonella heidelberg CFU in vitro. Different inhibition levels probiotic strains were assessed and compared in the presence and absence of 2.5% prebiotic compounds using cross-streaking and agar well diffusion assays. Recommendations for the growth of selected microorganisms such as temperature and oxygen conditions were taken into consideration. All the analysis was conducted in triplicates. The results showed that all the probiotics strains except S. thermophiles were able to significantly (P < 0.05) inhibit the growth of S. heidelberg in at least one of the assays. The difference in inhibition percentage confirms that probiotic strains have multiple inhibition mechanisms, such as production of antimicrobials, lower pH by producing organic acids (acetic acid, lactic acid, etc.), and inhibition of pathogen’s virulence factor expression, and production of lipopolysaccharide solubilizing compounds.

Methods for in vitro evaluating antimicrobial activity： A review

In recent years, there has been a growing interest in researching and developing new antimicrobial agents from various sources to combat microbial resistance. Therefore, a greater attention has been paid to antimicrobial activity screening and evaluating methods. Several bioassays such as disk-diffusion, well diffusion and broth or agar dilution are well known and commonly used, but others such as flow cy- tofluorometric and bioluminescent methods are not widely used because they require specified equip- ment and further evaluation for reproducibility and standardization, even if they can provide rapid re- sults of the antimicrobial agent＇s effects and a better understanding of their impact on the viability and cell damage inflicted to the tested microorganism. In this review article, an exhaustive list of in vitro antimicrobial susceptibility testing methods and detailed information on their advantages and limita- tions are reported.

Antimicrobial and cytotoxic potential of seeds and flowers crude extracts of sunflower

Oil-producing crops,including Helianthus annuus L.(H.annuus,Sunflower),have been used for the treatment of different human diseases since ancient times.This study aims to determine the antimicrobial and cytotoxic potential of different polarities of seeds and flower extracts of locally grown H.annuus by well-established bioassays.To achieve the objects,the plant extracts were prepared from both flowers and seeds powder samples individually with methanol solvent by using a Soxhlet extraction method.The antimicrobial and cytotoxic potential of the flowers and seed extracts were determined by the in vitro diffusion and brine shrimp method(BSL).The results of the antimicrobial potential of both prepared flower and seed extracts of various polarities at different concentrations showed promising potential against the gram-positive and gram-negative bacterial strains,within the range of inhibition zone 0–20 mm.Among the flower extracts,the highest potential was obtained in water extract and the lowest potential was in butanol extract followed by water>ethyl acetate>hexane>methanol>chloroform>butanol extracts.However,from the seed extracts,the highest potential was obtained in water extract and the lowest potential was in the methanol extract followed by water>hexane>butanol>ethyl acetate>chloroform>methanol extract.On the other hand,the results of cytotoxicity among the flowers extracts against the brine shrimp method where the highest LC50 against brine shrimp nauplii were found to be 31.25μg/mL in water extract and the lowest LC50 was 1345.86μg/mL in chloroform extract followed by water>ethyl acetate>hexane>butanol>methanol>chloroform extract.Similarly,different polarities of seed extracts,the highest LC50 against brine shrimp nauplii was shown to be 109.56μg/mL in butanol extract and the lowest LC50 was 1513.56μg/mL in ethyl acetate extract followed by butanol>hexane>water>methanol>chloroform>ethyl acetate extract.In conclusion,the results showed that both flowers and seeds extracts at various concentrations have promising potential against the positive and negative bacterial strains and the highest potential water extract could be used as natural broad-spectrum antibiotics as herbal medicine to treat different human infectious diseases.

Evaluation of the Antimicrobial Activity of Endophytic Fungus Isolated from <i>Cocos nucifera</i>(L.) Cotyledon against Medically-Important Pathogens

The study aimed to evaluate the endophytic fungi isolated from Cocos nucifera (L.) cotyledon for their possible antimicrobial activity. A total of twelve endophytic fungal species were successfully isolated, from which five were selected and presumptively identified, including Aspergillus niger, Raffaelea sp., Aspergillus flavus, Cladosporium sp. and Pseudallescheria spp. based on their macro and microscopic characters. The agar blocks of actively growing pure culture were assessed for antibacterial activity against five human pathogenic bacterial and fungal strains: Escherichia coli (BIOTECH 1634), Bacillus subtilis (BIOTECH 1697), Staphylococcus aureus (BIOTECH 1592), Serratia marcescens (BIOTECH 1748), and Candida tropicalis (BIOTECH 2085). Among the selected five endophytic used for antimicrobial activity screening, the Raffaelea sp. (CN-EF 2) has shown a high zone of inhibition of 11 mm against Bacillus subtilis, 18 mm against Escherichia coli, 15 mm against Staphylococcus aureus, and 11 mm against Candida tropicalis. Correspondingly, Aspergillus niger (CN-EF 1) has shown a zone of inhibition of 16 mm against C. tropicalis. The endophytic fungus Raffaelea sp. was effective against all test organisms except for Serratia marcescens, while Aspergillus niger was only effective for pathogenic fungi C. albicans. Hence, further in-depth studies of the isolated endophytic fungi are needed to determine the mechanisms behind the endophytes’ inhibitory effects.

Phytochemical Screening, Antibacterial Effect, and Essential Oil Extract from the Leaf of Artemisia afra against on Selected Pathogens

Background: Artemisia afra is an indigenous plant used in Africa. It is used in traditional medicine to treat a variety of diseases caused by bacterial infections. As a result, developing innovative therapeutic methods using natural ingredients to combat pathogenic germs is critical. Methodology: Artemisia afra leaves were extracted using maceration extraction utilizing three solvents (petroleum ether, ethanol, and dichloromethane). Fresh leaves were extracted using hydrodistillation. The agar well diffusion method was used to test the crude extract and essential oil leaves of Artemisia afra against different human pathogenic bacteria strains (E. coli, S. aureus, E. faecalis, and K. pneumonia) at different concentrations in the presence of a positive (ciprofloxacin 5.0 μg) and negative (DMSO) control. Results: Artemisia afra revealed the presence of alkaloids, flavonoids, tannins, saponins, anthraquinones, terpenoids, coumarins, phenolic quinones, cardiac glycosides, and steroids. It showed that the highest antibacterial activity given by the ethanol extract had highly inhibition zones against gram-positive and gram-negative bacteria. The essential oil extract was effective against all tested bacteria. Conclusion: Plant crude extracts and essential oils may have antibacterial properties due to the synergistic activity of two or more active secondary metabolites.

Evaluation of the Antifungal Activity of Aqueous and Alcoholic Extracts of Six Spices

Background: Nigeria is plagued with a variety of socioeconomic problems mainly poverty, poor access to quality healthcare and poor hygienic conditions resulting in the myriad of fungal infections that are frequently encountered in clinical practice. Method: The antifungal activity of aqueous, methanolic, propanolic and benzyl alcohol extracts of Capsicum annuum fruits and seeds, Capsicum chinense fruits and seeds, Aframomum melegueta pods and seeds, Allium sativum bulbs, Allium cepa bulbs and Zingiber officinale rhizomes on Candida albicans (yeast), Aspergillus niger (mould) and Trichophyton rubrum (dermatophyte) were evaluated by the agar well diffusion method. The aqueous and alcoholic filtered plant extracts were obtained by maceration, and also methanolic extracts were also obtained by Soxhlet extraction. The results were obtained by measuring the inhibition zone diameter in millimeters and were presented by subtracting the activity of the control. Results: Aqueous and methanolic extracts of Allium sativum gave the highest inhibition of the growth of Candida albicans (22 mm), followed closely by its propanolic extract with inhibition zone diameter of 15 mm, and also, propanolic extracts of Aframomum melegueta and Allium cepa gave inhibition zone diameters of 12 mm each. Soxhlet methanolic extract of Allium sativum had the highest inhibition of the growth of Aspergillus niger with an inhibition zone diameter of 25 mm, followed closely by Zingiber officinale Soxhlet methanolic extract with an inhibition zone diameter of 22 mm, also, the propanolic extract of Allium sativum gave an inhibition zone diameter of 21 mm, whereas Soxhlet methanolic extracts of Aframomum melegueta and Allium cepa gave an inhibition zone diameter of 19 mm each. The highest activity against Trichophyton rubrum was obtained with the Soxhlet methanolic extract of Allium sativum (39 mm), followed closely by its propanolic extract with an inhibition zone diameter of 27 mm. An inhibition zone diameter of 22 mm was recorded with the benzyl alcohol extract of Allium cepa, 22 mm with the Soxhlet methanolic extract of Aframomum melegueta and 19mm with the aqueous extract of Capsicum chinense seeds. Conclusion: The in-vitro inhibitory effects of these spice extracts indicated that the test spices could serve as potential candidates for developing new systemic and topical antifungal drugs against the wide range of pathogenic fungal strains, and they could also serve as natural prophylaxis against the fungal infections.

Antitumor and antimicrobial activities of endophytic fungi from medicinal parts of Aquilaria sinensis

The purpose of this study was to isolate and characterize endophytic fungi from the stem tissue which can produce fragrant ingredients in Aquilaria sinensis(also called agarwood) to determine their antitumor and antimicrobial activities.Twenty-eight fungal endophytes were isolated from agarwood by strict sterile sample preparation and were classified into 14 genera and 4 taxonomic classes(Sordariomycetes,Dothideomycetes,Saccharomycetes,and Zy-gomycetes) based on molecular identification.Of the 28 isolates,13(46.4%) showed antimicrobial activity against at least one of the test strains by the agar well diffusion method,and 23 isolates(82.1%) displayed antitumor activity against at least one of five cancer cell lines by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay.The diameters of inhibition zones of YNAS07,YNAS14,HNAS04,HNAS05,HNAS08,and HNAS11 were equal to or higher than 14.0 mm against Staphylococcus aureus,Escherichia coli,Bacillus subtilis,B.subtilis,Aspergillus fumigatus,and B.subtilis,respectively.The inhibition rates of YNAS06,YNAS08,and HNAS06 were not less than 60% to 293-T,293-T,and SKVO3 cells,respectively.These results suggest that the endophytic fungi associated with agarwood will provide us with not only useful micro-ecological information,but also potential antimicrobial and anti-tumor agents.

Antibacterial and antifungal activity of Terminalia arjuna Wight&Arn.bark against multi-drug resistant clinical isolates

Objective:To evaluate antimicrobial activity of Terminalia arjuna(T.arjuna)bark against clinical strains of multi-drug resistant bacteria,and Candida spp.isolated from patients,as well as the corresponding reference strains.Methods:The antimicrobial activity of water,methanol and chloroform extracts of T.arjuna bark were evaluated by agar-well diffusion method,followed by determination of minimum inhibitory concentration(MIC)by broth micro-dilution method.The clinical isolates were studied for antibacterial susceptibility by Kirby and Bauer disk diffusion technique.Results:The water and methanolic extracts of T.arjuna bark produced significant zones of inhibition against twenty-two tested bacteria including eight uropathogens.MIC values against the bacteria were found in the range of 0.16 to 2.56 mg/mL.The chloroform extract did not exhibit antibacterial activity.The polar extracts of T.arjuna also demonstrated strong antifungal effect against eight species of Candida,with MIC between 0.16 and 0.64 mg/mL.The antimicrobial efficacy of the polar extracts was found to be commensurate with high polyphenol content in contrast to the non-polar(chloroform fraction).Conclusions:This study has revealed the therapeutic prospect of T.arjuna bark for the treatment of microbial diseases.The polar fraction of the bark could be used for development of novel antimicrobial agents,particularly against urinary tract infections,and candidiasis/candidaemia.

Antimicrobial activity of crude and semi-purified fractions of Warburgia ugandensis against some pathogens

Objective:To investigate in vitro antimicrobial activities of leaves and heartwood crude and semi-purified fractions of Warburgia ugandensis(Canellaceae)(W.ugandensis)on some pathogens.Methods:were prepared.Six bacteria[Klebsiella pneumoniae(K.pneumoniae),Escherichia coli(E.coli),Pseudomonas aeruginosa Crude and semi-purified fractions of the leaves and heartwood of W.ugandensis(P.aeruginosa),Shigella boydii(S.boydii),Staphylococcus aureus(S.aureus)and Streptococcus pneumonia]and one fungus(Candida albicans)were tested by agar well diffusion and broth dilution method to determine minimum inhibitory concentration(MIC).Results:S.boydii and S.aureus were found to be the most susceptible bacterial isolated in agar well diffusion and broth dilution method of both the crude and petroleum ether extracts,while K.pneumoniae was the most resistant bacterium isolated under the same condition except in chloroform fraction.K.pneumoniae had shown MIC value of 10 mg/mL in the leaves and heartwood in both the crude and petroleum ether extract.S.boydii and S.aureus had shown the MIC value of 1.0 mg/mL in the crude extract for the both leaves and heartwood;Whereas the petroleum ether semi-purified fraction had shown 0.5 mg/mL in the heartwood.In the crude extract,E.coli and P.aeruginosa exhibits similar MIC value of 1.75 mg/mL.In semi purified petroleum ether extract,E.coli had MIC value of 1.0 mg/mL;Whereas P.aeruginosa had shown no change in crude extract.Candida albicans revealed equal MIC value of 1.0 mg/mL for the both crude and semi-purified fractions of the leaves and heartwood.Conclusions:The crude and semi-purified fractions of W.ugandensis have considerable effect on pathogens.Semi-purified petroleum ether fraction has better antimicrobial activity in both agar well diffusion and broth dilution method.This study further shows the potential of W.ugandensis for further study in order to be use as a modern drug.