Search results for: E.coli O157:H7

Authors: Sihame Amakrane, Zineb Ouahdi, Mohammed Salah, Said Belaaouad

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\This research explores the efficacy of novel pyrimidine derivatives on bacterial strains such as Escherichia coli, Staphylococcus aureus, and Myccobacterium tuberculosis, utilizing bending energy calculations. Of the 25 compounds examined, 13 displayed potent activity against all the bacterial strains under study, exhibiting bending energy measurements between -7.4 and -10.7 kcal/mol. The -7.4 kcal/mol value corresponds to the bending energy of the SA12 and SA13 compounds with the 2xct protein (Staphylococcus aureus), whereas the -10.7 kcal/molis linked with the bending energy of SA6 and SA11 compounds with the 6GAV protein (Myccobacterium tuberculosis). Further research will involve a QSAR (Quantitative Structure-Activity Relationship) study aimed at constructing a reliable model to combat the aforementioned bacterial strains and a molecular dynamics study to evaluate the stability of ligand-protein complexes.

Keywords: docking, QSAR, bending energy, e. coli

Procedia PDF Downloads 87

Authors: Yu Yoshihara, Chika Tada, Moe Takada, Nyam-Osor Purevdorj, Khorolmaa Chimedtseren, Yutaka Nakai

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Water pollution from animal waste and its influence on grazing animals is a current concern regarding Mongolian grazing lands. We allocated 32 free-grazing lambs to four groups and provided each with water from a different source (upper stream, lower stream, well, and pond) for 49 days. We recorded the amount of water consumed by the lambs, as well as their body weight, behavior, white blood cell count, acute phase (haptoglobin) protein level, and fecal condition. We measured the chemical and biological qualities of the four types of water, and we detected enteropathogenic and enterohemorrhagic Escherichia coli in fecal samples by using a genetic approach. Pond water contained high levels of nitrogen and minerals, and well water contained high levels of bacteria. The odor concentration index decreased in order from pond water to upper stream, lower stream, and well. On day 15 of the experiment, the following parameters were the highest in lambs drinking water from the following sources: water intake (pond or lower stream), body weight gain (pond), WBC count (lower stream), haptoglobin concentration (well), and enteropathogenic E. coli infection rate (lower stream). Lambs that drank well water spent more time lying down and less time grazing than the others, and lambs that drank pond water spent more time standing and less time lying down. Lambs given upper or lower stream water exhibited more severe diarrhea on day 15 of the experiment than before the experiment. Mongolian sheep seemed to adapt to chemically contaminated water: their productivity benefited the most from pond water, likely owing to its rich mineral content. Lambs that drank lower stream water showed increases in enteropathogenic E. coli infection, clinical diarrhea, and WBC count. Lambs that drank well water, which was bacteriologically contaminated, had increased serum acute phase protein levels and poor physical condition; they were thus at increased risk of negative health and production effects.

Keywords: DNA, Escherichia coli, fecal sample, lower stream, well water

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Authors: Naureen Akhtar

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The stability of the protein in detergent-containing solution is the key for its successful crystallisation. Fluorescence-detection size-exclusion chromatography (FSEC) is a potential approach for screening monodispersity as well as the stability of protein in a detergent-containing-solution. In this present study, covalently linked Green Fluorescent Protein (GFP) to bacterial nitrate transporter, NarU from Escherichia coli was studied for pre-crystallisation trials by FSEC. Immobilised metal ion affinity chromatography (IMAC) and gel filtration were employed for their purification. The main objectives of this study were over-expression, detergent screening and crystallisation of nitrate transporter proteins. This study could not produce enough proteins that could realistically be taken forward to achieve the objectives set for this particular research. In future work, different combinations of variables like vectors, tags, creation of mutant proteins, host cells, position of GFP (N- or C-terminal) and/or membrane proteins would be tried to determine the best combination as the principle of technique is still promising.

Keywords: transporters, detergents, over-expression, crystallography

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Authors: Ali Bagherpour, Masoud Soltanialvar

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Campylobacter species are common bacterial pathogens associated with human gastroenteritis which are generally transmitted through foods of animal origin. This study was carried out to determine the prevalence of Campylobacter species in poultry meat and by products in the city of Dezful in Iran. Since April 2012 to July 2013, a total of 400 samples including meat (n = 100), liver (n = 100), gizzard (n = 100), and poultry heart (n = 100), were randomly collected from Dezful industrial poultry abattoir and were experimented in order to investigate presence of Campylobacter species. According to culture test, 251 samples out of 400 samples under study (69%) were contaminated with Campylobacter species. The highest prevalence of Campylobacter species was observed in poultry's liver (78.3%) and then in gizzard (75.8%), heart (65%) and meat (56.7%). The most common isolated Campylobacter were C. jejuni (90.9%) and the rest were C. coli (9.1%). There was a significant difference (P < 0.05) in the prevalence of Campylobacter species between the meat samples taken in the summer (86.7%). The results of this study indicate the importance of edible offal of poultries as the potential source of Campylobacter infections.

Keywords: Campylobacter jejuni, Campylobacter coli, poultry, meat, products

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Authors: Mudawi M. Nour

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Background: Now a days, the multidisciplinary scientific research conception in the field of nanotechnology has witnessed development with regard to the numerous applications and synthesis of nanomaterials. Objective: The current investigation has been conducted with the main focus on the green synthesis of silver nanoparticles from the leaves of Salvadora persica and its antibacterial activity against MDR pathogens E. coli and S. aureus. Methodology: Silver nanoparticles (AgNPs) were prepared after addition of aqueous extract of Salvadora persica leaves. The UV-Vis spectrophotometer, Transmission Electron Microscopy (TEM), zeta potential and Scanning Electron Microscopy (SEM) were employed to detect the particle size and morphology, besides Fourier transform infra-red spectrometer (FTIR) analysis was performed to determine the capping and stabilizing agents in the extract. Antibacterial assay for the biogenic AgNPs was conducted against E. coli and S. aureus. Results: Color change of the mixture from yellow to dark brown is the first indication to AgNPs formation. Furthermore, 420 nm was the peak value for UV-Vis spectroscopy absorption of the mixture. Besides, TEM and SEM micrographs showed wide variability in the diameter of smaller NPs aggregated together with spherical shapes, and zeta sizer showed about 153.3 nm as an average size of nanoparticles. Microbial suppression was noticed for the tested microorganisms. Furthermore, with the help of FTIR analysis, the biomolecules that act as capping and stabilizing agents of AgNPs are proteins and phenols present in the plant extract. Conclusion: Salvadora persica leaves extract act as a reducing and stabilizing agent for the synthesis of AgNPs, keeping its ability to suppress the MDR pathogen.

Keywords: green synthesis, FTIR, MDR pathogen, salvadora persica

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Authors: Yuriy A. Knirel

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Enteric bacteria Escherichia coli is the predominant facultative anaerobe of the colonic flora, and some specific serotypes are associated with enteritis, hemorrhagic colitis, and hemolytic uremic syndrome. Shigella spp. are human pathogens that cause diarrhea and bacillary dysentery (shigellosis). They are in effect E. coli with a specific mode of pathogenicity. Strains of Salmonella enterica are responsible for a food-borne infection (salmonellosis), and specific serotypes cause typhoid fever and paratyphoid fever. All these bacteria are closely related in respect to structure and genetics of the lipopolysaccharide, including the O-polysaccharide part (O‑antigen). Being exposed to the bacterial cell surface, the O antigen is subject to intense selection by the host immune system and bacteriophages giving rise to diverse O‑antigen forms and providing the basis for typing of bacteria. The O-antigen forms of many bacteria are unique, but some are structurally and genetically related to others. The sequenced O-antigen gene clusters between conserved galF and gnd genes were analyzed taking into account the O-antigen structures established by us and others for all S. enterica and Shigella and most E. coli O-serogroups. Multiple genetic mechanisms of diversification of the O-antigen forms, such as lateral gene transfer and mutations, were elucidated and are summarized in the present paper. They include acquisition or inactivation of genes for sugar synthesis or transfer or recombination of O-antigen gene clusters or their parts. The data obtained contribute to our understanding of the origins of the O‑antigen diversity, shed light on molecular evolutionary relationships between the O-antigens of enteric bacteria, and open a way for studies of the role of gene polymorphism in pathogenicity.

Keywords: enteric bacteria, O-antigen gene cluster, polysaccharide biosynthesis, polysaccharide structure

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Authors: Mesfin Angaw Tesfay

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L-Xylulose is a potentially valuable rare sugar used as starting material for antiviral and anticancer drug development in pharmaceutical industries. L-Xylulose exist in a very low concentration in nature and have to be synthesized from cheap starting materials such as xylitol through biotechnological approaches. In this study, cofactor engineering and deep eutectic solvent were applied to improve the efficiency of L-xylulose production from xylitol. A water-forming NAD+ regeneration enzyme (NADH oxidase) from Streptococcus mutans ATCC 25175 was introduced into E. coli with xylitol-4-dehydrogenase (XDH) of Pantoea ananatis resulting in recombinant cells harboring the vector pETDuet-xdh-SmNox. Further, three deep eutectic solvents (DES) including, Choline chloride/glycerol (ChCl/G), Choline chloride/urea (ChCl/U), and Choline chloride/ethylene glycol (ChCl/EG) have been employed to facilitate the conversion efficiency of L-xylulose from xylitol. The co-expression system exhibited optimal activity at a temperature of 37 ℃ and pH 8.5, and the addition of Mg2+ enhanced the catalytic activity by 1.19-fold. Co-expression of NADH oxidase with XDH enzyme resulted in increased L-xylulose concentration and productivity from xylitol as well as the intracellular NAD+ concentration. Two of the DES used (ChCl/U and ChCl/EG) show positive effects on product yield and the ChCl/G has inhibiting effects. The optimum concentration of ChCl/U was 2.5%, which increased the L-xylulose yields compared to the control without DES. In a 1 L fermenter the final concentration and productivity of L-xylulose from 50 g/L of xylitol reached 48.45 g/L, and 2.42 g/L.h respectively, which was the highest report. Overall, this study is a suitable approach for large-scale production of L-xylulose from xylitol using the engineered E. coli cell.

Keywords: Xylitol-4-dehydrogenase, NADH oxidase, L-xylulose, Xylitol, Coexpression, DESs

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Authors: Satwik Majumder, Dongyun Jung, Jennifer Ronholm, Saji George

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Bovine mastitis is the most common infectious disease in dairy cattle, with major economic implications for the dairy industry worldwide. Continuous monitoring for the emergence of antimicrobial resistance (AMR) among bacterial isolates from dairy farms is vital not only for animal husbandry but also for public health. In this study, the prevalence of AMR in 113 Escherichia coli isolates from cases of bovine clinical mastitis in Canada was investigated. Kirby-Bauer disk diffusion test with 18 antibiotics and microdilution method with three heavy metals (copper, zinc, and silver) was performed to determine the antibiotic and heavy-metal susceptibility. Resistant strains were assessed for efflux and ß-lactamase activities besides assessing biofilm formation and hemolysis. Whole-genome sequences for each of the isolates were examined to detect the presence of genes corresponding to the observed AMR and virulence factors. Phenotypic analysis revealed that 32 isolates were resistant to one or more antibiotics, and 107 showed resistance against at least one heavy metal. Quinolones and silver were the most efficient against the tested isolates. Among the AMR isolates, AcrAB-TolC efflux activity and ß-lactamase enzyme activities were detected in 13 and 14 isolates, respectively. All isolates produced biofilm but with different capacities, and 33 isolates showed α-hemolysin activity. A positive correlation (Pearson r = +0.89) between efflux pump activity and quantity of biofilm was observed. Genes associated with aggregation, adhesion, cyclic di-GMP, quorum sensing were detected in the AMR isolates, corroborating phenotype observations. This investigation showed the prevalence of AMR in E. coli isolates from bovine clinical mastitis. The results also suggest the inadequacy of antimicrobials with a single mode of action to curtail AMR bacteria with multiple mechanisms of resistance and virulence factors. Therefore, it calls for combinatorial therapy for the effective management of AMR infections in dairy farms and combats its potential transmission to the food supply chain through milk and dairy products.

Keywords: antimicrobial resistance, E. coli, bovine mastitis, antibiotics, heavy-metals, efflux pump, ß-lactamase enzyme, biofilm, whole-genome sequencing

Procedia PDF Downloads 216

Authors: Georgios Psakis, Imren Rahbay, David Spiteri, Jeanice Mallia, Martin Polidano, Vasilis P. Valdramidis

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Absence of surface water and progressive groundwater quality deterioration have exacerbated scarcity rapidly, making the Mediterranean island of Malta one of the most water-stressed countries in Europe. Water scarcity challenges have been addressed by reverse osmosis desalination of seawater, 60% of which is blended with groundwater to form the current potable tap-water supply. Chlorination has been the adopted method of water disinfection prior to distribution. However, with the Malteseconsumer chlorine sensory-threshold being as low as 0.34 ppm, presence of chorine residuals and chlorination by-products in the distributed tap-water impacts negatively on its organoleptic attributes, deterring the public from consuming it. As part of the PURILMA initiative, and with the aim of minimizing the impact of chlorine residual on the quality of the distributed water, UV-C, and hydrosonication, have been identified as cost- and energy-effective decontamination alternatives, paving the way for more sustainable water management. Bench-scale assessment of the decontamination efficiency of UV-C (254 nm), revealed 4.7-Log10 inactivation for both Escherichia coli and Enterococcus faecalis at 36 mJ/cm2. At >200 mJ/cm2fluence rates, there was a systematic 2-Log10 difference in the reductions exhibited by E. coli and E. faecalis to suggest that UV-C disinfection was more effective against E. coli. Hybrid treatment schemes involving hydrosonication(at 9.5 and 12.5 dm3/min flow rates with 1-5 MPa maximum pressure) and UV-C showed at least 1.1-fold greater bactericidal activity relative to the individualized UV-C treatments. The observed inactivation appeared to have stemmed from additive effects of the combined treatments, with hydrosonication-generated reactive oxygen species enhancing the biocidal activity of UV-C.

Keywords: disinfection, groundwater, hydrosonication, UV-C

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Authors: I. Sani, A. Abdulhamid, F. Bello, Isah M. Fakai

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This research has focused on the application of green fluorescent protein (GFP) as a new technique for direct monitoring of fermentation processes involving cultured bacteria. To use GFP as a sensor for pH and oxygen, percentage ratio of red fluorescence to green (% R/G) was evaluated. Assessing the magnitude of the % R/G ratio in relation to low or high pH and oxygen concentration, the bacterial strains were cultivated under aerobic and anaerobic conditions. SCC1 strains of E. coli were grown in a 5 L laboratory fermenter, and during the fermentation, the pH and temperature were controlled at 7.0 and 370C respectively. Dissolved oxygen tension (DOT) was controlled between 15-100% by changing the agitation speed between 20-500 rpm respectively. Effect of reducing the DOT level from 100% to 15% was observed after 4.5 h fermentation. There was a growth arrest as indicated by the decrease in the OD650 at this time (4.5-5 h). The relative fluorescence (green) intensity was decreased from about 460 to 420 RFU. However, %R/G ratio was significantly increased from about 0.1% to about 0.25% when the DOT level was decreased to 15%. But when the DOT was changed to 100%, a little increase in the RF and decrease in the %R/G ratio were observed. Therefore, GFP can effectively detect and indicate any change in pH and oxygen level during fermentation processes.

Keywords: Escherichia coli SCC1, fermentation process, green fluorescent protein, red fluorescence

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Authors: Phanwipa Pangsri, Yawariyah Weahayee

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The lactic acid bacteria (LAB) were isolated from 10 samples of fermented foods (Sa-tor-dong and Bodo) in South locality of Thailand. The 23 isolates of lactic acid bacteria were selected, which were exhibited a clear zone and growth on MRS agar supplemented with CaCO3. All of lactic acid bacteria were tested on morphological and biochemical. The result showed that all isolates were Gram’s positive, non-spore forming but only 10 isolates displayed catalase negative. The 10 isolates including BD 1.1, BD 1.2, BD 2.1, BD2.2, BD 2.3, BD 3.1, BD 4.1, BD 5.2, ST4.1, and ST 5.2 were selected for inhibition activity determination. Only 2 strains (ST 4.1 and BD 2.3) showed inhibition zone on agar, when using Escherichia coli sp. as target strain. The ST 4.1 showed highest inhibition zone on agar, which was selected for probiotic property testing. The ST4.1 isolate could grow in MRS broth containing a high concentration of sodium chloride 6%, bile salts 7%, pH 4-10 and vary temperature at 15-45^oC.

Keywords: lactic acid bacteria, probiotic, antimicrobial, probiotic property testing

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Authors: Imana Shahrin Tania, Mohammad Ali

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The present study reports the synthesis, characterization and application of nano-sized zinc-oxide (ZnO) particles on a cotton fabric surface. The aim of the investigations is to impart the antimicrobial activity on textile cloth. Nanoparticle is synthesized by wet chemical method from zinc sulphate and sodium hydroxide. SEM (scanning electron micrograph) images are taken to demonstrate the surface morphology of nanoparticles. XRD analysis is done to determine the crystal size of the nanoparticle. With the conformation of nanoformation, the cotton woven fabric is treated with ZnO nanoparticle by mechanical thermo-fixation (pad-dry-cure) technique. To increase the wash durability of nano treated fabric, an acrylic binder is used as a fixing agent. The treated fabric shows up to 90% bacterial reduction for S. aureus (Staphylococcus aureus) and 87% for E. coli (Escherichia coli) which is appreciable for bacteria protective clothing.

Keywords: nanoparticle, zinc oxide, cotton fabric, antibacterial activity, binder

Procedia PDF Downloads 132

Authors: Arun Jyothi Bheemagani, Chakrapani Pullagummi, Anupalli Roja Rani

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Exploration of the chemical constituents of the plants and pharmacological screening may provide us the basis for the development of novel agents. Plants have provided us some of the very important life saving drugs used in the modern medicine. The aim of our work was to screen the phytochemical constituents and antimicrobial and antioxidant activities of methanol extract of leaves of Annona cherimola Mill plant from Tirumala forest, Tirupathi. It was originally called Chirimuya by the Inca people who lived where it was growing in the Andes of South America, is an edible fruit-bearing species of the genus Annona from the family Annonaceae. Annona cherimola Mill is a multipurpose tree with edible fruits and is one of the sources of the medicinal products. The antibacterial activity was measured by agar well diffusion method; the diameter of the zone of bacterial growth inhibition was measured after incubation of plates. The inhibitory effect was studied against the pathogenic bacteria (Klebsiella pneumonia, Bacillus subtilis, Staphylococcus aureus and Escherichia coli (E. coli). Antioxidant assays were also performed for the same extracts by spectrophotometric methods using known standard antioxidants as reference. The studied plant extracts were found to be very effective against the pathogenic microorganisms tested. The methanolic extract of Annona cherimola Mill from showed maximum activity against Escherichia coli and Staphylococcus aureus and the least concentration required showing the activity was 0.5mg/ml. Phytochemical screening of the plants revealed the presence of flavonoids, alkaloids, steroids and carbohydrates. Good presence of antioxidants was also found in the methanolic extracts.

Keywords: annona cherimola, phytochemicals, antioxidant and antibacterial activity, methanol extract

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Authors: Mohammed Jibrin Ndejiko

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Modern and current models such as flow cell technology which enhances a non-destructive growth and inspection of the sessile microbial communities revealed a great understanding of biofilms. A continuous flow system was designed to evaluate possibility of biofilm formation by Escherichia coli DH5α on the stainless steel (type 304) under continuous nutrient supply. The result of the colony forming unit (CFU) count shows that bacterial attachment and subsequent biofilm formation on stainless steel coupons with average surface roughness of 1.5 ± 1.8 µm and 2.0 ± 0.09 µm were both significantly higher (p ≤ 0.05) than those of the stainless steel coupon with lower surface roughness of 0.38 ± 1.5 µm. These observations support the hypothesis that surface profile is one of the factors that influence biofilm formation on stainless steel surfaces. The SEM and FESEM micrographs of the stainless steel coupons also revealed the attached Escherichia coli DH5α biofilm and dehydrated extracellular polymeric substance on the stainless steel surfaces. Thus, the fabricated flow system represented a very useful tool to study biofilm formation under continuous nutrient supply.

Keywords: biofilm, flowcell, stainless steel, coupon

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Authors: Mahmoud H. Abu Elella, Riham R. Mohamed, Magdy W. Sabaa

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Introduction: Microbial pollution is global problem threatening the human health. It is resulted by pathogenic microorganisms such as Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and other pathogenic strains. They cause a dangerous effect on human health, so great efforts have been exerted to produce new and effective antimicrobial agents. Nowadays, natural polysaccharides, such as chitosan and its derivatives are used as antimicrobial agents. The aim of our work is to synthesize of a biodegradable polymer such as N-quaternized chitosan (NQC) then Characterization of NQC by using different analysis techniques such as Fourier transform infrared (FTIR) and Scanning electron microscopy (SEM) and using it as an antibacterial agent against different pathogenic bacteria. Methods: Synthesis of NQC using dimethylsulphate. Results: FTIR technique exhibited absorption peaks of NQC, SEM images illustrated that surface of NQC was smooth and antibacterial results showed that NQC had a high antibacterial effect. Discussion: NQC was prepared and it was proved by FTIR technique and SEM images antibacterial results exhibited that NQC was an antibacterial agent.

Keywords: antimicrobial agent, N-quaternized chitosan chloride, silver nanocomposites, sodium polyacrylate

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Authors: Annisa I. Reza, Jasril Karim

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Despite the availability of antimicrobial agents in the market, the urge to study and find other chemical compounds with the better potential of replacing them still tempting the scientists. This experiment is in the aim to explore a novel brominated pyrazoline ring which was made from intermediate chalcone as a candidate to answer the challenge. Using green chemistry approach by microwave irradiation from domestic oven, both known chalcone and 5-(2-bromophenyl)-3-(naphthalen-1-yl)-4,5-dihydro-1H-pyrazole were successfully synthesized. Pyrazoline’s structure was confirmed based on UV, IR, ¹H-NMR, ¹³C-NMR and MS and together with its intermediate were examined against some microorganisms (Bacillus subtilis, Escherichia coli, and Candida albicans) under agar diffusion method. The results collected during experiment revealed that both tested compounds showed weak activity on B.subtilis which was proven by a zone of inhibitions, while there was no zone of inhibitions observed in E. coli and C. albicans. This is suggested because of the bulky structure around pyrazoline could not provide the main ring to interact with microbial’s cell wall. The study shows that the proposed compound had the low capability as a promising antimicrobial agent, yet it still enriches the information about pyrazoline ring.

Keywords: antimicrobial, chalcone, microwave irradiation, pyrazoline

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Authors: David D. Adams, Ibrahim B. Bello

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Spent oil contaminated Soil from ten selected mechanic workshops were investigated for their bacteria and bioremediation potentials. The bacterial isolates were morphologically and molecularly identified as Enterobacter hormaechei, Escherichia coli, Klebsiella pneumoniae, Shigella flexneri , Wesiella cibaria, Lactobacillus planetarium. The singles and a consortium of these bacteria incubated in the minimal salt medium incorporated with 1% engine oil exhibited various biodegradation rates, with the mixed consortium exhibiting the highest for this oil. The gene for the hydrocarbon enzyme Catechol 2, 3 dioxygenase (C2,30) was detected and amplified in Enterobacter hormaechei, Escherichia coli and Shigella flexneri using PCR and Agarose gel electrophoresis. The detection of the (C2,30) enzyme gene in, and the spent oil biodegradation activity exhibited by these bacteria suggest their possible possession of bioremediating potentials for the spent engine oil. It is therefore suggested that a pilot study on the field application of these bacteria for bioremediation and restoration of spent oil polluted environment should be done in mechanic workshops.

Keywords: spent engine oil, pollution, bacteria, enzyme, bioremediation, mechanic workshop

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Authors: Tomasz Borowski, Dawid Sołoducha, Agata Markowska-Szczupak, Aneta Wesołowska, Marian Kordas, Rafał Rakoczy

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Essential oils (EOs) are fragrant volatile oils obtained from plants. These are used for cooking (for flavor and aroma), cleaning, beauty (e.g., rosemary essential oil is used to promote hair growth), health (e.g. thyme essential oil cures arthritis, normalizes blood pressure, reduces stress on the heart, cures chest infection and cough) and in the food industry as preservatives and antioxidants. Rosemary and thyme essential oils are considered the most eminent herbs based on their history and medicinal properties. They possess a wide range of activity against different types of bacteria and fungi compared with the other oils in both in vitro and in vivo studies. However, traditional uses of EOs are limited due to rosemary and thyme oils in high concentrations can be toxic. In light of the accessible data, the following hypothesis was put forward: Low frequency rotating magnetic field (RMF) increases the antimicrobial potential of EOs. The aim of this work was to investigate the antimicrobial activity of commercial Salvia Rosmarinus L. and Thymus vulgaris L. essential oil from Polish company Avicenna-Oil under Rotating Magnetic Field (RMF) at f = 25 Hz. The self-constructed reactor (MAP) was applied for this study. The chemical composition of oils was determined by gas chromatography coupled with mass spectrometry (GC-MS). Model bacteria Escherichia coli K12 (ATCC 25922) was used. Minimum inhibitory concentrations (MIC) against E. coli were determined for the essential oils. Tested oils in very small concentrations were prepared (from 1 to 3 drops of essential oils per 3 mL working suspensions). From the results of disc diffusion assay and MIC tests, it can be concluded that thyme oil had the highest antibacterial activity against E. coli. Moreover, the study indicates the exposition to the RMF, as compared to the unexposed controls causing an increase in the efficacy of antibacterial properties of tested oils. The extended radiation exposure to RMF at the frequency f= 25 Hz beyond 160 minutes resulted in a significant increase in antibacterial potential against E. coli. Bacteria were killed within 40 minutes in thyme oil in lower tested concentration (1 drop of essential oils per 3 mL working suspension). Rapid decrease (>3 log) of bacteria number was observed with rosemary oil within 100 minutes (in concentration 3 drops of essential oils per 3 mL working suspension). Thus, a method for improving the antimicrobial performance of essential oil in low concentrations was developed. However, it still remains to be investigated how bacteria get killed by the EOs treated by an electromagnetic field. The possible mechanisms relies on alteration in the permeability of ionic channels in ionic channels in the bacterial cell walls that transport in the cells was proposed. For further studies, it is proposed to examine other types of essential oils and other antibiotic-resistant bacteria (ARB), which are causing a serious concern throughout the world.

Keywords: rotating magnetic field, rosemary, thyme, essential oils, Escherichia coli

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Authors: Mohsen Padervand

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Functionalized magnetic core-zeolitic shell nanostructures were prepared by the hydrothermal and coprecipitation methods. The products were characterized by Vibrating Sample Magnetometer (VSM), X-ray powder diffraction (XRD), Fourier Transform Infrared spectra (FTIR), nitrogen adsorption-desorption isotherms (BET) and Transmission Electron Microscopy (TEM). The growth of mordenite nanoparticles on the surface of silica coated nickel ferrite nanoparticles at the presence of organic templates was well approved. The antibacterial activity of prepared samples was investigated by the inactivation of E.coli as a gram negative bacterium. A new mechanism was proposed to inactivate the bacterium over the prepared samples. Minimum Inhibitory Concentration (MIC) and reuse ability were studied too. TEM images of the destroyed microorganism after the treatment time were applied to illustrate the inactivation mechanism. The interaction of the noble metals with organic components on the surface of nanostructures studied theoretically and the results were used to interpret the experimental results.

Keywords: nickel ferrite nanoparticles, magnetic core-zeolitic shell, antibacterial activity, E. coli

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Authors: Muhammad Shahzad Tufail, Iram Liaqat, Umer Sohail Meer, Muhammad Ishtaiq, Muhammad Sattar

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Nanotechnology is a vibrant field with numerous applications in many different branches of science and technology. Several methods are used to synthesize nanoparticles (NPs), which have multiple range of applications. Comparatively, the biogenic synthesis of NPs is a more economical and environmentally favourable method than the traditional chemical method. The current study aims to synthesize biogenically silver nanoparticles (AgNPs) using bacterial isolates. Four bacterial strains Escherichia coli (MT448673), Pseudomonas aeruginosa (MN900691), Bacillus subtilis (MN900684) and Bacillus licheniformis (MN900686) were used for the synthesis of AgNPs from silver nitrate (AgNO3) solution. The biofilm time kinetics of four bacterial isolates (P. aeruginosa, E. coli, B. licheniformis and B. subtilis) was analysed by incubating bacterial cultures at 37◦C in test tubes over a period of different time intervals i.e., 2, 3, 5 and 7 days following crystal violet staining method. All the four strains had ability to form strong biofilms between 48 to 72 hours of incubation. Two strains (B. subtilis and B. licheniformis) formed significant (p < 0.05) biofilm after 3 days of incubation period. The other two strains (E. coli and P. aeruginosa) showed strong biofilm formation after 2 days of incubation. Next, the antibiofilm activity of biogenically synthesized AgNPs (10 - 100 µgmL-1) was analysed against biofilm forming human pathogenic bacteria. Findings of the work revealed that 60-90% inhibition was observed at 60 µgmL-1 of AgNPs, while maximum inhibition (i.e.,100%) was found at highest concentration (90 µgmL-1). It was evident that highly significant (p < 0.05) decrease in biofilm formation was observed with increasing concentration of AgNPs.

Keywords: antibiofilm, biofilm formation, nanotechnology, pathogenic bacteria, silver nanoparticles

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Authors: Shubham Sharma, Sandra Barkauskaite, Brendan Duffy, Swarna Jaiswal, Amit K. Jaiswal

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Food packaging protects food from temperature, light, and humidity; preserves food and guarantees the safety and the integrity of the food. Advancement in packaging research leads to development of active packaging system with numerous properties such as oxygen scavengers, carbon-dioxide generating systems, antimicrobial active packaging, moisture control packaging, ethylene scavengers etc. In the active packaging, several additives such as essential oils, polyphenols etc. are incorporated into packaging film or within the packaging material to achieve the desired properties. This study investigates the effect on the structural, thermal and functional properties of different poly(lactide) – poly (butylene adipate-co-terephthalate) (PLA-PBAT) blend films incorporated with clove essential oil. The PLA-PBAT films were prepared by a solution casting method and then characterized based on their optical, mechanical properties, surface hydrophobicity, chemical composition, antimicrobial activity against S. aureus and E. coli, and inhibition of biofilm formation of E. coli. Results showed that, the developed packaging film containing clove oil has significant UV-blocking property (80%). However, incorporation of clove oil resulted in reduced transparency and tensile strength of the film as the concentration of clove oil increased. The surface hydrophobicity of packaging film was improved with the increasing concentration of essential oil. Similarly, thickness of the clove oil containing films increased from 36.71 µm to 106.67 µm as the concentration increases. The antimicrobial activity and biofilm inhibition study showed that the clove-incorporated PLA-PBAT composite film was effective against tested bacteria E. coli and S. aureus. This study showed that the PLA-PBAT – Clove oil composite film has significant antimicrobial and UV-blocking properties and can be used as an active food packaging film.

Keywords: active packaging, clove oil, poly(butylene adipate-co-terephthalate), poly(lactide)

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Authors: Vytautas Galvanauskas, Rimvydas Simutis, Donatas Levisauskas, Vykantas Grincas, Renaldas Urniezius

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The paper deals with model-based development and implementation of efficient control strategies for recombinant protein synthesis in fed-batch E.coli cultivation processes. Based on experimental data, a kinetic dynamic model for cultivation process was developed. This model was used to determine substrate feeding strategies during the cultivation. The proposed feeding strategy consists of two phases – biomass growth phase and recombinant protein production phase. In the first process phase, substrate-limited process is recommended when the specific growth rate of biomass is about 90-95% of its maximum value. This ensures reduction of glucose concentration in the medium, improves process repeatability, reduces the development of secondary metabolites and other unwanted by-products. The substrate limitation can be enhanced to satisfy restriction on maximum oxygen transfer rate in the bioreactor and to guarantee necessary dissolved carbon dioxide concentration in culture media. In the recombinant protein production phase, the level of substrate limitation and specific growth rate are selected within the range to enable optimal target protein synthesis rate. To account for complex process dynamics, to efficiently exploit the oxygen transfer capability of the bioreactor, and to maintain the required dissolved oxygen concentration, adaptive control algorithms for dissolved oxygen control have been proposed. The developed model-based control strategies are useful in scale-up of cultivation processes and accelerate implementation of innovative biotechnological processes for industrial applications.

Keywords: adaptive algorithms, model-based control, recombinant E. coli, scale-up of bioprocesses

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Authors: Vijay H. Ingole, Efthimia Lioliou

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Indirubin, a key bioactive component of traditional Chinese medicine, has gained increasing recognition for its potential in modern biomedical applications, particularly in pharmacology and therapeutics. The present work aimed to harness the potential by engineering an Escherichia coli strain capable of high-yield indirubin production. Through meticulous genetic engineering, we optimized the metabolic pathways in E. coli to enhance indirubin synthesis. Further, to explored the optimization of culture media and indirubin yield via batch and fed-batch fermentation techniques. By fine-tuning upstream process (USP) parameters, including nutrient composition, pH, temperature, and aeration, we established conditions that maximized both cell growth and indirubin production. Additionally, significant efforts were dedicated to refining downstream process (DSP) conditions for the extraction, purification, and quantification of indirubin. Utilizing advanced biochemical methods and analytical techniques such as UHPLC, we ensured the production of high purity indirubin. This approach not only improved the economic viability of indirubin bioproduction but also aligned with the principles of green production and sustainability.

Keywords: indirubin, bacterial strain, fermentation, HPLC

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Authors: Elizabeth G. Hereford, Geoffrey W. Gearner

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Logan’s Branch, within the Triplett Creek Watershed of Rowan County, Kentucky, is a waterway located near important agricultural and residential areas. Part of Logan’s Branch flows over an exposed black shale formation with elevated radioactivity and heavy metals. Three sites were chosen in relation to the formation and sampled five times over a thirty-day period during the recreational season. A fourth site in North Fork in Rowan County, Kentucky was also sampled periodically as it too has contact with the shale formation. These sites were then sampled monthly. All samples are analyzed for concentrations of Escherichia coli, heterotrophic bacteria, and total coliform bacteria utilizing the membrane filtration method and various culture media. Current data suggests that the radioactivity of the shale formation influences the bacteriological growth present in the waterway; however, further data will be collected and compared with that of my colleagues to confirm this trend.

Keywords: bacteriological analysis, Escherichia coli, heterotrophic bacteria, radioactive black shale formation, water quality

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Authors: Yuliya Y. Gavrichenko

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Background and Aims: The increasing bacterial resistance to almost all the available antibiotics has encouraged scientists to search for alternative sources of antibacterial agents. Nowadays, it is known that many plant secondary metabolites have diverse biological activity. These compounds can be potentially active against human bacterial and viral infections. Extended research has been carried out to explore the use of the luminescent bacterial test as a rapid, accurate and inexpensive method to assess the antibacterial properties and to predict the biological activity spectra for plant origin substances. Method: Botanical material of fifteen species was collected from their natural and cultural habitats on the Crimean peninsula. The aqueous extracts of following plants were tested: Robinia pseudoacacia L., Sideritis comosa, Cotinus coggygria Scop., Thymus serpyllum L., Juglans regia L., Securigera varia L., Achillea millefolium L., Phlomis taurica, Corylus avellana L., Sambucus nigra L., Helichrysum arenarium L., Glycyrrhiza glabra L., Elytrigia repens L., Echium vulgare L., Conium maculatum L. The test was carried out using luminous strains of marine bacteria Photobacterium leiognathi, which was isolated from the Sea of Azov as well as four Escherichia coli MG1655 recombinant strains harbouring Vibrio fischeri luxCDABE genes. Results: The bactericidal capacity of plant extracts showed significant differences in the study. Cotinus coggygria, Phlomis taurica, Juglans regia L. proved to be the most toxic to P. leiognathi. (EC50 = 0.33 g dried plant/l). Glycyrrhiza glabra L., Robinia pseudoacacia L., Sideritis comosa and Helichrysum arenarium L. had moderate inhibitory effects (EC50 = 3.3 g dried plant/l). The rest of the aqueous extracts have decreased the luminescence of no more than 50% at the lowest concentration (16.5 g dried plant/l). Antibacterial activity of herbal extracts against constitutively luminescent E. coli MG1655 (pXen7-lux) strain was observed at approximately the same level as for P. leiognathi. Cotinus coggygria and Conium maculatum L. extracts have increased light emission in the mutant E. coli MG1655 (pFabA-lux) strain which is associated with cell membranes damage. Sideritis comosa, Phlomis taurica, Juglans regia induced SOS response in E. coli (pColD-lux) strain. Glycyrrhiza glabra L. induced protein damage response in E. coli MG1655 (pIbpA-lux) strain. Conclusion: The received results have shown that the plants’ extracts had nonspecific antimicrobial effects against both E. coli (pXen7-lux) and P. leiognathi biosensors. Mutagenic, cytotoxic and protein damage effects have been observed. In general, the bioluminescent inhibition test result correlated with the traditional use of screened plants. It leads to the following conclusion that whole-cell luminescent biosensors could be the indicator of overall plants antibacterial capacity. The results of the investigation have shown a possibility of bioluminescent method in medicine and pharmacy as an approach to research the antibacterial properties of medicinal plants.

Keywords: antibacterial property, bioluminescence, medicinal plants, whole-cell biosensors

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Authors: Lotem Sarid, Meirav Trebicz-Geffen, Serge Ankri

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Queuosine (Q) is a naturally occurring modified nucleoside that occurs in the first position of transfer RNA anticodons such as Asp, Asn, His, and Tyr. As eukaryotes lack pathways to synthesize queuine, the nucleobase of queuosine, they must obtain it from their diet or gut microbiota. Our previous work investigated the effects of queuine on the physiology of the eukaryotic parasite Entamoeba histolytica and defined the enzyme EhTGT responsible for its incorporation into tRNA. To our best knowledge, it is unknown how E. histolytica salvages Q from gut bacteria. We used N-acryloyl-3-aminophenylboronic acid (APB) PAGE analysis to demonstrate that E. histolytica trophozoites can salvage queuine from Q or E. coli K12 but not from the modified E. coli QueC strain, which cannot produce queuine. Next, we examined the role of EhDUF2419, a protein with homology to DNA glycosylase, as a queuine salvage enzyme in E. histolytica. When EhDUF2419 expression is silenced, it inhibits Q's conversion to queuine, resulting in a decrease in Q-tRNA levels. We also observed that Q protects control trophozoites from oxidative stress (OS), but not siEhDUF2419 trophozoites. Overall, our data reveal that EhDUF2419 is central for the salvaging of queuine from bacteria and for the resistance of the parasite to OS.

Keywords: entamoeba histolytica, epitranscriptomics, gut microbiota, queuine, queuosine, response to oxidative stress, tRNA modification.

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Authors: Mohsina Akhter

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Cancer has become a wide spread disease around the globe and takes many lives every year. Different treatments are being practiced but all have potential side effects with somewhat less specificity towards target sites. Pseudomonas aeruginosa is known to secrete a protein azurin with special anti-cancer function. It has unique cell penetrating peptide comprising of 18 amino acids that have ability to enter cancer cells specifically. Reported function of Azurin is to stabilize p53 inside the tumor cells and induces apoptosis through Bax mediated cytochrome c release from mitochondria. At laboratory scale, we have made recombinant azurin through cloning rpTZ57R/T-azu vector into E.coli strain DH-5α and subcloning rpET28-azu vector into E.coli BL21-CodonPlus (DE3). High expression was ensured with IPTG induction at different concentrations then optimized high expression level at 1mM concentration of IPTG for 5 hours. Purification has been done by using Ni+2 affinity chromatography. We have concluded that azurin can be a remarkable improvement in cancer therapeutics if it produces on a large scale. Azurin does not enter into the normal cells so it will prove a safe and secure treatment for patients and prevent them from hazardous anomalies.

Keywords: azurin, pseudomonas aeruginosa, cancer, therapeutics

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Authors: M.Ayazi, N. Golshan Ebrahimi

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The development of antibacterial surface has devoted extensive researches and important field due to the growing antimicrobial resistance strains. The superhydrophobic surface has raised attention because of reducing bacteria adhesion in the absence of antibiotic agents. Evaluating the current development antibacterial surface has to be investigating to consider the potential of applying superhydrophobic surface to reduce bacterial adhesion or role of patterned surfaces on it. In this study, we present different samples with bio-inspired hierarchical and microstructures to consider their ability in reducing bacterial adhesion. The structures have inspired from rice-like pattern and lotus-leaf that developed on the polydimethylsiloxane (PDMS) and polypropylene (PP). The results of the attachment behaviors have considered on two bacteria strains of gram-negative Escherichia coli (E. coli) bacteria and gram-positive Staphylococcus aureus (S. aureus). The reduction of bacteria adhesion on these roughness surfaces demonstrated the effectiveness of rinsing ability on removing bacterial cells on structured plastic surfaces. Results have also offered the important role of bacterial species, material chemistry and hierarchical structure to prevent bacterial adhesion.

Keywords: hierarchical structure, self-cleaning, lotus-effect, bactericidal

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Authors: Lee A. Browne, K. Rumbold

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The first fluorinating enzyme, 5'-fluoro-5'-deoxyadenosine synthase (fluorinase) was isolated from the soil bacterium Streptomyces cattleya. Such an enzyme, with the ability to catalyze a C-F bond, presents great potential as a biocatalyst. Naturally fluorinated compounds are extremely rare in nature. As a result, the number of fluorinases identified remains relatively few. The field of fluorination is almost completely synthetic. However, with the increasing demand for fluorinated organic compounds of commercial value in the agrochemical, pharmaceutical and materials industries, it has become necessary to utilize biologically based methods such as biocatalysts. A key step in this crucial process is the large-scale production of the fluorinase enzyme in considerable quantities for industrial applications. Thus, this study aimed to optimize expression of the fluorinase enzyme in both prokaryotic and eukaryotic expression systems in order to obtain high protein yields. The fluorinase gene was cloned into the pET 41b(+) and pPinkα-HC vectors and used to transform the expression hosts, E.coli BL21(DE3) and Pichia pastoris (PichiaPink™ strains) respectively. Expression trials were conducted to select optimal conditions for expression in both expression systems. Fluorinase catalyses a reaction between S-adenosyl-L-Methionine (SAM) and fluoride ion to produce 5'-fluorodeoxyadenosine (5'FDA) and L-Methionine. The activity of the enzyme was determined using HPLC by measuring the product of the reaction 5'FDA. A gradient mobile phase of 95:5 v/v 50mM potassium phosphate buffer to a final mobile phase containing 80:20 v/v 50mM potassium phosphate buffer and acetonitrile were used. This resulted in the complete separation of SAM and 5’-FDA which eluted at 1.3 minutes and 3.4 minutes respectively. This proved that the fluorinase enzyme was active. Optimising expression of the fluorinase enzyme was successful in both E.coli and PichiaPink™ where high expression levels in both expression systems were achieved. Protein production will be scaled up in PichiaPink™ using fermentation to achieve large-scale protein production. High level expression of protein is essential in biocatalysis for the availability of enzymes for industrial applications.

Keywords: biocatalyst, expression, fluorinase, PichiaPink™

Procedia PDF Downloads 552

Authors: Jun Hyung Lee, Robin B. Guevarra, Jin Ho Cho, Bo-Ra Kim, Jiwon Shin, Doo Wan Kim, Young Hwa Kim, Minho Song, Hyeun Bum Kim

Abstract:

Colibacillosis is one of the major health problems in young piglets ultimately resulting in their death, and it is common especially in young piglets. For the swine industry, colibacillosis is one of the important economic burdens. Therefore, it is necessary for the swine industries to prevent Colibacillosis in piglets in order to reduce economic losses. Thus, we tested three types of natural plant extracts (PEs) to evaluate antibacterial effects against Shiga toxin-producing Escherichia coli (STEC) isolated from the piglet. Three PEs including turmeric oleoresin (containing curcumin 79 to 85%), capsicum oleoresin (containing capsaicin 40%-40.1%), and garlic essential oil (100% natural garlic) were tested using the direct contact agar diffusion test, minimum inhibitory concentration test, growth curve assay, and heat stability test. The tests were conducted with PEs at each concentration of 2.5%, 5%, and 10%. For the heat stability test, PEs with 10% concentration were incubated at each 4, 20, 40, 60, 80, and 100 °C for 1 hour, then the direct contact agar diffusion test was used. For the positive and negative controls, 0.5N HCl and 1XPBS were used. All the experiments were duplicated. In the direct contact agar diffusion test, garlic essential oil with 2.5%, 5%, and 10% concentration showed inhibit zones of 1.1cm, 3.0cm, and 3.6 cm in diameters compared to that of 3.5cm diameter for 0.5N HCl. The minimum inhibited concentration of garlic essential oil was 2.5%. Growth curve assay showed that the garlic essential oil was able to inhibit STEC growth significantly after 4 hours. The garlic essential oil retained the ability to inhibit STEC growth after heat treatment at each temperature. However, turmeric and capsicum oleoresins were not able to significantly inhibit STEC growth by all the tests. Even though further tests using the piglets will be required to evaluate effects of garlic essential oil for the Colibacillosis prevention for piglets, our results showed that the garlic essential oil could be used as a potential natural agent to prevent Colibacillosis in swine.

Keywords: garlic essential oil, pig, Colibacillosis, Escherichia coli

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