Species of Agave with antimicrobial activity against selected pathogenic bacteria and fungi

The in vitro antimicrobial activity against pathogenic bacteria, yeast, and molds were examined in extracts of the Agave species A. lecheguilla, A. picta, A. scabra and A. lophanta using an agar diffusion technique. The extracts of A. picta produced zones of inhibition of 9–13 mm for E. coli, L. monocytogenes, S. aureus, and V. cholerae, while B. cereus and Y. enterocolitica were not inhibited. The other Agave species did not show any detectable inhibitory activity against the bacteria tested; however, all four Agave sp. were inhibitory against all yeast and molds analyzed as evident by 9–20 mm zones of inhibition. The minimum microbicidal concentration (MMC) of the active extract ranged from 1.8 to 7.0 mg/ml for the sensitive bacteria, and 2.0–3.0 mg/ml for yeast. In the case of molds, the minimum inhibitory concentration (MIC) of the active extracts ranged from 3.0 to 6.0 mg/ml. Together, these data suggest that the Agave sp. analyzed are potential antimicrobial candidates with a broad range of activity.     

Mitogenic response and probiotic characteristics of lactic acid bacteria isolated from indigenously pickled vegetables and fermented beverages

Lactic acid bacteria from indigenous pickled vegetables and fermented beverages (fermented rice and Madhuca longifolia flowers) were isolated and investigated for their functional characteristics in vitro as potential new probiotic strains. Four isolates (all Lactobacillus spp.) selected on the basis of high tolerance to bile (0.2%) were identified by standard and molecular methods (16S rDNA) as L. helveticus, L. casei, L. delbrueckii and L. bulgaricus from pickled vegetables and fermented beverages respectively. These selected strains had antibiotic resistance, tolerance to artificial gastric juice and phenol (0.4%), enzymatic profile, and antagonistic activity against enteric pathogens (Enterobacter sakazakii, Salmonella typhimurium, Shigella flexneri 2a, Listeria monocytogenes, Yersinia enterocolitica and Aeromonas hydrophila). All strains survived well in artificial gastric juice at low pH (3.0) values for 4 h, possessed bile salt hydrolase activity and were susceptible to most antibiotics including vancomycin. Additionally, the isolates exhibited high tolerance to phenol, high cell surface hydrophobicity (>60%) and induced proliferation of murine splenocytes. All the four strains of present study suppressed the Con A-stimulated proliferation of the mouse spleen cells, although L. casei had the strongest suppressive effect. The results of this study suggest a potential application of the strains (following human clinical trials), for developing probiotic foods.     

Antimicrobial activity of an endophytic <Emphasis Type="Italic">Xylaria</Emphasis> sp.YX-28 and identification of its antimicrobial compound 7-amino-4-methylcoumarin

An endophytic Xylaria sp., having broad antimicrobial activity, was isolated and characterized from Ginkgo biloba L. From the culture extracts of this fungus, a bioactive compound P3 was isolated by bioactivity-guided fractionation and identified as 7-amino-4-methylcoumarin by nuclear magnetic resonance, infrared, and mass spectrometry spectral data. The compound showed strong antibacterial and antifungal activities in vitro against Staphylococcus aureus [minimal inhibitory concentrations (MIC) 16 μg·ml⁻¹], Escherichia coli (MIC, 10 μg·ml⁻¹), Salmonella typhia (MIC, 20 μg·ml⁻¹), Salmonella typhimurium (MIC, 15 μg·ml⁻¹), Salmonella enteritidis (MIC, 8.5 μg·ml⁻¹), Aeromonas hydrophila (MIC, 4 μg·ml⁻¹), Yersinia sp. (MIC, 12.5 μg·ml⁻¹), Vibrio anguillarum (MIC, 25 μg·ml⁻¹), Shigella sp. (MIC, 6.3 μg·ml⁻¹), Vibrio parahaemolyticus (MIC, 12.5 μg·ml⁻¹), Candida albicans (MIC, 15 μg·ml⁻¹), Penicillium expansum (MIC, 40 μg·ml⁻¹), and Aspergillus niger (MIC, 25 μg·ml⁻¹). This is the first report of 7-amino-4-methylcoumarin in fungus and of the antimicrobial activity of this metabolite. The obtained results provide promising baseline information for the potential use of this unusual endophytic fungus and its components in the control of food spoilage and food-borne diseases.     

Possible use of <Emphasis Type="Italic">ail</Emphasis> and <Emphasis Type="Italic">foxA</Emphasis> polymorphisms for detecting pathogenic <Emphasis Type="Italic">Yersinia enterocolitica</Emphasis>

Background  

Yersinia enterocolitica is an enteric pathogen that invades the intestinal mucosa and proliferates within the lymphoid follicles (Peyer's patches). The attachment invasion locus (ail) mediates invasion by Y. enterocolitica and confers an invasive phenotype upon non-invasive E. coli; ail is the primary virulence factor of Y. enterocolitica. The ferrioxamine receptor (foxA) located on the Y. enterocolitica chromosome, together with its transport protein, transports a siderophore specific for ferric ion. Currently, ail is the primary target gene for nucleic acid detection of pathogenic Y. enterocolitica.     

Specific identification of pathogenic Yersinia enterocolitica by monoclonal antibodies generated against recombinant attachment invasion locus (rAil) protein

Classical pathogenic strains of Yersinia enterocolitica produce a 17 kDa outer membrane protein, Ail (attachment-invasion locus), which mediates bacterial attachment to some cultures epithelial cell lines and invasion of others. In the present study, hybridomas were developed for the production of monoclonal antibodies (MAbs) against Ail protein of Y. enterocolitica. A set of five stabilized hybridoma cell lines were generated, of which, two MAbs, YEA 302 and YEA 303, exhibited specific reaction to the native Ail protein (17 kDa) present in whole cell lysate of Y. enterocolitica strains beside having reaction with rAil. The other three MAbs, YEA 5, 17 and 32, had some cross reactions with proteins other than Ail. Two out of five MAbs were IgG1, two were IgG2b and one in IgM in nature. MAbs (YEA 302 and YEA 303) did not show any cross-reaction with whole cell lysate of Brucella abortus, Vibrio cholerae, Salmonella typhimurium and Escherichia coli and other species of Enterobacteriaceae including Y. pestis in ELISA and Western blot analysis. The presence of Ail protein among the strains recovered from pork and milk samples was evaluated by these sets of MAbs and the results were compared with the duplex PCR. Collectively, the data suggest that these MAbs may have the potential for their use in the detection of pathogenic Y. enterocolitica reliably, rapidly and at a relatively low cost.     

Antibacterial Flavonoids and Other Compounds from the Aerial Parts of Vernonia guineensis Benth. (Asteraceae)

An extensive phytochemical study of the aerial parts of Vernonia guineensis Benth. (Asteraceae) led to the isolation of a new flavone, vernoguinoflavone and a naturally isolated glycerol ester, eicosanoic acid 2‐hydroxy‐1,3‐propanediyl ester, together with eighteen known secondary metabolites including quercetin, luteolin, vernopicrin, vernomelitensin, β‐amyrin, oleanolic acid, ursolic acid, lupeol, betulinic acid, β‐carotene, a mixture of stigmasterol and β‐sitosterol, β‐sitosterol‐3‐O‐β‐D‐glucoside, 2,3‐dihydroxypropyl heptacosanoate, pentacosanoic acid, docosan‐1‐ol, tritriacontan‐1‐ol, and heptatriacontan‐1‐ol. Eleven compounds are reported herein for the first time from this species. The structures of these compounds were elucidated on the basis of extensive spectroscopic analyses, particularly 1D and 2D NMR, and HR‐ESI‐MS and by comparison of their data with those reported in the literature. The crude extract, fractions and some isolated compounds were evaluated for their antibacterial activity against Gram‐negative bacteria: Escherichia coli (ATCC 25922), Shigella flexineri (NR 518), Salmonella muenchen, Salmonella typhimurium and Salmonella typhi (ATCC 19430). All the tested compounds demonstrated inhibitory activities against the tested enteric bacteria with MIC values ranging from 3.12 to 100 μg/ml. Three flavonoids isolated from the most active fraction demonstrated the best bioactivities against Escherichia coli, Salmonella muenchen and Salmonella typhimurium with MIC values ranging from 3.12 to 25 μg/mL.     

Evaluation of in vitro antimicrobial activity of leaf and stem essential oils of <Emphasis Type="Italic">Chloroxylon swietenia</Emphasis> DC.

The essential oils obtained by hydrodistillation of leaves and stems of Chloroxylon swietenia DC. were analysed by GC and GC-MS. The main components in the leaf oil were limonene, pregeijerene, geijerene and germacrene D, while stem oil was rich in limonene, methyl eugenol, pregeijerene and geijerene. The essential oils were evaluated for antimicrobial activity against two gram-positive and two gram-negative bacteria and four pathogenic fungi using agar disc diffusion technique. Subsequently, the minimum inhibitory concentration (MIC) from oils was determined by broth microdilution. Both the oils exhibited moderate to strong activities against all the organisms tested. Bacillus subtilis was most susceptible at 100 μg/ml of leaf and stem oils with inhibition zones of 15.9 and 13.1 mm respectively. Among all the fungi tested, A. niger inhibited effectively with a zone of inhibition of 14.9 and 11.5 mm for leaf and stem oils respectively. The results obtained suggest that the essential oils of the plant possess antimicrobial properties and serve as a biofriendly source of antimicrobial ingredients for the food and pharmaceutical industries.     

36种中药材体外抗菌活性筛选研究

该文研究36种常用中药材80%乙醇提取物在体外抗临床常见致病菌的抗菌活性。采用药敏纸片法测耐药菌的耐药谱,中药粗粉用80%乙醇浸泡提取,提取液减压浓缩得浸膏,通过琼脂打孔法测定提取物抑菌圈,再通过微量倍比稀释法测定最低抑菌浓度(MIC)和最低杀菌浓度(MBC)。结果表明:36种中药材醇提物中,有15种具有广谱抗菌活性,对实验中各标准菌表现出不同程度的抑制作用,对MRSA抗菌活性也较强。其中,岩陀、卷柏、首乌藤、苏木、乌药、夏枯草6种药材的抗菌活性比较突出,抑菌圈均大于11 mm,细菌对其表现为中高度敏感;它们对7株标准菌的MIC/MBC值除个别为12.5 mg·m L~(-1)以外,均小于1.563 mg·m L~(-1),对16株MRSA的MIC/MBC值均小于1.563 mg·m L~(-1),它们的萃取层活性均小于1 mg·m L~(-1)。所筛选出的15种抗菌活性较强的中药材,可为后续研究其活性单体化合物和作用机制,研发有效的抗多重耐药菌的中药制剂以及解决细菌耐药性问题提供一定的参考。     

In-vitro evaluation of the antimicrobial activity of Bauhinia variegata,locally known as koiralo

Bauhinia variegata, commonly known as Koiralo is considered as medicinal plant in Nepal and India. The alcoholic extract of this plant was found to have antimicrobial activity against Bacillus subtilis (ATCC 6635) Pseudomonas aeruginosa (ATCC 27853), Salmonella typhi, Shigella dysenteriae, Staphylococcus aureus (ATCC 29213) and Vibrio cholerae. The largest zone of inhibition (18 mm) was found to be exhibited against B. subtilis. For this organism the minimum bactericidal concentration (MBC) of the crude extract was 0.39 mg/ml. The extract was found to be more effective against gram-positive than gram-negative bacteria. The antimicrobial activity of the extract was found to be decreased during purification.     

Spontaneous binding ofYersinia enterocolitica to murine leukocytes

Twelve strains ofYersinia enterocolitica were examined for their ability to bind spontaneously to murine leukocytes. Each of eight HeLa cell invasive strains exhibited nonselective binding to peritoneal leukocytes, lymph node leukocytes, and thymocytes, whereas four noninvasive strains lacked binding properties. Like the HeLa cell invasion, the binding ofY. enterocolitica to leukocytes was much less efficient for bacteria grown at 37°C than for bacteria grown at 22°C. The binding properties were not influenced by the virulence plasmid that codes for Vwa⁺ phenotype. This leukocyte binding test is proposed as a simple assay for invasive properties ofY. enterocolitica.     

Antimicrobial and anti-dust mite efficacy of Cinnamomum camphora chvar. Borneol essential oil using pilot-plant neutral cellulase-assisted steam distillation

Cinnamomum camphora chvar. Borneol essential oil (BEO) was efficiently extracted by using pilot-plant neutral cellulase-assisted steam distillation (NCSD). Borneol, β-cadinene and α-caryophyllene were identified as major components. Bacillus subtilis was the most sensitive bacteria to BEO with the lowest minimal inhibition concentration (MIC) and minimal bactericial concentration (MBC) at 1·75 and 3·50 mg ml⁻¹, respectively. Antimicrobial activity of the BEO was also reasonably high against Salmonella typhimurium, Escherichia coli and Staphylococcus aureus, but not sensitive against two fungi, i.e. Aspergillus niger and Penicillium aurantiogriseum. Changes in permeability and integrity of cell membrane, damage of cell wall and further leakage out of metabolites and ions were determined as bactericidal mechanisms of BEO against the two gram-positive bacteria. The BEO showed a reasonably high repelling activity of dust mite, which achieved higher than 95% repelling dust mite activity after the treatment of BEO solution at 0·50 mg ml⁻¹. When the concentration of BEO was higher than 0·50 mg ml⁻¹, it was B-grade miticide with miticidal activity higher than 95%. Miticidal procedures were characterized as excitation, contraction, relaxation and lastly leading to the death of dust mite. It is speculated that the BEO would cause dehydration and death of dust mite as neuromuscular toxicity.     

Molecular and biochemical characterization of urease and survival of Yersinia enterocolitica biovar 1A in acidic pH in vitro

Background  

Yersinia enterocolitica, an important food- and water-borne enteric pathogen is represented by six biovars viz. 1A, 1B, 2, 3, 4 and 5. Despite the lack of recognized virulence determinants, some biovar 1A strains have been reported to produce disease symptoms resembling that produced by known pathogenic biovars (1B, 2-5). It is therefore imperative to identify determinants that might contribute to the pathogeniCity of Y. enterocolitica biovar 1A strains. Y. enterocolitica invariably produces urease and the role of this enzyme in the virulence of biovar 1B and biovar 4 strains has been reported recently. The objective of this work was to study genetic organization of the urease (ure) gene complex of Y. enterocolitica biovar 1A, biochemical characterization of the urease, and the survival of these strains under acidic conditions in vitro.     

Evaluation of isolation methods for pathogenic Yersinia enterocolitica from pig intestinal content

Aims: The aim of this study was to evaluate the efficiency of four isolation methods for the detection of pathogenic Yersinia enterocolitica from pig intestinal content. Methods and Results: The four methods comprised of 15 isolation steps using selective enrichments (irgasan–ticarcillin–potassium chlorate and modified Rappaport broth) and mildly selective enrichments at 4 or 25°C. Salmonella–Shigella‐desoxycholate–calcium chloride agar, cefsulodin–irgasan–novobiocin agar were used as plating media. The most sensitive method detected 78% (53/68) of the positive samples. Individual isolation steps using cold enrichment as the only enrichment or as a pre‐enrichment step with further selective enrichment showed the highest sensitivities (55–66%). All isolation methods resulted in high numbers of suspected colonies not confirmed as pathogenic Y. enterocolitica. Conclusions: Cold enrichment should be used in the detection of pathogenic Y. enterocolitica from pig intestinal contents. In addition, more than one parallel isolation step is needed. Significance and Impact of the Study: The study shows that depending on the isolation method used for Y. enterocolitica, the detected prevalence of Y. enterocolitica in pig intestinal contents varies greatly. More selective and sensitive isolation methods need to be developed for pathogenic Y. enterocolitica.     

Synergistic effect of green tea extract and probiotics on the pathogenic bacteria, <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> and <Emphasis Type="Italic">Streptococcus pyogenes</Emphasis>

The aim of this study was to assess the effect of a commercial green tea extract (TEAVIGO™) on the microbial growth of three probiotic strains (Lactobacillus and Bifidobacterium), as well as three pathogenic bacteria. MIC and co-culture studies were performed. The MICs of the green tea extract against Staphylococcus aureus and Streptococcus pyogenes (100 μg ml⁻¹) were considerably lower than those against the probiotic strains tested (>800 μg ml⁻¹) and Escherichia coli (800 μg ml⁻¹). In co-culture studies, a synergistic effect of the probiotic strains and the green tea extract was observed against both Staph. aureus and Strep. pyogenes. Green tea extract in combination with probiotics significantly reduced the viable count of both pathogens at 4 h and by 24 h had completely abolished the recovery of viable Staph. aureus and Strep. pyogenes. These reductions were more significant than the reductions induced by probiotics or green tea extracts used separately. These results demonstrate the potential for combined therapy using the green tea extract plus probiotics on microbial infections caused by Staph. aureus and Strep. pyogenes. As probiotics and the green tea extract are derived from natural products, treatment with these agents may represent important adjuncts to, or alternatives to, conventional antibiotic therapy.     

Purification of bioactive natural product against human microbial pathogens from marine sea weed <Emphasis Type="Italic">Dictyota acutiloba</Emphasis> J. Ag.

Herbal extracts play an essential role in treating various diseases. The threats in drug resistant pathogenic microbial strains can be prevented by the un-tapped medicinal principles from plants. The present study has been focused to search for powerful antimicrobial natural products from Dictyota acutiloba J. Ag. against human enteric pathogens and dermatophytic fungi. Chloroform and acetone extracts of Dictyota acutiloba exhibited antimicrobial activity against methicillin resistant Staphylococcus aureus (MRSA), methicillin susceptible Staphylococcus aureus (MSSA), Enterobacter sp., Pseudomonas aeruginosa MTCC741, Salmonella typhi MTCC733, Bacillus subtilis, Klebsiella pneumoniae MTCC109, Candida albicans and Aspergillus niger MTCC281. Purified compounds A₁ and C₁ by column chromatography, TLC and HPLC inhibited the gram positive, gram negative bacteria and fungi. MIC of C₁ and A₁ ranged between 0.5 and 0.9 μg ml⁻¹. The absorption maximum of C₁ and A₁ was 355 nm. Structural characterization of these purified molecules can lead to the new therapeutic molecule to fight the pathogenic microorganisms.     

Effect of garlic and allium-derived products on the growth and metabolism of Spironucleus vortens

Spironucleus is a genus of small, flagellated parasites, many of which can infect a wide range of vertebrates and are a significant problem in aquaculture. Following the ban on the use of metronidazole in food fish due to toxicity problems, no satisfactory chemotherapies for the treatment of spironucleosis are currently available. Using membrane inlet mass spectrometry and automated optical density monitoring of growth, we investigated in vitro the effect of Allium sativum (garlic), a herbal remedy known for its antimicrobial properties, on the growth and metabolism of Spironucleus vortens, a parasite of tropical fish and putative agent of hole-in-the-head disease. The allium-derived thiosulfinate compounds allicin and ajoene, as well as an ajoene-free mixture of thiosulfinates and vinyl-dithiins were also tested. Whole, freeze-dried garlic and allium-derived compounds had an inhibitory effect on gas metabolism, exponential growth rate and final growth yield of S. vortens in Keister’s modified, TY-I-S33 culture medium. Of all the allium-derived compounds tested, the ajoene-free mixture of dithiins and thiosulfinates was the most effective with a minimum inhibitory concentration (MIC) of 107 μg ml⁻¹ and an inhibitory concentration at 50% (IC_50%) of 58 μg ml⁻¹. It was followed by ajoene (MIC = 83 μg ml⁻¹, IC_50% = 56 μg ml⁻¹) and raw garlic (MIC >20 mg ml⁻¹, IC_50% = 7.9 mg ml⁻¹); allicin being significantly less potent with an MIC and IC_50% above 160 μg ml⁻¹. All these concentrations are much higher than those reported to be required for the inhibition of most bacteria, protozoa and fungi previously investigated, indicating an unusual level of tolerance for allium-derived products in S. vortens. However, chemically synthesized derivatives of garlic constituents might prove a useful avenue for future research.     

苹果渣乙醇提取物抗菌活性及防腐作用研究

为提高苹果渣资源利用率,探究苹果渣乙醇提取物的抗菌活性和防腐性能,该文采用微波辅助提取法制取苹果渣乙醇提取物,用抑菌圈实验测定其抗菌活性,并研究了其防腐作用。结果表明:(1)苹果渣乙醇提取物对酵母菌抑制作用不明显(抑菌圈直径<1 mm),对金黄色葡萄球菌和大肠杆菌的抑菌作用较明显(抑菌圈直径为6～9 mm),最佳抑菌浓度为4.0 g·L^-1。(2)pH值和盐浓度对其抑菌效果有影响,pH值为6～7,盐浓度为5.0 g·L^-1,抑菌效果最好。(3)对百香果有较好的保鲜防腐效果,最佳使用浓度为0.2%。在该浓度下贮藏后的百香果腐烂率为6.7%(对照组为67%),失重率为5.5%(对照组为36.3%),可溶性固形物、总酸含量均与贮藏前差异不显著(P> 0.05)(对照组P< 0.05),且果实较饱满,硬度较高,鲜艳有光泽,酸甜适中。综上所述,苹果渣乙醇提取物对大肠杆菌和金黄色葡萄球菌具有良好的抑制作用,对百香果的保鲜防腐效果佳,可应用到天然食品的保鲜防腐。     

In Vitro Antimicrobial Activity of <Emphasis Type="Italic">Acacia catechu</Emphasis> and Its Phytochemical Analysis

Acacia catechu, commonly known as catechu, cachou and black cutch is an important medicinal plant and an economically important forest tree. The methanolic extract of this plant was found to have antimicrobial activities against six species of pathogenic and non-pathogenic microorganisms: Bacillus subtilis, Staphylococcus aureus, Salmonella typhi, Escherichia coli, Pseudomonas aeruginosa and Candida albicans. The maximum zone of inhibition (20 mm) was found to be exhibited against S. aureus. For this organism the minimum bactericidal concentration (MBC) of the crude extract was 1,000 μg/ml. The extract was found to be equally effective against gram positive and gram negative bacteria. The antimicrobial activity of the extract was found to be decreased during purification. The chemical constituents of organic plant extracts were separated by thin layer chromatography (TLC) and the plant extracts were purified by column chromatography and were further identified by Gas chromatography–mass selection (GC–MS) analysis. The composition of A. catechu extract had shown major components of terpene i.e. camphor (76.40%) and phytol (27.56%) along with other terpenes in minor amounts which are related with their high antibacterial and antifungal properties.     

Probiotic Characterization of Lactobacillus paracasei subsp. paracasei KNI9 Inhibiting Adherence of Yersinia enterocolitica on Caco-2 Cells In Vitro

The study was aimed to characterize the probiotic properties of Lactobacillus paracasei subsp. paracasei strain KNI9 and its antagonistic activity against Yersinia enterocolitica subsp. enterocolitica. The strain KNI9 was susceptible to antibiotics such as chloramphenicol, tetracycline, erythromycin, and streptomycin recommended by European food safety authority (EFSA). Strain KNI9 exhibited tolerance to simulated oro-gastrointestinal (OGT) condition, adherence to Caco-2 cells, and antimicrobial activity against intestinal enteric pathogens such as Yersinia enterocolitica subsp. enterocolitica, Shigella boydii, and Listeria monocytogenes. Furthermore, the strain KNI9 inhibited the adherence and invasiveness of Y. enterocolitica subsp. enterocolitica to Caco-2 cell line. These results indicate that the L. paracasei subsp. paracasei KNI9 could be further developed into a potential probiotic strain after appropriate in vivo studies.

     

Investigation of Chondrus crispus as a potential source of new antifouling agents

The search for environment-friendly and non-toxic antifouling (AF) paint components has led to the investigation of natural products from seaweeds. The defence metabolites used by algae to deter unwanted epibiosis have potential for harnessing and use in AF applications. Crude algal extracts may provide a suitable mixture of compounds with AF potency. Crude ethanol extracts of the macroalgae Chondrus crispus (Rhodophyceae), from both dried and fresh sources were tested and compared using bioassays based on five marine bacterial strains, five phytoplankton strains and two macroalgae to assess the AF efficacy. Dried extract from the algae had a lower minimum inhibitory concentration at 25 μg mL⁻¹ against the growth of bacteria and phytoplankton species than that from the fresh source. Macroalgae tests indicated that the extracts had an anti-germination activity 25–50 μg mL⁻¹ against both Undaria pinnatifida and Ulva intestinalis spores. A field trial of AF paint incorporating crude extract indicated an initial AF potency lasting six weeks.