FUNKCIONALIZIRANA NANOTELESCA KOT DIAGNOSTIČNI REAGENTI ZA DOLOČANJE TOKSIČNIH MIKROALG

Early detection and monitoring of toxic microalgae remains a challenge that will be necessary to overcome for understanding harmful algal bloom events and their consequent ecological and environmental impacts. Although there are diagnostic techniques based on microscopy, fluorescence in situ hybridization (FISH), DNA microarrays, real-time PCR, sandwich hybridization assays employed for the detection of harmful algal species, such techniques are not suitable for field monitoring and especially for the quantification of Alexandrium minutum due to abundance of cryptic species that cannot be differentiated based on morphology or genomic traits. These approaches are also time-consuming and laborious, hence there is need for alternative simple, rapid and cost-effective methods. In this study, our aim was to develop different nanobody-based capture strategies for the detection of A. minutum. Nanobodies were successfully displayed on the surface of Escherichia coli to facilitate detection of A. minutum. The method is simple and cost effective as no antibody purification step is necessary. As an alternative, A. minutum was quantified by exploiting the peroxidase activity of a G-quadruplex DNAzyme covalently bound to nanobodies and by means of a label free electrochemical immunosensor which exploit nanobodies bound via an irreversible SpyTag-SpyCatcher system as the specific immunocapture element. Such immunosensor was prepared by immobilizing the SpyTagged anti-A. minutum nanobody (SpyTagged C1) by means of a selfassembled monolayer (SAMs) of L- cysteine (L-Cys) displayed onto a gold nanoparticle-modified glassy carbon electrode. Electrochemical Impedance Spectroscopy (EIS) was performed to measure the electrical response of the circuit as the function of A. minutum immunocaptured cells. The results of EIS studies confirmed that the sensing chip detected A. minutum selectively, exhibited a wide dynamic range spanning from 103 to 109 cell L-1 and a limit of detection of 3.1× 103 cell L-1. The immunosensor data can be recorded and then analyzed with a portable potentiostat. Such device can be interfaced with a smart phone, a condition suitable for the rapid A. minutum quantification in situ.Zgodnje odkrivanje in monitoring strupenih mikroalg ostaja izziv, ki ga bo treba premagati za boljše razumevanje cvetenja alg in posledičnih ekoloških in okoljskih vplivov. Za odkrivanje škodljivih vrst alg obstajajo številne diagnostične metode, kot na primer mikroskopija, fluorescenčna hibridizacija in situ (FISH), mikromreže DNA, PCR v realnem času in hibridizacijske metode. Vendar te tehnike niso primerne za monitoring na terenu. Zlasti pri mikroalgi Alexandrium minutum te tehnike tudi ne omogočajo ustrezne kvantifikacije, saj obstajajo številne kriptične vrste, ki jih ni mogoče razlikovati na podlagi morfoloških ali genetskih lastnosti. Ker so te metode tudi zamudne in tehnično zahtevne, je še kako dobrodošel razvoj alternativnih metod, ki bi bile preproste, hitre in stroškovno učinkovite. V doktorskem raziskovalnem delu smo želeli razviti različne detekcijske metode na osnovi nanotelesc za odkrivanje mikroalge A. minutum. Nanotelesca za specifično detekcijo A. minutum smo uspešno izrazili na površini bakterije Escherichia coli. Ta metoda je preprosta in stroškovno učinkovita, saj čiščenje protiteles ni potrebno. Zatem smo razvili postopek za količinsko ovrednotenje A. minutum, kjer smo spremljali peroksidazno aktivnosti G-kvadrupleksnega DNA-zyma, ki je bil kovalentno vezan na nanotelesca. Peroksidazno aktivnost smo ugotavljali s pomočjo elektrokemičnega imunosenzorja brez oznake (label-free), na katerega so bila nanotelesca, kot specifičen imunodetekcijski element, vezana preko ireverzibilnega sistema SpyTag-SpyCatcher. Imunosenzor smo pripravili tako, da smo nanotelesca proti A. minutum, označena z molekulo Spy (SpyTagged C1), imobilizirali na enoplastne sloje (SAMs) L-cisteina (L-Cys), ki so bili nanešeni na ogljikovo elektrodo, modificirano z zlatimi nano-delci. Merjenje električnega odziva tokokroga kot funkcije vezave celic A. minutum smo merili z elektrokemijsko impedančno spektroskopijo (EIS). Rezultati merjenja EIS so potrdili, da je senzorski čip selektivno zaznal prisotnost A. minutum, da je imel širok dinamični razpon od 103 do 109 celic L-1 ter da je bila meja zaznavnosti 3,1 × 103 celice L-1. Podatke z imunosenzorja lahko zabeležimo in nato analiziramo s prenosnim potenciostatom. Takšno napravo je mogoče povezati s pametnim telefonom, kar omogoča hitro in učinkovito spremljanje A. minutum in situ

Antibacterial activity of seed extracts of Okra (Abelmoschus esculentus) against selected pathogen

The aim of the study was to determine the antibacterial potency of aqueous and ethanoic extracts of Abelmoschus esculentus seeds. Antibacterial activity of aqueous and ethanoic extracts of A. esculentus seeds were evaluated against selected foodborne pathogensListeria monocytogenes, Bacillus cereus, Salmonella enterica, Staphylococcus aureus and Escherichia coli) using agar well diffusion method by measuring the clear zone of inhibition diameter in (mm). It was observed that the aqueous and ethanoic extracts of the four samples of A. esculentus seeds showed broad range of inhibitory activity against the test bacteria especially L. monocytogenes. Thus, A. esculentus seed has a great potential as effective antilisterial and antibacterial activity. Practical Application: Consumption of okra seeds is highly recommended for its nutritive value and antibacterial activity against pathogens, which are most commonly attributed to foodborne diseases such as listeriosis, diarrhea, and salmonellosis. Also, further study could be conducted by isolating and identifying the active ingredient responsible for antibacterial potency for pharmaceutical purpose

Antimicrobial Efficacy and Phytochemical Screening of Mushrooms, Lenzites Betulinus, and Coriolopsis Gallica Extracts.]

Objective: This study was conducted to evaluate the antimicrobial activities of Lenzites betulinus and Coriolopsis gallica extracts against some bacterial isolates of medical importance. The organisms are Pseudomonas aeruginosa, Staphylococcus aureus, Proteus vulgaris, Klebsiella pneumonia and Escherichia coli. Methods: Agar well diffusion assay was used, the ethanolic, petroleum ether and aqueous extract of C. gallica was assayed against the bacterial isolates. Results: Petroleum ether extract of C. gallica and L. betulinus was able to inhibit P. vulgaris with inhibition zones of 26.0 mm and 20.0 mm respectively. Ethanolic extract of C. gallica also inhibited P. vulgaris with inhibition zone of 22.0 mm while aqueous extract and petroleum ether of L. betulinus inhibited P. aeruginosa with inhibition zones of 20.0 mm and 23.0 mm respectively. E. coli and S. aureus were also inhibited by the aqueous and ethanolic extract of L. betulinus with zones of inhibition 15.0 mm and 26.0 mm against E. coli while 19.0 mm and 22.0 mm against S. aureus respectively. This study also showed the presence of some phytochemicals like tannins, phenolics, flavonoids, steroids and saponin in the mushrooms with antimicrobial effects against the bacterial isolates used. The active components present in ethanolic, aqueous and petroleum ether extracts of C. gallica include phenolics, flavonoids and steroids while phytochemicals obtained from L. betulinus are phenolics, tannins, flavonoids and saponin respectively. Conclusion: The efficacy of these mushrooms as antimicrobial agents was attributed to the presence of various photochemicals present; hence they could be used in the simulation of drugs against some multi resistant strain of microorganisms. [TAF Prev Med Bull 2012; 11(6.000): 695-698

A future perspective on neurodegenerative diseases: Nasopharygneal and gut microbiota

Neurodegenerative diseases are considered a serious life‐threatening issue regardless of age. Resulting nerve damage progressively affects important activities, such as movement, coordination, balance, breathing, speech and the functioning of vital organs. Reports on the subject have concluded that neurodegenerative disease can be caused by mutations of susceptible genes, alcohol consumption, toxins, chemicals and other unknown environmental factors. Although several diagnostic techniques can be used to determine aetiologies, the process is difficult and often fails. Research shows that nasopharyngeal and gut microbiota play important roles in brain to spinal cord coordination. However, no conclusive epidemiologic evidence is available on the roles played by respiratory and gut microbiota in the development of neurodegenerative diseases. Thus, understanding the connection between respiratory and gut microbiota and the nervous system could provide information on causal links. The present review describes future perspectives on the role played by nasopharyngeal and gut microbiota in the development of neurodegenerative diseases

Strategies of biofilm inhibition and virulence attenuation of food borne pathogen-Escherichia coli O157:H7

Enterohemorrhagic Escherichia coli (E. coli) O157:H7, a gram-negative bacteria identified as a foodborne pathogen causing severe disease is of great concern worldwide. The pathogenicity of E. coli O157:H7 is due to the presence of some virulence factors and its ability to form biofilm which resist antimicrobial compounds, withstand harsh environmental condition and protects from the host immune responses. Formation of biofilm is a multistep process such as adhesion, cellular aggregation and productions of extracellular matrix in which colonies are embedded. There are high numbers of research in the discovery of natural and synthetic compounds which can attenuate the E. coli O157:H7 biofilm formation as well as suppress virulence-related genes. The present review article focuses on the steps involved in E. coli O157:H7 biofilm formation, factors associated with virulence and attenuation

Inhibition strategies of Listeria monocytogenes biofilms- Current knowledge and future outlooks.

There is an increasing trend in the food industry on the Listeria monocytogenes biofilm formation and inhibition. This is attributed to its easy survival on contact surfaces, resistance to disinfectants or antibiotics and growth under the stringent condition used for food processing and preservation thereby leading to food contamination products by direct or indirect exposure. Though, there is a lack of conclusive evidences about the mechanism of biofilm formation, in this review, the concept of biofilm formation and various chemical, physical, and green technology approaches to prevent or control the biofilm formed is discussed. State-of-the-art approaches ranging from the application of natural to synthetic molecules with high effectiveness and non-toxicity targeted at the different steps of biofilm formation could positively influence the biofilm inhibition in the future

Antimicrobial efficacy and phytochemical screening of Mushrooms, Lenzites betulinus, and Coriolopsis gallica extracts.

AIM: The spread of multidrug-resistant strains of bacteria makes it necessary to discover new classes of antibacterial and compounds that inhibit these resistant mechanisms. Hence, this study was conducted to evaluate the antimicrobial activities of Lenzites betulinus and Coriolopsis gallica extracts against some bacterial isolates of medical importance. METHOD: The organisms are Pseudomonas aeruginosa, Staphylococcus aureus, Proteus vulgaris, Klebsiella pneumoniae, Escherichia coli. Using agar well diffusion assay, the ethanolic, petroleum ether and aqueous extract of C. gallica was assayed against the bacterial isolates and the result showed that Petroleum ether extract of C. gallica and L. betulinus was able to inhibit P. vulgaris with inhibition zones of 26.0 mm and 20.0 mm respectively. RESULTS: Ethanolic extract of C. gallica also inhibited P. vulgaris with inhibition zone of 22.0 mm while aqueous extract and petroleum ether of L. betulinus inhibited P. aeruginosa with inhibition zones of 20.0 mm and 23.0 mm respectively. E. coli and S. aureus were also inhibited by the aqueous and ethanolic extract of L. betulinus with zones of inhibition 15.0 mm and 26.0 mm against E. coli while 19.0 mm and 22.0 mm against S. aureus respectively. This study also showed the presence of some phytochemicals like tannins, phenolics, flavonoids, steroids, and saponin in the mushrooms with antimicrobial effects against the bacterial isolates used. CONCLUSION: The active components present in ethanolic, aqueous and petroleum ether extracts of C. gallica include phenolics, flavonoids, and steroids while phytochemicals obtained from L. betulinus are phenolics, tannins, flavonoids, and saponin respectively

Diversity of bacteria and bacterial products as antibiofilm and antiquorum sensing drugs against pathogenic bacteria

The increase in antibiotic resistance of pathogenic bacteria has led to the development of new therapeutic approaches to inhibit biofilm formation as well as interfere quorum sensing (QS) signaling systems. The QS system is a phenomenon in which pathogenic bacteria produce signaling molecules that are involved in cell to cell communication, production of virulence factors, biofilm maturation, and several other functions. In the natural environment, several non-pathogenic bacteria are present as mixed population along with pathogenic bacteria and they control the behavior of microbial community by producing secondary metabolites. Similarly, non-pathogenic bacteria also take advantages of the QS signaling molecule as a sole carbon source for their growth through catabolism with enzymes. Several enzymes are produced by bacteria which disrupt the biofilm architecture by degrading the composition of extracellular polymeric substances (EPS) such as exopolysaccharide, extracellular- DNA and protein. Thus, the interference of QS system by bacterial metabolic products and enzymatic catalysis, modification of the QS signaling molecules as well as enzymatic disruption of biofilm architecture have been considered as the alternative therapeutic approaches. This review article elaborates on the diversity of different bacterial species with respect to their metabolic products as well as enzymes and their molecular modes of action. The bacterial enzymes and metabolic products will open new and promising perspectives for the development of strategies against the pathogenic bacterial infections