Acidophilic and Acid Tolerant Actinobacteria as New Sources of Antimicrobial Agents against Helicobacter Pylori.

About half of the world's population is infected by Helicobacter pylori, which is related to various diseases. The increase in the resistance of H. pylori to antibiotics is alarming and requires new medication candidates. In this study, 83 acidic soil samples (pH 3.9-6.8) were collected from tea and rice farms, located in the semitropical strip in the north of Iran (Lahijan and Fooman cities, Gilan Province). After various pretreatments, including dry heating (120 oC, 10 min), exposure to electromagnetic waves (800 Hz, 3 min), and centrifuging (2950 g, 15 min), 33 acidophilic or acid-tolerant actinobacteria were isolated and their potentials as a source of active metabolites against H. pylori were investigated. According to phenotypic and molecular identification tests, the actinobacterial isolates were classified into Streptomyces and Kitasatospora genera. Among 10 strains that had anti-H. pylori activity, the highest potentials were seen in the strains UTMC 3061 and UTMC 3318. The minimum inhibitory concentrations (MIC) of the related metabolites were 125 and 62.5 µg/ml, respectively. In the checkerboard test, the metabolites of these actinobacteria showed synergism with clarithromycin and reduced its MIC from 1 to 0.5 µg/ml. However, no synergism was seen between the metabolites and amoxicillin or metronidazole. The gas chromatography-mass spectrometry (GC-MS) analysis of the metabolites showed some antimicrobial agents, including carbamic acid, maltol, 2.4-di-tert-butylphenol, methyl dimendone, prolylleucyl, and oleamide. The strains UTMC 3061 and UTMC 3318 showed 99.41 and 100% similarity in 16S rRNA gene sequence to Streptomyces spinoverrucosus and Streptomyces cirratus, respectively. Their metabolites showed good antibiotic activity and limited toxicity and can be considered as promising sources of natural products against H. pylori.

Biobleaching of mechanical paper pulp using Streptomyces rutgersensis UTMC 2445 isolated from a lignocellulose-rich soil.

AIMS: The utilization of micro-organisms in pulp and paper industries has proved biobleaching technology as an environmentally friendly alternative to the conventional approach. In this paper, the effect of actinobacterial fermentation broth on pulp biobleaching has been investigated. METHODS AND RESULTS: Actinobacterial colonies were isolated from lignocellulose-rich soil samples and screened for xylanase production and bleaching activity. The most efficient isolate in bleaching activity showed 100% similarity to Streptomyces rutgersensis. Pulp treatment with 5-day fermentation broth of this strain showed up to 7% increase in brightness (30°C for 6 h, pH (5-7)) compared to untreated (control) pulp. Also, after 60 min biotreatment, significant reduction (12·5%) in consumption of bleaching chemicals was achieved to obtain final brightness of 55%. CONCLUSION: Actinobacterial fermentation broth can be considered as a rich source of effective biobleaching agents which may be considered as environmental friendly and cost-effective technique in comparison with traditional method. SIGNIFICANCE AND IMPACT OF THE STUDY: Our findings showed ability of S. rutgersensis UTMC 2445 in bleaching chemomechanical paper pulp. Also, two strains of Saccharothrix, a rare actinobacterium, with biobleaching activity were introduced. In the proposed method, there is no need to use purified enzymes, and biobleaching process can be done using the fermentation broth.

Introduction of marine-derived Streptomyces sp. UTMC 1334 as a source of pyrrole derivatives with anti-acetylcholinesterase activity.

AIM: Alzheimer's disease (AD) is the most common cause of dementia. The acetylcholinesterase (AChE) inhibitors are the most viable therapeutic target for its symptomatic treatment. The present study was aimed at exploring anti-AChE metabolite producing marine Actinobacteria. METHODS AND RESULTS: Of 220 isolates, 34 Actinobacteria extracts were tested for the presence of AChE inhibitors. The obtained results showed that bacterial strain UTMC 1334, inhibited AChE activity in a dose-dependent manner (IC50  = 0·36 ± 0·019 mg ml-1 ). Based on anti-oxidant and cytotoxicity studies, the most potent extract was able to scavenge DPPH radicals with an IC50 value of 45·67 µg ml-1 with the least cytotoxicity. The GC-MS analysis demonstrated that the mentioned activities could be related to pyrrole-derived compounds as were found the predominant constituents in the extract. The most active extract belonged to the strain that shows 99·41% similarity with Streptomyces lateritius based on 16S rRNA gene sequencing. CONCLUSIONS: Our results show anti-AChE activity is prevalent in marine Actinobacteria, and even in rather comparable prevalence with the antibiotics. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights that exploring new lead anti-AChE compounds may result in discovering novel adjuvant candidates with potency in the treatment of cognitive diseases such as AD.

Simultaneous anti-diabetic and anti-vascular calcification activity of Nocardia sp. UTMC 751.

Alpha-amylase can act as a significant player in causing hyperglycaemia, leading to protein glycation, which is the main complication in this condition, besides causing vascular calcification (VC), an important vascular failure caused due to this. In order to find a natural source of the biocompounds with inhibitory effects on α-amylase, 15 fermentation broth extracts of actinobacteria (FBEA) (200 µg ml-1 ) have been screened. Finally, the effects of the most efficient FBE have been investigated on osteopontin (OPN, a VC marker) mRNA level in the vascular smooth muscle cells under the calcification conditions, and the chemical constituents of the most efficient FBE were analysed using gas chromatography and mass spectrometry (GC-MS) analysis. The tested FBEA showed anti-amylase (7·2-21%) and anti-denaturation (7·5-37%) activities. Among the tested FBEA, Nocardia sp. UTMC 751 FBE showed the highest anti-amylase activity (21%). This treatment group also displayed the minimum fructosamine and the maximum thiol groups content. In addition, this FBE reduced the mRNA level of the OPN (fourfold). The GC-MS analysis demonstrated the existence of three volatile and known antioxidants including pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl)-, pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(phenylmethyl)- and methyl ester of 3-(3,5-di-tert-butyl-4-hydroxyphenyl)-propionic acid in the FBE of Nocardia sp. UTMC 751. The results indicated that Nocardia sp. UTMC 751 is a considerable source of bioactive compounds that are effective against the direct and indirect pathological targets involved in diabetes. This study highlights the significant potential of rare Actinomycetes in producing pharmaceutically important biocompounds. SIGNIFICANCE AND IMPACT OF THE STUDY: Actinobacteria are one of the best natural libraries for discovering drugs. Various commercial drugs have been developed against infectious and metabolic disorders from actinobacteria; however, there is no report on their simultaneous inhibitory effect against diabetes, a life-threatening disease, and its related pathological processes, like inflammation and vascular calcification (VC). In this research, after several screening, Nocardia sp. UTMC 751 was introduced as the first microbial source exhibiting a simultaneous inhibitory activity on the targets, including hyperglycaemia and protein glycation, and other involved pathological processes like inflammation and VC.

Isolation and screening of rare Actinobacteria, a new insight for finding natural products with antivascular calcification activity.

AIM: Vascular calcification (VC) is a significant pathological process in some life-threatening diseases. Several pathological mechanisms, including transdifferentiation of vascular smooth muscle cells to osteoblast-like cells and apoptosis are involved in VC. Compounds with an inhibitory effect on these processes are potentially efficient medications. In consideration of the multiple biological activities of Actinobacteria, this research was aimed at finding anti-VC metabolite-producing Actinobacteria. METHODS AND RESULTS: After the isolation and identification of Actinobacteria, the effect of their fermentation broth extracts on the apoptosis rate was measured using various methods, for example, ethidium bromide/acridine orange staining, DNA laddering and diphenylamine assays. The effect of the most effective fermentation broth extract of Actinobacteria (FBEA) on the mRNA expression of runt-related transcription factor 2 (Runx2) and osteopontin (OPN) was examined. Finally, the most effective FBEA was fractionated and the chemical composition of anti-VC fractions was analysed using GC-MS. Various VC inhibition rates were observed in the tested FBEA (20 µg ml-1 ; 17·9-60·15%). The inhibition of DNA fragmentation was 7-48%. The FBE with the greatest anticalcification activity belonged to Kribbella sp. UTMC 267 and, according to 16S rRNA analysis, Kribbella sancticallisti with a similarity of 98·53% is its nearest neighbour. The FBE of Kribbella sp. UTMC 267 reduced Runx2 mRNA expression by 2·95-fold and OPN mRNA expression by 28·57-fold, both of which are considered significant (P < 0·05). Finally, GC-MS analysis showed the existence of potent anti-oxidative and anti-inflammation agents in FBE of Kribbella sp. UTMC 267. CONCLUSIONS: Actinobacterial metabolites can provide a new strategy for treating VC diseases by reducing the expression of osteogenic genes, the apoptosis rate and oxidative stress. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights the therapeutic potential of Kribbella sp. metabolites and Actinobacteria as a new natural source for drug discovery programs in the nonantibiotic bioactivity field.

Isolation and screening of proangiogenic and antiangiogenic metabolites producing rare actinobacteria from soil.

AIMS: Angiogenesis is a physiological process that has important impacts on the pathology and healing of various diseases, and its induction or inhibition by bioactive actinobacterial metabolites can help the treatment of some diseases. In this study, the effects of actinobacterial extract in the process of angiogenesis have been explored. METHODS AND RESULTS: In this research, proangiogenic and antiangiogenic metabolites producing actinobacteria were isolated from soil samples and their fermentation broth were extracted and after evaluation of their toxicity by MTT assay, antiangiogenic and proangiogenic activities were screened against human umbilical vein endothelial cells (HUVECs) by in vitro tube formation and migration assay. Isolated strains were identified through molecular techniques. The results showed that Nocardiopsis arvandica UTMC 103 and Nonomuraea sp. UTMC 2180 extracts had a high potential of anti-angiogenic activity on HUVECs. CONCLUSIONS: For the first time proangiogenic potency of a rare actinobacterium, Kribbella sp. UTMC 522, was reported, and N. arvandica UTMC 103 and Nonomuraea sp. UTMC 2180 extracts inhibits the proliferation, migration and angiogenesis activity of HUVECs with reasonable potency. SIGNIFICANCE AND IMPACT OF THE STUDY: Metabolites of the introduced rare actinobacteria are potent proangiogenic and angiogenic inhibitors. Identification of angiogenic-antiangiogenic mechanisms and purification of the extracts would be useful in therapeutic angiogenesis.

A cylinder-plate method for microbiological assay of clavulanic acid.

Clavulanic acid is a natural occurring beta-lactam product of Streptomyces clavuligerus and is a potent inhibitor of bacterial beta-lactamases. The present work reports a microbiological assay based on the cylinder-plate method for determination of clavulanic acid. The assay is based on the inhibitory effect of clavulanic acid in combination with penicillin G upon Escherichia coli ATCC 35218, which is used as the test organism. The correlation between clavulanic acid concentration and the inhibitory effect on E. coli was linear (r > 0.99) and in the range of 8-20 microg/ml. These results indicate that the proposed method is appropriate for the determination of clavulanic acid in commercial samples and can be used in routine quality control.

Increased erythromycin production by alginate as a medium ingredient or immobilization support in cultures of Saccharopolyspora erythraea.

Erythromycin production by Saccharopolyspora erythraea immobilized in 2% (w/v) calcium alginate or grown in medium containing 20 g sodium alginate/l inoculated with free cells was almost twice more than that of the control. S. erythraea did not consume alginate, agar, dextran, silicon antifoaming agent or cyclodextrin as a carbon source, although, all of these increased the production of erythromycin. Highest titer of erythromycin (2.3 times more than that of the control) was achieved in medium containing 1 g agar/l.

Enhancing of erythromycin production by Saccharopolyspora erythraea with common and uncommon oils.

The enhancing effect of various concentrations of 18 oils and a silicon antifoam agent on erythromycin production by Saccharopolyspora erythraea was evaluated in a complex medium containing soybean flour and dextrin as the main substrates. The oils used consisted of sunflower, pistachio, cottonseed, melon seed, water melon seed, lard, corn, olive, soybean, hazelnut, rapeseed, sesame, shark, safflower, coconut, walnut, black cherry kernel and grape seed oils. The biomass, erythromycin, dextrin and oil concentrations and the pH value were measured. Also, the kinds and frequencies of fatty acids in the oils were determined. The productivity of erythromycin in the oil-containing media was higher than that of the control medium. However, oil was not suitable as a main carbon source for erythromycin production by S. erythraea. The highest titer of erythromycin was produced in medium containing 55 g/l black cherry kernel oil (4.5 g/l). The titers of erythromycin in the other media were also recorded, with this result: black cherry kernel > water melon seed > melon seed > walnut > rapeseed > soybean > (corn = sesame) > (olive = pistachio = lard = sunflower) > (hazelnut = cotton seed) > grape seed > (shark = safflower = coconut). In media containing various oils, the hyphae of S. erythraea were longer and remained in a vegetative form after 8 days, while in the control medium, spores were formed and hyphae were lysed.

Inhibition of nitrofurantoin reduction by menthol leads to enhanced antimicrobial activity.

Nitrofurantoin is a nitroaromatic compound used for the treatment of urinary tract infections. Nitrofurantoin activity is regulated by a nitroreduction process. It is first reduced by bacterial nitroreductases to active short-life intermediates, which are further converted to non-toxic molecules, which negatively affect its antibacterial activity. In this study we have shown that resistant strains of Klebsiella sp. inactivate the bactericidal activity of nitrofurantoin. Also we demonstrated a synergistic effect between menthol and nitrofurantoin bactericidal activities against nitrofurantoin susceptible Enterobacteriaceae.