Penicillin-Binding Proteins, β-Lactamases,and β-Lactamase Inhibitors in β-Lactam-Producing Actinobacteria: Self-Resistance Mechanisms

The human society faces a serious problem due to the widespread resistance to antibiotics in clinical practice. Most antibiotic biosynthesis gene clusters in actinobacteria contain genes for intrinsic self-resistance to the produced antibiotics, and it has been proposed that the antibiotic resistance genes in pathogenic bacteria originated in antibiotic-producing microorganisms. The model actinobacteria Streptomyces clavuligerus produces the β-lactam antibiotic cephamycin C, a class A β-lactamase, and the β lactamases inhibitor clavulanic acid, all of which are encoded in a gene supercluster; in addition, it synthesizes the β-lactamase inhibitory protein BLIP. The secreted clavulanic acid has a synergistic effect with the cephamycin produced by the same strain in the fight against competing microorganisms in its natural habitat. High levels of resistance to cephamycin/cephalosporin in actinobacteria are due to the presence (in their β-lactam clusters) of genes encoding PBPs which bind penicillins but not cephalosporins. We have revised the previously reported cephamycin C and clavulanic acid gene clusters and, in addition, we have searched for novel β-lactam gene clusters in protein databases. Notably, in S. clavuligerus and Nocardia lactamdurans, the β-lactamases are retained in the cell wall and do not affect the intracellular formation of isopenicillin N/penicillin N. The activity of the β-lactamase in S. clavuligerus may be modulated by the β-lactamase inhibitory protein BLIP at the cell-wall level. Analysis of the β-lactam cluster in actinobacteria suggests that these clusters have been moved by horizontal gene transfer between different actinobacteria and have culminated in S. clavuligerus with the organization of an elaborated set of genes designed for fine tuning of antibiotic resistance and cell wall remodeling for the survival of this Streptomyces species. This article is focused specifically on the enigmatic connection between β-lactam biosynthesis and β-lactam resistance mechanisms in the producer actinobacteria.     

Quorum Quenching in Culturable Phyllosphere Bacteria from Tobacco

Many Gram-negative plant pathogenic bacteria employ a N-acylhomoserine lactone (AHL)-based quorum sensing (QS) system to regulate their virulence traits. A sustainable biocontrol strategy has been developed using quorum quenching (QQ) bacteria to interfere with QS and protect plants from pathogens. Here, the prevalence and the diversity of QQ strains inhabiting tobacco leaf surfaces were explored. A total of 1177 leaf-associated isolates were screened for their ability to disrupt AHL-mediated QS, using the biosensor Chromobacterium violaceum CV026. One hundred and sixty-eight strains (14%) are capable of interfering with AHL activity. Among these, 106 strains (63%) of the culturable quenchers can enzymatically degrade AHL molecules, while the remaining strains might use other QS inhibitors to interrupt the chemical communication. Moreover, almost 79% of the QQ strains capable of inactivating AHLs enzymatically have lactonase activity. Further phylogenetic analysis based on 16S rDNA revealed that the leaf-associated QQ bacteria can be classified as Bacillus sp., Acinetobacter sp., Lysinibacillus sp., Serratia sp., Pseudomonas sp., and Myroides sp. The naturally occurring diversity of bacterial quenchers might provide opportunities to use them as effective biocontrol reagents for suppressing plant pathogen in situ.     

An Overview: The Toxicity of Ageratina adenophora on Animals and Its Possible Interventions

Ageratina adenophora is one of the major invasive weeds that causes instability of the ecosystem. Research has reported that A. adenophora produces allelochemicals that inhibit the growth and development of food crops, and also contain some toxic compounds that cause toxicity to animals that consume it. Over the past decades, studies on the identification of major toxic compounds of A. adenophora and their toxic molecular mechanisms have been reported. In addition, weed control interventions, such as herbicides application, was employed to reduce the spread of A. adenophora. However, the development of therapeutic and prophylactic measures to treat the various A. adenophora—induced toxicities, such as hepatotoxicity, splenotoxicity and other related disorders, have not been established to date. The main toxic pathogenesis of A. adenophora is oxidative stress and inflammation. However, numerous studies have verified that some extracts and secondary metabolites isolated from A. adenophora possess anti-oxidation and anti-inflammation activities, which implies that these extracts can relieve toxicity and aid in the development of drug or feed supplements to treat poisoning-related disorders caused by A. adenophora. Furthermore, beneficial bacteria isolated from rumen microbes and A. adenophora can degrade major toxic compounds in A. adenophora so as to be developed into microbial feed additives to help ameliorate toxicity mediated by A. adenophora. This review presents an overview of the toxic mechanisms of A. adenophora, provides possible therapeutic strategies that are available to mitigate the toxicity of A. adenophora and introduces relevant information on identifying novel prophylactic and therapeutic measures against A. adenophora—induced toxicity.     

Metabolomics Analysis Reveals Specific Novel Tetrapeptide and Potential Anti-Inflammatory Metabolites in Pathogenic Aspergillus species

Infections related to Aspergillus species have emerged to become an important focus in infectious diseases, as a result of the increasing use of immunosuppressive agents and high fatality associated with invasive aspergillosis. However, laboratory diagnosis of Aspergillus infections remains difficult. In this study, by comparing the metabolomic profiles of the culture supernatants of 30 strains of six pathogenic Aspergillus species (A. fumigatus, A. flavus, A. niger, A. terreus, A. nomius and A. tamarii) and 31 strains of 10 non-Aspergillus fungi, eight compounds present in all strains of the six Aspergillus species but not in any strain of the non-Aspergillus fungi were observed. One of the eight compounds, Leu–Glu–Leu–Glu, is a novel tetrapeptide and represents the first linear tetrapeptide observed in Aspergillus species, which we propose to be named aspergitide. Two other closely related Aspergillus-specific compounds, hydroxy-(sulfooxy)benzoic acid and (sulfooxy)benzoic acid, may possess anti-inflammatory properties, as 2-(sulfooxy)benzoic acid possesses a structure similar to those of aspirin [2-(acetoxy)benzoic acid] and salicylic acid (2-hydroxybenzoic acid). Further studies to examine the potentials of these Aspergillus-specific compounds for laboratory diagnosis of aspergillosis are warranted and further experiments will reveal whether Leu–Glu–Leu–Glu, hydroxy-(sulfooxy)benzoic acid and (sulfooxy)benzoic acid are virulent factors of the pathogenic Aspergillus species.     

Identification and Functional Analysis of MicroRNAs and Their Targets in Platanus acerifolia under Lead (Pb) Stress

MicroRNAs (miRNAs) play important regulatory roles in development and stress responses in plants. Lead (Pb) is a non-essential element that is highly toxic to living organisms. Platanus acerifolia is grown as a street tree in cities throughout temperate regions for its importance in improving the urban ecological environment. MiRNAs that respond to abiotic stresses have been identified in plants; however, until now, the influence of Pb stress on P. acerifolia miRNAs has not been reported. To identify miRNAs and predict their target genes under Pb stress, two small RNA and two degradome libraries were constructed from Pb-treated and Pb-free leaves of P. acerifolia seedlings. After sequencing, 55 known miRNAs and 129 novel miRNAs were obtained, and 104 target genes for the miRNAs were identified by degradome sequencing. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses were performed to predict the functions of the targets. The expressions of eight differentially expressed miRNAs were validated by quantitative real-time polymerase chain reaction (qRT-PCR). This is the first report about P. acerifolia miRNAs and their target genes under Pb stress. This study has provided data for further research into molecular mechanisms involved in resistance of P. acerifolia to Pb stress.     

Sanguinaria canadensis: Traditional Medicine,Phytochemical Composition,Biological Activities and Current Uses

Sanguinaria canadensis, also known as bloodroot, is a traditional medicine used by Native Americans to treat a diverse range of clinical conditions. The plants rhizome contains several alkaloids that individually target multiple molecular processes. These bioactive compounds, mechanistically correlate with the plant’s history of ethnobotanical use. Despite their identification over 50 years ago, the alkaloids of S. canadensis have not been developed into successful therapeutic agents. Instead, they have been associated with clinical toxicities ranging from mouthwash induced leukoplakia to cancer salve necrosis and treatment failure. This review explores the historical use of S. canadensis, the molecular actions of the benzophenanthridine and protopin alkaloids it contains, and explores natural alkaloid variation as a possible rationale for the inconsistent efficacy and toxicities encountered by S. canadensis therapies. Current veterinary and medicinal uses of the plant are studied with an assessment of obstacles to the pharmaceutical development of S. canadensis alkaloid based therapeutics.     

Biologie der Methan-Bildung

Biology of methane formation. It has long been known that methane is produced in nature where organic compounds are degraded by microorganisms under anaerobic conditions. On an industrial scale, this process has been used for more than 50 years in the stabilization of sewage sludge from municipal waste water treatment plants. Recently it could be demonstrated that at least three different groups of bacteria are involved in the degradation of organic material into methane and CO₂. Hydrolytic and fermentative bacteria first degrade the organic compounds into various alcohols, fatty acids, hydrogen and CO₂. The second group of bacteria convert these metabolites into acetic acid, hydrogen, and CO₂, which are then utilized by the methanogenic bacteria to produce methane and CO₂.     

Risks of using antifouling biocides in aquaculture

Biocides are chemical substances that can deter or kill the microorganisms responsible for biofouling. The rapid expansion of the aquaculture industry is having a significant impact on the marine ecosystems. As the industry expands, it requires the use of more drugs, disinfectants and antifoulant compounds (biocides) to eliminate the microorganisms in the aquaculture facilities. The use of biocides in the aquatic environment, however, has proved to be harmful as it has toxic effects on the marine environment. Organic booster biocides were recently introduced as alternatives to the organotin compounds found in antifouling products after restrictions were imposed on the use of tributyltin (TBT). The replacement products are generally based on copper metal oxides and organic biocides. The biocides that are most commonly used in antifouling paints include chlorothalonil, dichlofluanid, DCOIT (4,5-dichloro-2-n-octyl-4-isothiazolin-3-one, Sea-nine 211^®), Diuron, Irgarol 1051, TCMS pyridine (2,3,3,6-tetrachloro-4-methylsulfonyl pyridine), zinc pyrithione and Zineb. There are two types of risks associated with the use of biocides in aquaculture: (i) predators and humans may ingest the fish and shellfish that have accumulated in these contaminants and (ii) the development of antibiotic resistance in bacteria. This paper provides an overview of the effects of antifouling (AF) biocides on aquatic organisms. It also provides some insights into the effects and risks of these compounds on non-target organisms.     

Constituents and Pharmacological Activities of Myrcia (Myrtaceae): A Review of an Aromatic and Medicinal Group of Plants

Myrcia is one of the largest genera of the economically important family Myrtaceae. Some of the species are used in folk medicine, such as a group known as “pedra-hume-caá” or “pedra-ume-caá” or “insulina vegetal” (insulin plant) that it is used for the treatment of diabetes. The species are an important source of essential oils, and most of the chemical studies on Myrcia describe the chemical composition of the essential oils, in which mono- and sesquiterpenes are predominant. The non-volatile compounds isolated from Myrcia are usually flavonoids, tannins, acetophenone derivatives and triterpenes. Anti-inflammatory, antinociceptive, antioxidant, antimicrobial activities have been described to Myrcia essential oils, while hypoglycemic, anti-hemorrhagic and antioxidant activities were attributed to the extracts. Flavonoid glucosides and acetophenone derivatives showed aldose reductase and α-glucosidase inhibition, and could explain the traditional use of Myrcia species to treat diabetes. Antimicrobial and anti-inflammatory are some of the activities observed for other isolated compounds from Myrcia.     

Discovery of a Potent Anti-Yeast Triterpenoid Saponin,Clematoside-S from Urena lobata L.

Urena lobata has been used as a traditional medicinal plant in India and China. In this study, we investigated the antimicrobial activity and isolated the active compound from the leaves of U. lobata. The 80% ethanol extract from U. lobata leaves showed an effective anti-yeast activity against Saccharomyces cerevisiae (S. cerevisiae) strains. Using a combination of chromatographic methods, (−)-trachelogenin (1) and clematoside-S (2) were isolated from this plant for the first time, and their chemical structure was identified by mass spectrometry (MS) and extensive nuclear magnetic resonance (NMR) data analysis. In addition, 1 was found to be inactive against all of the test microorganisms in the antimicrobial assay, whereas 2 exhibits a specific anti-yeast activity against S. cerevisiae strains with diameter of inhibition zones in the range from 11 to 20 mm. Furthermore, the MIC (minimum inhibitory concentration) and MBC (minimum bactericidal concentration) values of 2 against S. cerevisiae strains were detected to be in the ranges of 0.61 to 9.8 μg/mL and 2.42 to 9.8 μg/mL, respectively. This is the first report of 2 with a specific anti-yeast activity. The above result suggests the potential application of U. lobata to be used as a natural anti-yeast agent in food preservation.     

Antimicrobial and Hypoglycemic Activities of Novel N-Mannich Bases Derived from 5-(1-Adamantyl)-4-substituted-1,2,4-triazoline-3-thiones

The reaction of 5-(1-adamantyl)-4-ethyl or allyl-1,2,4-triazoline-3-thione with formaldehyde solution and various 1-substituted piperazines yielded the corresponding N-Mannich bases. The newly synthesized N-Mannich bases were tested for in vitro inhibitory activities against a panel of Gram-positive and Gram-negative bacteria and the yeast-like pathogenic fungus Candida albicans. Six compounds showed potent antibacterial activity against one or more of the tested microorganisms, while two compounds exhibited moderate activity against the tested Gram-positive bacteria. None of the newly synthesized compounds were proved to possess marked activity against Candida albicans. The oral hypoglycemic activity of six compounds was determined in streptozotocin (STZ)-induced diabetic rats. Four compounds produced significant strong dose-dependent reduction of serum glucose levels, compared to gliclazide at 10 mg/kg dose level (potency ratio > 75%).     

PG-2, a Potent AMP against Pathogenic Microbial Strains,from Potato (Solanum tuberosum L cv. Gogu Valley) Tubers Not Cytotoxic against Human Cells

In an earlier study, we isolated potamin-1 (PT-1), a 5.6-kDa trypsin-chymotrypsin protease inhibitor, from the tubers of a potato strain (Solanum tuberosum L cv. Gogu Valley). We established that PT-1 strongly inhibits pathogenic microbial strains, but not human bacterial strains, and that its sequence shows 62% homology with a serine protease inhibitor. In the present study, we isolated an antifungal and antibacterial peptide with no cytotoxicity from tubers of the same potato strain. The peptide (peptide-G2, PG-2) was isolated using salt-extraction, ultrafiltration and reverse-phase high performance liquid chromatography (RP-HPLC). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) showed the protein to have a molecular mass of 3228.5 Da, while automated Edman degradation showed the N-terminal sequence of PG-2 to be LVKDNPLDISPKQVQALCTDLVIRCMCCC-. PG-2 exhibited antimicrobial activity against Candida albicans, a human pathogenic yeast strain, and Clavibacter michiganensis subsp. michiganensis, a plant late blight strain. PG-2 also showed antibacterial activity against Staphylococcus aureus, but did not lyse human red blood cells and was thermostable. Overall, these results suggest PG-2 may be a good candidate to serve as a natural antimicrobial agent, agricultural pesticide and/or food additive.     

Supercritical Carbon Dioxide Extraction of Carotenoids from Pumpkin (Cucurbita spp.): A Review

Carotenoids are well known for their nutritional properties and health promoting effects representing attractive ingredients to develop innovative functional foods, nutraceutical and pharmaceutical preparations. Pumpkin (Cucurbita spp.) flesh has an intense yellow/orange color owing to the high level of carotenoids, mainly α-carotene, β-carotene, β-cryptoxanthin, lutein and zeaxanthin. There is considerable interest in extracting carotenoids and other bioactives from pumpkin flesh. Extraction procedures able to preserve nutritional and pharmacological properties of carotenoids are essential. Conventional extraction methods, such as organic solvent extraction (CSE), have been used to extract carotenoids from plant material for a long time. In recent years, supercritical carbon dioxide (SC-CO₂) extraction has received a great deal of attention because it is a green technology suitable for the extraction of lipophylic molecules and is able to give extracts of high quality and totally free from potentially toxic chemical solvents. Here, we review the results obtained so far on SC-CO₂ extraction efficiency and quali-quantitative composition of carotenoids from pumpkin flesh. In particular, we consider the effects of (1) dehydration pre-treatments; (2) extraction parameters (temperature and pressure); the use of water, ethanol and olive oil singularly or in combination as entrainers or pumpkin seeds as co-matrix.     

Removal of neurotoxic ethyl parathion pesticide by two-stage chemical/enzymatic treatment system using Fenton’s reagent and organophosphorous hydrolase

Organophosphate compounds, which are essential ingredients of pesticide, plasticizer, air fuel, and chemical warfare agents, are serious neurotoxic hazardous materials. Therefore, diverse physical-chemical treatments have been attempted to degrade organophosphate compounds. In the present work, we propose a two-stage chemical and enzymatic treatment system. As a first stage, pretreatment of oxidation and coagulation using Fenton’s reagent utilizing iron and hydrogen peroxide was employed. Preferentially, 1 mM ethyl parathion (EP) pesticide was largely (∼80%) removed by Fenton’s reagent reaction within 15 min. To remove residual EP, enzymatic treatment with organophosphorous hydrolase (OPH) from recombinant Escherichia coli was employed as a second stage. We successfully demonstrated that the proposed two-stage hybrid treatment process removed 1mM environmental toxic EP efficiently (∼98%) within 30 min.     

Isolation of the Autoinducer-Quenching Strain that Inhibits LasR in Pseudomonas aeruginosa

Quorum sensing (QS) has been recognized as a general phenomenon in microorganisms and plays an important role in many pathogenic bacteria. In this report, we used the Agrobacterium tumefaciens biosensor strain NT1 to rapidly screen for autoinducer-quenching inhibitors from bacteria. After initial screening 5389 isolates obtained from land and beach soil, 53 putative positive strains were identified. A confirmatory bioassay was carried out after concentrating the putative positive culture supernatant, and 22 strains were confirmed to have anti-LasR activity. Finally, we determined the strain JM2, which could completely inhibit biofilm formation of Pseudomonas aeruginosa PAO1, belonged to the genus Pseudomonas by analysis of 16S rDNA. Partially purified inhibitor factor(s) F5 derived from culture supernatants specifically inhibited LasR-controlled elastase and protease in wild type P. aeruginosa PAO1 by 68% and 73%, respectively, without significantly affecting growth; the rhl-controlled pyocyanin and rhamnolipids were inhibited by 54% and 52% in the presence of 100 μg/mL of F5. The swarming motility and biofilm of PAO1 were also inhibited by F5. Real time RT-PCR on samples from 100 μg/mL F5-treated P. aeruginosa showed downregulation of autoinducer synthase (LasRI and rhlI) and cognate receptor (lasR and rhlR) genes by 50%, 28%, 48%, and 29%, respectively. These results provide compelling evidence that the F5 inhibitor(s) interferes with the las system and significantly inhibits biofilm formation.     

Isolation,Bioactivity, and Production of ortho-Hydroxydaidzein and ortho-Hydroxygenistein

Daidzein and genistein are two major components of soy isoflavones. They exist abundantly in plants and possess multiple bioactivities. In contrast, ortho-hydroxydaidzein (OHD) and ortho-hydroxygenistein (OHG), including 6-hydroxydaidzein (6-OHD), 8-hydroxydaidzein (8-OHD), 3′-hydroxydaidzein (3′-OHD), 6-hydroxygenistein (6-OHG), 8-hydroxygenistein (8-OHG), and 3′-hydroxygenistein (3′-OHG), are rarely found in plants. Instead, they are usually isolated from fermented soybean foods or microbial fermentation broth feeding with soybean meal. Accordingly, the bioactivity of OHD and OHG has been investigated less compared to that of soy isoflavones. Recently, OHD and OHG were produced by genetically engineering microorganisms through gene cloning of cytochrome P450 (CYP) enzyme systems. This success opens up bioactivity investigation and industrial applications of OHD and OHG in the future. This article reviews isolation of OHD and OHG from non-synthetic sources and production of the compounds by genetically modified microorganisms. Several bioactivities, such as anticancer and antimelanogenesis-related activities, of OHD and OHG, are also discussed.     

Responses to Oxidative and Heavy Metal Stresses in Cyanobacteria: Recent Advances

Cyanobacteria, the only known prokaryotes that perform oxygen-evolving photosynthesis, are receiving strong attention in basic and applied research. In using solar energy, water, CO₂ and mineral salts to produce a large amount of biomass for the food chain, cyanobacteria constitute the first biological barrier against the entry of toxics into the food chain. In addition, cyanobacteria have the potential for the solar-driven carbon-neutral production of biofuels. However, cyanobacteria are often challenged by toxic reactive oxygen species generated under intense illumination, i.e., when their production of photosynthetic electrons exceeds what they need for the assimilation of inorganic nutrients. Furthermore, in requiring high amounts of various metals for growth, cyanobacteria are also frequently affected by drastic changes in metal availabilities. They are often challenged by heavy metals, which are increasingly spread out in the environment through human activities, and constitute persistent pollutants because they cannot be degraded. Consequently, it is important to analyze the protection against oxidative and metal stresses in cyanobacteria because these ancient organisms have developed most of these processes, a large number of which have been conserved during evolution. This review summarizes what is known regarding these mechanisms, emphasizing on their crosstalk.