Search results for: E. coli

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

Procedia PDF Downloads 119

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: S. I. Enem, S. I. Oboegbulem

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Objective: The most frequently implicated E. coli serotype causing haemorrhagic colitis and haemorrhagic uraemic syndrome (HUS) is VTEC 0157. However, non-O157 VTEC is now known to be as prevalent as VETC O157 infection (or even more) in most parts of the world. The objective of the study was to establish the occurrence of non-O157 VTEC serotypes in cattle in the Federal Capital Territory (FCT) Abuja, Nigeria. The level of significance of the infection with sex, age and season were also tested. Methods: The study was carried out in the FCT, Abuja, Nigeria which is located between latitude 8o and 90 25` North of the equator and longitude 60 45` and 7045` East of the Greenwich meridian. The cross sectional epidemiological method and multi-staged sampling technique were used in this study. Samples were collected from the freshly voided faeces of both apparently healthy and diarrhoeic cattle in selected abattoirs and cattle herds. Enriched samples were analyzed bacteriologically and biochemically after which they were characterised using commercially prepared latex agglutination test kits. Results: A total of 718 faecal samples from cattle were analyzed for the presence of VTEC non-O157. Thirty eight (5.23%) were positive for non-O157. There was no significant association (p > 0.05) between sex and infection with non-O157 VTEC in cattle. There was a significant association (P < 0.05) between age and infection with non-O157 VTEC in cattle. Calves were more associated than the adults. There was also a significant association (P < 0.05) between season and infection with non-O157 VTEC in cattle. The dry season was more associated than the wet season. Conclusion: The study established the occurrence and prevalence of non-O157 VTEC in cattle in FCT, Abuja, Nigeria. As a major food animal in Nigeria, infection in cattle provides an epidemiological causal association to the infection in humans. The result showed that warmer seasons (dry season) stimulate the presence of VTEC infection in animals and thus, as a consequence, increases the number of human cases. The prevalence was also higher in younger calves (< 6 months) probably as a result of undeveloped immune system.

Keywords: prevalence, distribution, Verocytotoxigenic escherichia coli (VTEC), non-O157 serotypes, cattle

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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: 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™

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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

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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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Authors: Sajida Rasheed, Imran Hashmi, Luiza Campos, Qizhi Zhou, Kim Keu

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A quadratic model (p ˂ 0.0001) was developed by using central composite design of 50 experimental runs (42 non-center + 8 center points) to assess efficiency of background chlorine residuals in combating accidental microbial episode in a prototype distribution network (DN) (rig). A known amount of background chlorine residuals were maintained in DN and a required number of bacteria, Escherichia coli K-12 strain were introduced by an injection port in the pipe loop system. Samples were taken at various time intervals at different pipe lengths. Spread plate count was performed to count bacterial number. The model developed was significant. With microbial concentration and time (p ˂ 0.0001), pipe length (p ˂ 0.022), background chlorine residuals (p ˂ 0.07) and time^2 (p ˂ 0.09) as significant factors. The ramp function of variables shows that at the microbial count of 10^6, at 0.76 L/min, and pipe length of 133 meters, a background residual chlorine 0.16 mg/L was enough for complete inactivation of microbial episode in approximately 18 minutes.

Keywords: central composite design (CCD), distribution network, Escherichia coli, residual chlorine

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Authors: Ruta Mickiene, Audrius Maruska, Ona Ragazinskiene

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This research focuses on phytochemistry and antimicrobial activities of compounds isolated and identified from species Rosmarinus officinalis L. This is a study of synergistic effects between phenolic fraction and essential oils. The antimicrobial activity of extracts from Rosmarinus officinalis L. originated from the sector of medicinal plants, Kaunas botanical garden of Vytautas Magnus University Lithuania, were tested by the method of series dilutions, against different bacteria species. Investigated microorganisms were Escherichia coli, Proteus vulgaris and Staphylococcus aureus with and without antibiotic resistances originating from livestock. The antimicrobial activities of extracts were described by determination of the Minimal Inhibitory Concentration (MIC). Preliminary results show that the MIC range between 9.0 % and 12.0 % for the different Rosmarinus officinalis L. extracts and bacterial species. The total amounts of phenolic compounds and total amounts of flavonoids were tested in the methanolic extracts of the plants. The chemical composition for essential oils analysed by GC/MS. Predominant components were alpha pinene (20%), camphor (10%), 1.8‐cineole (5%), phellandrene (6%), camphene (5%), beta pinene (4%), bornylacetate (4%), limonene (2%), borneol (3%), alpha terpineol (3%), cymene (2%), caryophyllene (15%), verbenone (7%), alpha terpinene (3%), eucalyptol (11%).

Keywords: antimicrobial activity, essential oil, Rosmarinus officinalis L., escherichia coli

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Authors: Daria Olkiewicz, Maciej Walczak

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In this paper, the biocidal effects of trans-cinnamaldehyde (a main component of cinnamon oil) and geraniol (a constituent of Pelargonium graveolens essential oil) are presented. The activity of the combination of trans-cinnamaldehyde and geraniol was tested against 3 bacterial strains: Staphylococcus aureus ATCC6538 (Gramm+), Escherichia coli ATCC8739 (Gramm-, Lac+) and Pseudomonas aeruginosa KKP 991(Gramm-, Lac-). The biocidal activity of trans-cinnamaldehyde-geraniol mixture against bacteria mentioned above was evaluated by disk-diffusion method. The model strains were exposed on 1, 2.5, 5 and 10 mg of trans-cinnamaldehyde-geraniol mixture per disk, and all strains were susceptible to this combination of plant compounds. For all microorganisms, also Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC) were estimated. For Staphylococcus aureus MIC was 0.0625 mg/ml of the trans-cinnamaldehyde and geraniol mixture, and MBC was 1.25 mg/ml; For Escherichia coli MIC=0.5 mg/ml, MBC=1 mg/ml, and finally Pseudomonas aeruginosa was inhibited in 0.5 mg/ml, and minimal biocidal concentration of tested mixture for it was 1.25 mg/ml. There are also reports about the synergistic working of trans-cinnamaldehyde and geraniol against microorganisms and the antimicrobial activity of polymers enriched with trans-cinnamaldehyde or geraniol, therefore the successful development and introduction to the today life of biocidal polymer enriched with trans-cinnamaldehyde and geraniol are possible.

Keywords: antibacterial activity, biocidal polymers, geraniol, trans-cinnamaldehyde

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Authors: Mansur Abdulrasheed, Ibrahim I. Hussein, Ahmed M. Mubarak, Ahmed F. Umar

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This study was aimed at evaluating the phytochemical constituents and the antimicrobial activity of the seed oil of Moringa oleifera and garlic against some selected food-borne microorganisms (Staphylococcus aureus, Escherichia coli, Salmonella spp and Pseudomonas aeruginosa) using disc diffusion method. The results of the phytochemical screening revealed differences in the presence of the phytochemicals among the extracts. Saponins were detected in both Moringa oleifera and garlic seed oil, while alkaloid and tannins were observed in seed oil of garlic. Furthermore, the antibacterial assay results show that the seed oil of Moringa oleifera was inactive against all the tested organisms, even at 100 % concentration. In contrast, garlic oil was found to be active against all the tested organisms. The highest inhibition was observed in E. coli (12 mm) at 100 % concentration, while at 20 % concentration, Salmonella Sp and P. aeruginosa showed the least inhibiton (6 mm). The antimicrobial activity of the seed oil of garlic may be attributed to its phytochemicals components which were not detected in the seed oil of Moringa oleifera. The results of this study have shown the potentials of the seed oil of garlic as an antimicrobial agent more especially in foods, by inhibiting the growth of the test organisms, which range from food-borne pathogens to food spoilage organisms.

Keywords: antimicrobial, garlic, Moringa oleifera, food borne pathogens

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Authors: Zhijun Ren, Zhang Lin, Zhao Ye, Zuo Xiangyu, Mei Dongxing

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As a pretreatment process of ballast water treatment, the performance of high gradient magnetic separation (HGMS) technology for the removal of particulates and microorganisms was studied. The results showed that HGMS process could effectively remove suspended particles larger than 5 µm and had ability to resist impact load. Microorganism could also be effectively removed by HGMS process, and the removal effect increased with increasing magnetic field strength. The maximum removal rates for Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were 4016.1% and 9675.3% higher, respectively, than without the magnetic field. In addition, the superoxide dismutase (SOD) activity of the microbes decreased by 32.2% when the magnetic field strength was 15.4 mT for 72 min. The microstructure of the stainless steel wool was investigated, and the results showed that particle removal by HGMS has common function by the magnetic force of the high-strength, high-gradient magnetic field on weakly magnetic particles in the water, and on the stainless steel wool.

Keywords: HGMS, particulates, superoxide dismutase (SOD) activity, steel wool magnetic medium

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Authors: Mansur Abdulrasheed, Ibrahim I. Hussein, Ahmed M. Mubarak, Ahmed F. Umar

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This study was aimed at evaluating the phytochemical constituents and the antimicrobial activity of the seed oil of Moringa oleifera and garlic against some selected food-borne microorganisms (Staphylococcus aureus, Escherichia coli, Salmonella spp and Pseudomonas aeruginosa) using disc diffusion method. The results of the phytochemical screening revealed differences in the presence of the phytochemicals among the extracts. Saponins were detected in both Moringa oleifera and garlic seed oil, while alkaloid and tannins were observed in seed oil of garlic. Furthermore, the antibacterial assay results show that the seed oil of Moringa oleifera was inactive against all the tested organisms, even at 100 % concentration. In contrast, garlic oil was found to be active against all the tested organisms. The highest inhibition was observed in E. coli (12 mm)at 100 % concentration, while at 20 % concentration, Salmonella Sp and P. aeruginosa showed the least inhibit on (6 mm). The antimicrobial activity of the seed oil of garlic may be attributed to its phytochemicals components which were not detected in the seed oil of Moringa oleifera. The results of this study have shown the potentials of the seed oil of garlic as an antimicrobial agent more especially in foods, by inhibiting the growth of the test organisms, which range from food-borne pathogens to food spoilage organisms.

Keywords: antimicrobial, garlic, Moringa oleifera, food borne pathogens

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Authors: N. Meziou-Chebouti, A. Merabet, Y. Chebouti N. Behidj

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This work is a contribution to the knowledge of physicochemical characteristics of mature carob followed by evaluation of the activity, antimicrobial phenolics leaves and green pods of Ceratonia siliqua L. physicochemical study shows that mature carob it has a considerable content of sugar (50.90%), but poor in proteins (7%), fat (8%) and also has a high mineral content. The results obtained from phenolic extracts of leaves and green pods of Ceratonia siliqua L. show a wealth leaf phenolic extract especially flavonoids (0,545 mg EqQ/g) relative to the extract of green pods (0,226 mgEqQ/g). Polyphenols leaves have a slightly inhibitory effect on the growth of strains: Staphylococcus aureus, Escherichia coli, Klebsiella pneumoiae, Streptococcus sp and Sanmonella enteritidis, a strong inhibitory effect on the growth of Pseudomonas strain aerogenosa. Moreover, polyphenols pod have a slightly inhibitory effect on the growth of Streptococcus sp strains, Pseudomonas and aerogenosa Sanmonella enteritidis, a slightly inhibitory effect on the growth of Klebsiella pneumoniae strains, E. coli and Staphylococcus aureus.

Keywords: antimicrobial activity, bacteria, clove, Ceratonia siliqua, polyphenols

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Authors: Vivek Bhat, Rohini Kelkar, Sanjay Biswas

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Introduction: Cancer patients are at increased risk of bacterial infections. This may due to the disease process itself, the effect of chemotherapeutic drugs or invasive procedures such as catheterization. A wide variety of bacteria including some emerging pathogens are increasingly being reported from these patients. The incidence of multidrug-resistant organisms particularly in the Gram negative group is also increasing, with higher resistance rates seen to cephalosporins, β-lactam/β-lactam inhibitor combinations, and the carbapenems. This study documents the bacteriological spectrum of infections and their resistance patterns in cancer patients. Methods: This study includes all bacterial isolates recovered from infections cancer patients over a period of 18 months. Samples included Blood cultures, Pus/wound swabs, urine, tissue biopsies, body fluids, catheter tips and respiratory specimens such as sputum and bronchoalveolar lavage (BAL). All samples were processed in the microbiology laboratory as per standard laboratory protocols. Organisms were identified to species level and antimicrobial susceptibility testing was performed manually by the disc diffusion technique or in the Vitek-2 (Biomereux, France) instrument. Interpretations were as per Clinical laboratory Standards Institute (CLSI) guidelines. Results: A total of 1150 bacterial isolates were cultured from 884 test samples during the study period. Of these 227 were Gram-positive and 923 were Gram-negative organisms. Staphylococcus aureus (99 isolates) was the commonest Gram-positive isolate followed by Enterococcus (79) and Gr A Streptococcus (30). Among the Gram negatives, E. coli (304), Pseudomonas aeruginosa (201) and Klebsiella pneumoniae (190) were the most common. Of the Staphylococcus aureus isolates 27.2% were methicillin resistant. Only 5.06% enterococci were vancomycin resistant. High rates of resistance to cefotaxime and ciprofloxacin were seen amongst E. coli (84.8% & 83.55%) and Klebsiella pneumoniae (71 & 62.1%) respectively. Resistance to carbapenems (meropenem) was high at 70% in Acinetobacter spp.; however all isolates were sensitive to colistin. Among the aminoglycosides, amikacin retained good efficacy against Escherichia coli (82.9%) and Pseudomonas aeruginosa (78.1%). Occasional isolates of emerging pathogens such as Chryseobacterium indologens, Roseomonas, and Achromobacter xyloxidans were also recovered. Conclusion: The common infections in cancer patients include respiratory, wound, tract infections and sepsis. The commonest isolates include Staphylococcus aureus, Enterococci, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa. There is a high level of resistance to the commonly used antibiotics among Gram-negative organisms.

Keywords: bacteria, resistance, infection, cancer

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Authors: Yakup Ulusu, Numan Eczacıoğlu, İsa Gökçe, Helen Waller, Jeremy H. Lakey

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Besides having the appropriate amino acid sequence to perform the function of proteins, it is important to have correct conformation after this sequence to process. To consist of this conformation depends on the amino acid sequence at the primary structure, hydrophobic interaction, chaperones and enzymes in charge of folding etc. Misfolded proteins are not functional and tend to be aggregated. Cysteine originating disulfide cross-links make stable this conformation of functional proteins. When two of the cysteine amino acids come side by side, disulfide bond is established that forms a cystine bridge. Due to this feature cysteine plays an important role on the formation of three-dimensional structure of many proteins. There are two cysteine amino acids (C44, C69) in the Tol-A-III protein. Unlike protein disulfide bonds from within his own, any non-specific cystine bridge causes a change in the three dimensional structure of the protein. Proteins can be expressed in various host cells as directly or fusion (chimeric). As a result of overproduction of the recombinant proteins, aggregation of insoluble proteins in the host cell can occur by forming a crystal structure called inclusion body. In general fusion proteins are produced for provide affinity tags to make proteins more soluble and production of some toxic proteins via fusion protein expression system like pTolT. Proteins can be modified by using a site-directed mutagenesis. By this way, creation of non-specific disulfide crosslinks can be prevented at fusion protein expression system via the present cysteine replaced by another amino acid such as serine, glycine or etc. To do this, we need; a DNA molecule that contains the gene that encodes for the target protein, required primers for mutation to be designed according to site directed mutagenesis reaction. This study was aimed to be replaced cysteine encoding codon TGT with serine encoding codon AGT. For this sense and reverse primers designed (given below) and used site-directed mutagenesis reaction. Several new copy of the template plasmid DNA has been formed with above mentioned mutagenic primers via polymerase chain reaction (PCR). PCR product consists of both the master template DNA (wild type) and the new DNA sequences containing mutations. Dpn-l endonuclease restriction enzyme which is specific for methylated DNA and cuts them to the elimination of the master template DNA. E. coli cells obtained after transformation were incubated LB medium with antibiotic. After purification of plasmid DNA from E. coli, the presence of the mutation was determined by DNA sequence analysis. Developed this new plasmid is called PtolT-δ.

Keywords: site directed mutagenesis, Escherichia coli, pTolT, protein expression

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Authors: Caspar S. Carson, Gabriel W. Prather, Nicholas E. Wong, Jeffery R. Anton, William H. McCoy

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Cutibacterium acnes is a commensal bacterium found on human skin that has been linked to acne. C. acnes can also be an opportunistic pathogen when it infiltrates the body during surgery. This pathogen can cause dangerous infections of medical implants, such as shoulder replacements, leading to life-threatening blood infections. Compounding this issue, C. acnes resistance to many antibiotics has become an increasing problem worldwide, creating a need for special forms of treatment. C. acnes expresses the protein RoxP, and it requires this protein to colonize human skin. Though this protein is required for C. acnes skin colonization, its function is not yet understood. Inhibition of RoxP function might be an effective treatment for C. acnes infections. To develop such reagents, the McCoy Laboratory generated four unique anti-RoxP antibodies. Preliminary studies in the McCoy Lab have established that each antibody binds a distinct site on RoxP. To assess the potential of these antibodies as therapeutics, it is necessary to specifically characterize these antibody epitopes and evaluate them in assays that assess their ability to inhibit RoxP-dependent C. acnes growth. To provide material for these studies, an antibody expression construct, Fv-clasp(v2), was adapted to encode anti-RoxP antibody sequences. The author hypothesizes that this expression strategy can produce sufficient amounts of >95% pure antibody fragments for further characterization of these antibodies. Four anti-RoxP Fv-clasp(v2) expression constructs (pET vector-based) were transformed into E. coli BL21-Gold(DE3) cells and a small-scale expression and purification trial was performed for each construct to evaluate anti-RoxP Fv-clasp(v2) yield and purity. Successful expression and purification of these antibody constructs will allow for their use in structural studies, such as protein crystallography and cryogenic electron microscopy. Such studies would help to define the antibody binding sites on RoxP, which could then be leveraged in the development of certain methods to treat C. acnes infection through RoxP inhibition.

Keywords: structural biology, protein expression, infectious disease, antibody, therapeutics, E. coli

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Authors: Neslihan Taskale Karatug, Mustafa Akcelik

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Some literature data show that misL protein play a role on host immune response formed against Salmonella Typhimurium. The aim of the present study is to learn the role of the protein in S. Typhimurium pathogenicity. To describe certain functions of the protein, primarily recombinant misL protein was produced and purified. PCR was performed using a primer set targeted to passenger domain of the misL gene on S. Typhimurium LT2 genome. Amplicon and pet28a vector were enzymatically cleaved with EcoRI and NheI. The digested DNA materials were purified with High Pure PCR Product Purification Kit. The ligation reaction was achieved with the pure products. After preparation of competent Escherichia coli Dh5α, ligation mix was transformed into the cell by electroporation. To confirm the existence of insert gene, recombinant plasmid DNA of Dh5α was isolated with high pure plasmid DNA kit. Proved the correctness of recombinant plasmid was electroporated to BL21. The cell was induced by IPTG. After induction, the presence of recombinant protein was checked by SDS-PAGE. The recombinant misL protein was purified using HisPur Ni-NTA spin colon. The pure protein was shown by SDS-PAGE and western blot immünoassay. The concentration of the protein was measured BCA Protein Assay kit. In the wake of ligation with digested products (2 kb misL and 5.4 kb pet28a) visualised on gel size of the band was about 7.4 kb and was named as pNT01. The pNT01 recombinant plasmid was transformed into Dh5α and colonies were chosen in selective medium. Plasmid DNA isolation from them was carried out. PCR was achieved on the pNT01 to check misL and 2 kb band was observed on the agarose gel. After electroporation of the plasmid and induction of the cell, 68 kDa misL protein was seen. Subsequent to the purification of the protein, only a band was observed on SDS-PAGE. Association of the pure protein with anti-his antibody was verified by the western blot assay. The concentration of the pure misL protein was determined as 345 μg/mL. Production of polyclonal antibody will be achieved by using the obtained pure recombinant misL protein as next step. The role of the protein will come out on the immune system together some assays.

Keywords: cloning, Escherichia coli, recombinant protein purification, Salmonella Typhimurium

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Authors: I. L. Ibrahim, A. Mann, B. A. Adam

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This project involved screening for antibacterial activity, phytochemical and mineral properties of Leptadenia hastata by flame photometry. The result of phytochemical screening reveals that the presence of flavonoids, tannins, saponins, alkaloids, steroidal, and anthraquinones while the cardiac glycoside was absent. This justifies the plant been used as anti-bleeding and anti-inflammatory agents. The result of flame photometry revealed that 1.85 % (Na), 0.65% (K) and 1.85 % (Ca) which indicates the safe nature of the plant extract as such could be used to lower high blood pressure. The antibacterial properties of both the aqueous and ethanolic extract were studied against some bacteria, Escherichia coli, Bacillus Cercus, Pseudomonas aeruginas, and Enterobacter aerogegens, by disc diffusion method and the result reveals that there are very good activities against the organism while the ethanolic extract at concentration 1.0 – 1.2 mg/ml. the ethanolic extract showed in considerable zone inhibition against bacteria’s; Escherichia coli, Bacillus Cercus, pseudomonas aeruginosa andklebsellapnemuoniae. Minimum inhibitory concentration (MIC) and minimum Bacterial concentration (MBC) were conducted with fairly good significant effect of inhibition on the organism, therefore, plant extract could be a potential source of antibacterial agent.

Keywords: antibacterial activity, Leptadenia hastata, infectious diseases, phytochemical screening

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Authors: Niranjan Koirala, Sumangala Darsandhari, Hye Jin Jung, Jae Kyung Sohng

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Sakuranetin, an antifungal agent and genkwanin, an anti-inflammatory agent, are flavonoids with several potential pharmaceutical applications. To produce such valuable flavonoids in large quantity, an Escherichia coli cell factory has been created. E. coli harboring O-methyltransferase (SaOMT2) derived from Streptomyces avermitilis was employed for regiospecific methylation of naringenin and apigenin. In order to increase the production via biotransformation, metK gene was overexpressed and the conditions were optimized. The maximum yield of sakuranetin and genkwanin under optimized conditions was 197 µM and 170 µM respectively when 200 µM of naringenin and apigenin were supplemented in the separate cultures. Furthermore, sakuranetin was purified in large scale and used as a substrate for in vitro glycosylation by YjiC to produce glucose and galactose derivatives of sakuranetin for improved solubility. We also found that unlike naringenin, sakuranetin effectively inhibits α-melanocyte stimulating hormone (α-MSH)-stimulated melanogenesis in B16F10 melanoma cells. In addition, genkwanin more potently inhibited angiogenesis than apigenin. Based on our findings, we speculate that these compounds warrant further investigation in vivo as potential new therapeutic anti-carcinogenic, anti-melanogenic and anti-angiogenic agents.

Keywords: anti-carcinogenic, anti-melanogenic, glycosylation, methylation

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Authors: Elham A. Kwildi, Yahia S. Abugnah, Nuri S. Madi

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This study was designed to evaluate the microbiological quality and safety of two types of salads prepared at a catering center affiliated with Libyan Airlines in Tripoli, Libya. Two hundred and twenty-one (221) samples (132 economy-class and 89 first- class) were used in this project which lasted for ten months. Biweekly, microbiological tests were performed which included total plate count (TPC) and total coliforms (TCF), in addition to enumeration and/or detection of some pathogenic bacteria mainly Escherichia coli, Staphylococcus aureus, Bacillus cereus, Salmonella sp, Listeria sp and Vibrio parahaemolyticus parahaemolyticus, By using conventional as well as compact dry methods. Results indicated that TPC of type 1 salad ranged between (<10 – 62 x 103 cfu/gm) and (<10 to 36 x103 cfu/g), while TCF were (<10 – 41 x 103 cfu/gm) and (< 10 to 66 x102 cfu/g) using both methods of detection respectively. On the other hand, TPC of type 2 salad were: (1 × 10 – 52 x 103) and (<10 – 55 x 103 cfu/gm) and in the range of (1 x10 to 45x103 cfu/g), and the (TCF) counts were between (< 10 to 55x103 cfu/g) and (< 10 to 34 x103 cfu/g) using the 1st and the 2nd methods of detection respectively. Also, the pathogens mentioned above were detected in both types of salads, but their levels varied according to the type of salad and the method of detection. The level of Staphylococcus aureus, for instance, was 17.4% using conventional method versus 14.4% using the compact dry method. Similarly, E. coli was 7.6% and 9.8%, while Salmonella sp. recorded the least percentage i.e. 3% and 3.8% with the two mentioned methods respectively. First class salads were also found to contain the same pathogens, but the level of E. coli was relatively higher in this case (14.6% and 16.9%) using conventional and compact dry methods respectively. The second rank came Staphylococcus aureus (13.5%) and (11.2%), followed by Salmonella (6.74%) and 6.70%). The least percentage was for Vibrio parahaemolyticus (4.9%) which was detected in the first class salads only. The other two pathogens Bacillus cereus and Listeria sp. were not detected in either one of the salads. Finally, it is worth mentioning that there was a significant decline in TPC and TCF counts in addition to the disappearance of pathogenic bacteria after the 6-7th month of the study which coincided with the first trial of the HACCP system at the center. The ups and downs in the counts along the early stages of the study reveal that there is a need for some important correction measures including more emphasis on training of the personnel in applying the HACCP system effectively.

Keywords: air travel, vegetable salads, foodborne outbreaks, Libya

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Authors: Jessica C. Bannon, Cleso M. Jordao Jr., Mohammad Melebari, Carlos Leon-Velarde, Roger Johnson, Keith Warriner

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There has been an increased incidence of non-O157 Shiga Toxin Escherichia coli (STEC) with six serotypes (Top 6) being implicated in causing haemolytic uremic syndrome (HUS). Beef has been suggested to be a significant vehicle for non-O157 STEC although conclusive evidence has yet to be obtained. The following aimed to determine the prevalence of the Top 6 non-O157 STEC in beef processing using three different diagnostic platforms then characterize the recovered isolates. Hide, carcass and environmental swab samples (n = 60) were collected from a beef processing facility over a 12 month period. Enriched samples were screened using Biocontrol GDS, BAX or PALLgene molecular diagnostic tests. Presumptive non-O157 STEC positive samples were confirmed using conventional PCR and serology. STEC was detected by GDS (55% positive), BAX (85% positive), and PALLgene (93%). However, during confirmation testing only 8 of the 60 samples (13%) were found to harbour STEC. Interestingly, the presence of virulence factors in the recovered isolates was unstable and readily lost during subsequent sub-culturing. There is a low prevalence of Top 6 non-O157 STEC associated with beef although other serotypes are encountered. Yet, the instability of the virulence factors in recovered strains would question their clinical relevance.

Keywords: beef, food microbiology, shiga toxin, STEC

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