A novel source of biologically active compounds - The leaves of Serbian herbaceous peonies.

In order to gain further insight into how various extraction techniques (maceration, microwave-, and ultrasound-assisted extractions) affect the chemical profile and biological activities of leaf extracts from Paeonia tenuifolia L., Paeonia peregrina Mill., and Paeonia officinalis L., this research was performed. The targeted chemical characterization of the extracts was achieved using the Ultra-High-Performance-Liquid-Chromatography-Linear-Trap-Mass-Spectrometry OrbiTrap instrumental technique, while Fourier Transform Infrared Spectroscopy was conducted to investigate the structural properties of the examined leaf extracts. According to the results, the species P. officinalis, Bozurna locality as the origin of the plant material, and microwave-assisted extraction produced the maximum polyphenol yield, (491.9 ± 2.7 mg gallic acid equivalent (GAE)/mL). The ethanolic extracts exhibited moderate antioxidant activity as evaluated by DPPH (2,2-diphenyl-1-picrylhydrazyl) and phosphomolybdenum tests. With MIC values of 0.125 mg/mL, the leaf extracts produced by ultrasound-assisted extraction and maceration (Deliblato sands and Bogovo gumno) had the best antibacterial activity against Pseudomonas aeruginosa and Salmonella Typhimurium. Ultrasound-assisted extraction has proven to produce the most effective antimicrobial agents. Inhibitory potential towards glucosidase, amylase, cholinesterases, and tyrosinase was evaluated in enzyme inhibition assays and molecular docking simulations. Results show that leaves of P. tenuifolia L. obtained by ultrasound-assisted extraction had the highest acetylcholinesterase and butyrylcholinesterase inhibitory activity. Namely, the complexity of the polyphenol structures, the extraction method, the used locality, and the different mechanisms of the reactions between bioactives from leaf extracts and other components (free radicals, microorganisms, and enzymes) are the main factors that influence the results of the antioxidant tests, as well as the antibacterial and enzyme-inhibitory activities of the extracts. Hydroxymethyl-phenyl pentosyl-hexoside and acetyl-hydroxyphenyl-hexoside were the first time identified in the leaf extract of the Paeonia species. Due to their proven biological activities and the confirmed existence of bioactive compounds, leaf extracts may find use in foodstuffs, functional foods, and pharmaceutical products.

Phytochemical Analysis, Biological Activities, and Molecular Docking Studies of Root Extracts from Paeonia Species in Serbia.

Without being aware of their chemical composition, many cultures have used herbaceous peony roots for medicinal purposes. Modern phytopreparations intended for use in human therapy require specific knowledge about the chemistry of peony roots and their biological activities. In this study, ethanol-water extracts were prepared by maceration and microwave- and ultrasound-assisted extractions (MAE and UAE, respectively) in order to obtain bioactive molecules from the roots of Paeonia tenuifolia L., Paeonia peregrina Mill., and Paeonia officinalis L. wild growing in Serbia. Chemical characterization; polyphenol and flavonoid content; antioxidant, multianti-enzymatic, and antibacterial activities of extracts; and in vitro gastrointestinal digestion (GID) of hot water extracts were performed. The strongest anti-cholinesterase activity was observed in PT extracts. The highest anti-ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) radical potential was observed in PP extracts, whereas against DPPH (2,2-diphenyl-1-picrylhydrazyl radicals), the best results were achieved with PO extracts. Regarding antibacterial activity, extracts were strongly potent against Bacillus cereus. A molecular docking simulation was conducted to gather insights into the binding affinity and interactions of polyphenols and other Paeonia-specific molecules in the active sites of tested enzymes. In vitro GID of Paeonia teas showed a different recovery and behavior of the individual bioactives, with an increased recovery of methyl gallate and digallate and a decreased recovery of paeoniflorin and its derivatives. PT (Gulenovci) and PP (Pirot) extracts obtained by UAE and M were more efficient in the majority of the bioactivity assays. This study represents an initial step toward the possible application of Paeonia root extracts in pharmacy, medicine, and food technologies.

Liposomal and Liposomes-Film Systems as Carriers for Bioactives from Paeonia tenuifolia L. Petals: Physicochemical Characterization and Biological Potential.

Paeonia tenuifolia L. (steppe peony) petal extract was proficiently encapsulated into liposomes and biopolymer films in the current work, both times utilizing a single-step procedure. The encapsulation efficiency, size of the particles, and index of polydispersity (PDI), as well as the ζ potential of the obtained liposomes were determined, whereas in the case of films, the test included moisture content and mechanical property assessment. Fourier transform infrared spectroscopy (FT-IR) was used to evaluate the chemical composition and existence of numerous interactions in the systems. All the obtained encapsulates were subjected to antibacterial, antifungal and antibiofilm activity testing of the pathogens associated with human skin. The results indicated that the liposomes prepared using Phospholipon had the highest encapsulation efficiency (72.04%), making them the most favorable ones in the release study as well. The biological assays also revealed that Phospholipon was the most beneficial phospholipid mixture for the preparation of liposomes, whereas the film containing these liposomes did not have the ability to inhibit pathogen growth, making the double encapsulation of P. tenuifolia L. petal extract needless. These findings may be a first step toward the potential use of steppe peony extract-loaded films and liposomes in pharmaceutical and cosmetical industries.

A detailed survey of agroecological status of Allium ursinum across the republic of Serbia: Mineral composition and bioaccumulation potential.

The purpose of this study was to determine the content of twenty-two biogenic elements (BEs) and potentially toxic elements (PTEs) in the soil and fresh Allium ursinum leaves from 43 different locations, in order to examine their bioaccumulation potential. Analyses of soil and plant material were carried out by using Inductively Coupled Plasma coupled with Optical Emission Spectroscopy (ICP-OES), a mercury analyzer (Hg), liquid chromatography (Cr), and AAS hybrid technique (As). The obtained results of the investigated elements were compared with the proposed limit values. The soil contamination factor (CF) as well as plant bioaccumulation factor (BAF) were calculated and the correlation analysis was performed. The results showed that the content of some BEs/PTEs in the soil were above the limit values, with two locations highly contaminated (CF > 6) with five (Cr(VI), Cu, Mn, Ni, V) and four (As, Co, Pb, V) elements. The content of As, Cd, Cr, and Pb in the leaves was higher than the permitted levels at some locations. The BAF was high (K, Ca, Zn, As), medium (Mg, Cu, B, Ni, Na, Pb), and low (Fe, Mn, Cr). The correlation between BEs/PTEs content in the leaves and soil was not significant, except for the following elements: Cd (0.37), Ca (0.34), As (0.36), Pb (0.30), and Na (0.25). The observed medium correlation suggested that the detected elements originated both from the atmosphere and the soil. Although A. ursinum at examined locations seemed to be mostly safe for consumption, a selective mechanism of adoption of certain BEs/PTEs requires continuous monitoring of their content in the future, to avoid quantities that can jeopardize human health through its consumption.

Paeonia peregrina Mill Petals as a New Source of Biologically Active Compounds: Chemical Characterization and Skin Regeneration Effects of the Extracts.

Paeonia peregrina Mill. is a perennial herbaceous plant species, known for the medicinal value of all of its plant parts, although the chemical composition of the petals is unknown. This study aimed to determine the chemical fingerprint of the petals and also establish the optimal extraction parameters, extraction medium, and extraction method for petals collected from different localities in Serbia. The optimization was performed in order to acquire extracts that are rich in the contents of total polyphenol content (TPC) and total flavonoid content (TFC), and also exhibit strong antioxidant activity. In addition, the influence of the extracts on several human skin pathogens was evaluated, as well as their ability to aid wound closure and act as anti-inflammatory agents. Both the extraction medium and the applied technique significantly influenced the skin-beneficial biological activities, while methanol proved to be a more favorable extraction medium. In conclusion, the extraction conditions that yielded the extract with the richest phenolic content with satisfactory biological potential varied between the assays, while the most promising locality in Serbia for the collection of P. peregrina petals was Pancevo (South Banat).

Comparative transcriptome sequencing analysis to postulate the scheme of regulated leaf coloration in Perilla frutescens.

Perilla as herb, ornamental, oil and edible plant is widely used in East Asia. Until now, the mechanism of regulated leaf coloration is still unclear. In this study, four different kinds of leaf colors were used to measure pigment contents and do transcriptome sequence to postulate the mechanism of leaf coloration. The measurements of chlorophyll, carotenoid, flavonoid, and anthocyanin showed that higher contents of all the aforementioned four pigments were in full purple leaf 'M357', and they may be determined front and back leaf color formation with purple. Meanwhile, the content of anthocyanin was controlled back leaf coloration. The chromatic aberration analysis and correlative analysis between different pigments and L*a*b* values analysis also suggested front and back leaf color change was correlated with the above four pigments. The genes involved in leaf coloration were identified through transcriptome sequence. The expression levels of chlorophyll synthesis and degradation related genes, carotenoid synthesis related genes and anthocyanin synthesis genes showed up-/down-regulated expression in different color leaves and were consistent of accumulation of these pigments. It was suggested that they were the candidate genes regulated perilla leaf color formation, and genes including F3'H, F3H, F3',5'H, DFR, and ANS are probably important for regulating both front and back leaf purple formation. Transcription factors involved in anthocyanin accumulation, and regulating leaf coloration were also identified. Finally, the probable scheme of regulated both full green and full purple leaf coloration and back leaf coloration was postulated.

Chemical Profile and Skin-Beneficial Activities of the Petal Extracts of Paeonia tenuifolia L. from Serbia.

Without being aware of its chemical makeup, many ancient societies have used Steppe peony in their traditional medicine. Given that modern phytopreparation intended for use on human skin requires, above all, knowledge of its chemical composition, the goal of this study was to make a screening of the composition of aqueous and methanolic extracts of the petals of P. tenuifolia L. and to examine them for various skin-beneficial properties. The extracts were prepared by maceration, ultrasound-assisted, and microwave-assisted extraction procedures. The chemical profiling was conducted by the use of UHPLC-LTQ-OrbiTrap MS and UHPLC/MS, and spectrophotometric methods for the determination of total polyphenol and total flavonoid contents. The biological activities entailed antioxidant ABTS, DPPH, CUPRAC (Cupric Ion Reducing Antioxidant Capacity), and FRAP (Ferric Reducing Antioxidant Power) assays, antimicrobial (antibacterial and antifungal) and antibiofilm activities, cytotoxicity, wound healing potential, as well as the adhesion and invasion of Staphylococcus lugdunensis. The results showed that the petals are rich in phenolic acids and flavonoids, which are commonly associated with numerous biological activities. The aqueous extracts were more efficient in the majority of the bioactivity assays then the methanolic ones, whereas the optimal extraction method varied between the assays. This study is the first step towards the safe use of the aqueous extracts of P. tenuifolia petals for therapeutic skin treatments.

Essential oils as adjuvants in endodontic therapy: myth or reality?

To enhance endodontics, the search for new antibacterials that can improve infected tooth root canal treatment is ongoing. As potent antibacterial and antibiofilm agents, essential oils (EOs) have been suggested as novel endodontic materials. Several studies indicate that EO-based irrigants and medicaments show promising reductive potential against the most important intracanal pathogen, Enterococcus faecalis, and notably contribute to intracanal biofilm eradication. In terms of additional benefits that EO-based endodontic materials can provide, their antioxidant and anti-inflammatory potential are also important, but they have only scarcely been explored in research. Investigations into the benefits of EO-based endodontic materials together with their biocompatibility are needed. The results presented in this review strongly encourage further research on this topic.

DNA Demethylation Induces Tree Peony Flowering with a Low Deformity Rate Compared to Gibberellin by Inducing PsFT Expression under Forcing Culture Conditions.

Gibberellin (GA) is frequently used in tree peony forcing culture, but inappropriate application often causes flower deformity. Here, 5-azacytidine (5-azaC), an efficient DNA demethylating reagent, induced tree peony flowering with a low deformity rate by rapidly inducing PsFT expression, whereas GA treatment affected various flowering pathway genes with strong pleiotropy. The 5-azaC treatment, but not GA, significantly reduced the methylation level in the PsFT promoter with the demethylation of five CG contexts in a 369 bp CG-rich region, and eight light-responsive related cis-elements were also predicted in this region, accompanied by enhanced leaf photosynthetic efficiency. Through GO analysis, all methylation-closer differentially expressed genes (DEGs) were located in the thylakoid, the main site for photosynthesis, and were mainly involved in response to stimulus and single-organism process, whereas GA-closer DEGs had a wider distribution inside and outside of cells, associated with 12 categories of processes and regulations. We further mapped five candidate DEGs with potential flowering regulation, including three kinases (SnRK1, WAK2, and 5PTase7) and two bioactive enzymes (cytochrome P450 and SBH1). In summary, 5-azaC and GA may have individual roles in inducing tree peony flowering, and 5-azaC could be a preferable regulation approach; DNA demethylation is suggested to be more focused on flowering regulation with PsFT playing a core role through promoter demethylation. In addition, 5-azaC may partially undertake or replace the light-signal function, combined with other factors, such as SnRK1, in regulating flowering. This work provides new ideas for improving tree peony forcing culture technology.

Potential of Essential Oils from Anise, Dill and Fennel Seeds for the Gypsy Moth Control.

The gypsy moth (Lymantria dispar L. (Lepidoptera: Erebidae)) is a serious pest of hardwood forests. In the search for an environmentally safe means of its control, we assessed the impact of different concentrations of essential oils (EOs) from the seeds of three Apiaceae plants (anise Pimpinella anisum, dill Anethum graveolens, and fennel Foeniculum vulgare) on behavior, mortality, molting and nutritional physiology of gypsy moth larvae (GML). EOs efficacy was compared with commercial insecticide NeemAzal®-T/S (neem). The main compounds in the Eos were trans-anethole in anise; carvone, limonene, and α-phellandrene in dill; and trans-anethole and fenchone in fennel seed. At 1% EOs concentration, anise and fennel were better antifeedants and all three EOs were more toxic than neem. Neem was superior in delaying 2nd to 3rd larval molting. In the 4th instar, 0.5%, anise and fennel EOs decreased relative consumption rate more than neem, whereas all three EOs were more effective in reducing growth rate, approximate digestibility and efficiency of conversion of food into body mass leading to higher metabolic costs to GML. Decrease in consumption and metabolic parameters compared to control GML confirmed that adverse effects of the EOs stem from both pre- and post-ingestive mechanisms. The results indicate the potential of three EOs to be used for gypsy moth control.

Cymbopogon citratus essential oil: an active principle of nanoemulsion against Enterococcus faecalis root canal biofilm.

Aim: The objective was to formulate and characterize the nanoemulsion based on Cymbopogon citratus oil, intended for use in infected teeth root canal therapy. The investigation of the antioxidant and antibiofilm potential toward Enterococcus faecalis was aimed as well. Materials & methods: Characterization of oil (by GC/MS analysis) and nanoemulsion (by dynamic light scattering instrument), and determination of antibacterial (by microdilution assay), antibiofilm (by crystal violet assay) and antioxidant properties (by 2,2-diphenyl-1-picryl-hydrazyl-hydrate and thiobarbituric acid assay methods) were provided. Antibiofilm efficacy of irrigation procedure including nanoemulsion was screened on extracted teeth (by CFU-counting assay). Results: Notable antibacterial and antibiofilm activity, both against forming and preformed biofilms of oil, was observed. Irrigation involved nanoemulsion showed remarkable antibiofilm potential. Both substances induced some antioxidant activity. Conclusion: Results encourage further research with the aim of application of the nanoemulsion in dental practice.

Antimicrobial potential of irrigants based on essential oils of Cymbopogon martinii and Thymus zygis towards in vitro multispecies biofilm cultured in ex vivo root canals.

OBJECTIVE: The objective was to determine the antibiofilm efficacy of Cymbopogon martinii and Thymus zygis essential oils and to estimate the disruption potential of oil-based endodontic irrigants on the multispecies biofilm formed in the root canals of extracted teeth. DESIGN: The essential oils were characterized (GC-MS), while their antibacterial and cytotoxic properties were detected by microdilution and MTT assays. Particles sizes and polydispersity indices were determined for the irrigants. The isolates from root canals of pediatric patients were identified (MALDI-TOF). The multispecies biofilms were formed from Streptococcus mitis, Streptococcus sanguinis and Enterococcus faecalis. Antibiofilm properties of the essential oils and irrigants were determined by crystal violet and plate counting assay. Statistical analysis was performed by one-way ANOVA. RESULTS: The screening of biofilm biomass revealed a disruption potential of C. martinii essential oil. The plate counting assay showed the efficacy of both oils in diminishing cell viability: high in biofilms (reduction of log10CFU was 2.75-2.87) and moderate in the planktons formed above. The essential oil-based irrigants showed the same antibiofilm activity as in the control (1.5 % sodium hypochlorite) for C. martini and almost 2-fold higher for T. zygis. Successive irrigations with 1.5 % sodium hypochlorite, saline and an oil-based irrigant was more efficient for C. martini than for the control (reductions of log10CFU was 1.69 and 1.14, respectively). CONCLUSIONS: Notable activities achieved by essential oils and oil-based irrigants, particularly regarding C. martini, against the biofilm consisting of S. mitis, S. sanguinis, and E. faecalis, are expected to encourage further research in endodontics.

Chamomile Floricolous Downy Mildew Caused by Peronospora radii.

Floricolous downy mildews (Peronospora, oomycetes) are a small, monophyletic group of mostly inconspicuous plant pathogens that induce symptoms exclusively on flowers. Characterization of this group of pathogens, and information about their biology, is particularly sparse. The recurrent presence of a disease causing flower malformation which, in turn, leads to high production losses of the medicinal herb Matricaria chamomilla in Serbia has enabled continuous experiments focusing on the pathogen and its biology. Peronospora radii was identified as the causal agent of the disease, and morphologically and molecularly characterized. Diseased chamomile flowers showed severe malformations of the disc and ray florets, including phyllody and secondary inflorescence formation, followed by the onset of downy mildew. Phylogeny, based on internal transcribed spacer and cox2, indicates clustering of the Serbian P. radii with other P. radii from chamomile although, in cox2 analyses, they formed a separate subcluster. Evidence pointing to systemic infection was provided through histological and molecular analyses, with related experiments validating the impact of soilborne and blossom infections. This study provides new findings in the biology of P. radii on chamomile, thus enabling the reconstruction of this floricolous Peronospora species' life cycle.

Antimicrobial synergism and cytotoxic properties of Citrus limon L., Piper nigrum L. and Melaleuca alternifolia (Maiden and Betche) Cheel essential oils.

OBJECTIVES: The chemical composition, antimicrobial and synergistic effect, and cytotoxic activity of Citrus limon (lemon), Piper nigrum (green pepper) and Melaleuca alternifoila (tea tree) essential oils (EOs) were investigated. METHODS: Chemical analyses of essential oils were tested by GC-FID and GC-MS spectroscopy. The antimicrobial activity assay was conducted using microdilution method against several oral bacteria and Candida spp. originating from the humans with oral disorders. The synergistic antimicrobial activity was evaluated using checkerboard method. The cytotoxicity evaluation of EOs was assessed using MTT test. KEY FINDINGS: Limonene (37.5%) and ß-pinene (17.9%) were the major compounds in C. limon oil, ß-pinene (34.4%), δ-3-carene (19.7%), limonene (18.7%) and α-pinene (10.4%) in P. nigrum oil and terpinen-4-ol (38.6%) and γ-terpinene (21.7%) in M. alternifolia oil. The broad-spectrum antimicrobial activity was achieved by tested three EOs, with C. limon oil being the strongest against bacteria and M. alternifolia oil strongest against fungi. The EOs demonstrated synergism; their combined application revealed an increase in antimicrobial activity. All tested essential oils showed lower cytotoxic activity in comparison with the positive control, and the obtained results confirmed a dose-dependent activity. CONCLUSIONS: The results of this study encourage use of tested EOs in development of a novel agent intended for prevention or therapy of corresponding oral disorders.

Sensitivity of clinical isolates of Candida to essential oils from Burseraceae family.

The aim of this study was to investigate the chemical composition and antifungal activity of four commercial essential oils from the Burseraceae family - two Boswellia carterii Flueck oils, Canarium luzonicum (Blume) A. Gray oil, and Commiphora myrrha (Nees) Engl oil, against most common Candida spp. recovered from the human oral cavity. The essential oil samples were analyzed by GC-FID and GC/MS. The analysis showed that major essential oils' components were α-pinene (23.04 % and 31.84 %), limonene (45.62 %) and curzerene (34.65 %), respectively. Minimum inhibitory (MIC) and minimum fungicidal (MFC) concentrations were determined using a microdilution standardized technique. All tested Candida spp. clinical isolates and ATCC strains showed susceptibility to tested essential oils in a dose dependent manner. The strongest antifungal activity was shown by essential oil of B. carterii, sample 2; the average MIC values ranged from 1.25 to 1.34 mg/ml, and MFC values ranged from 2.50 to 3.75 mg/ml, depending on the fungus. This study supports the possible use of essential oils from the Bursecaceae family in reduction and elimination of Candida spp. populations in patients with oral cavity fungal infections.

Could essential oils of green and black pepper be used as food preservatives?

Black and green pepper essential oils were used in this study in order to determine the chemical composition, in vitro antimicrobial activity against food spoilage microorganisms and in situ oils effect on food microorganism, after incorporation in chicken soup, by suggested methodology for calculation of Growth inhibition concentrations (GIC50). Chemical analysis revealed a total of 34 components. The major constituent of black pepper oil was trans-caryophyllene (30.33 %), followed by limonene (12.12 %), while ß-pinene (24.42 %), δ(3)-carene (19.72 %), limonene (18.73 %) and α-pinene (10.39 %) were dominant compounds in green pepper oil. Antimicrobial activity was determined by microdilution technique and minimal inhibitory (MIC) and minimal bactericidal/fungicidal concentrations (MBC/MFC) were determined. Green pepper oil showed stronger antibacterial and antifungal activity (MIC 0.50-1.87; MBC 0.63-2.5 mg/ml; MIC 0.07-0.16; MFC 0.13-1.25 mg/ml) against black pepper oil (MIC 0.07-3.75; MBC 0.60-10.00 mg/ml; MIC 0.63-5.00; MFC 1.25-10.00 mg/ml. Oils successfully inhibited the growth of S. aureus in chicken soup in a dose dependent manner. GIC50 values were calculated after 24, 48 and 72 h and were in range of 0.156-0.689 mg/ml. The 50 % inhibitory concentrations (IC50) of EOs were 36.84 and 38.77 mg/ml with in 2, 2-diphenyl-1-picrylhydrazyl (DPPH) assay respectively. The obtained results revealed that black and green pepper volatiles are efficient in controlling the growth of known food-spoilage microorganisms.

[Production of pili, hemolysin and siderophores in the urinary isolates of Escherichia coli].

INTRODUCTION: Escherichia coli (E. coli) are the most frequent cause of the urinary tract infections. Uropathogenic E. coli (UPEC) produce virulence factors which enable them to survive in the urinary tract and cause an infection. OBJECTIVE: The objective of this study was to determine phenotype characterization of E. coli isolated from outpatients' urine in the region of Banja Luka over three-year period. In line with the objective, the following research tasks were set up: determining the production of type 1 fimbriae, P-pili, alpha-hemolysin and siderophores. METHODS: A total of 417 urinary isolates and 100 control intestinal isolates were screened for virulence factors. Production of adhesions was confirmed by haemagglutination test. Plate haemolysis test was done for the detection of alpha-hemolysin, and siderophores production assay was carried out by using the method named chrome azurol sulfonate agar diffusion assay. RESULTS: In the group of urinary isolates, almost 60% of isolates produced two or three virulence factors; only 3.8% produced none of the virulence factors. In the group of intestinal isolates, even 43% of isolates produced none of the virulence factors while 48% of isolates produced a single virulence factor and 9% produced two virulence factors. CONCLUSION: Urinary isolates E. coli express significantly more P-pili, alpha-hemolysin and siderophore than intestinal isolates (p < 0.001).There was no significant difference in production of type 1 fimbriae among the urinary and intestinal isolates.

[Prevalence and antimicrobial susceptibility of extended-spectrum beta-lactamase producing urinary isolates of Escherichia coli in outpatients].

INTRODUCTION: In Gram-negative bacteria, the production of beta-lactamases is the most important mechanism of resistance to beta-lactam antibiotics. In the Banja Luka region, there were no extensive researches on the prevalence and antimicrobial resistance of the extended-spectrum beta-lactamase (ESBL) producing Escherichia coli (E. coli) isolates. OBJECTIVE: The aim of the present study was to determine the presence of ESBL producing E. coli isolates as the cause of the urinary tract infections in outpatients, the distribution of these ESBL isolates according to age and gender of patients and their susceptibility to antimicrobials. METHODS: Urine specimens obtained from outpatients were cultured on chromogenic CPS-ID3 media. All plates showing significant (> 10(5) cfu/ml) growth of E. coli in pure culture were further processed. Antimicrobial susceptibility testing was performed on VITEK TWO Compact using AST-GN27 cards for testing Gram negative bacteria and detection of ESBL producers. RESULTS: Out of 2,195 isolates, 177 (8.1%) were ESBL producers. Ninety-two isolates were obtained from female patients (5% of E. coli isolated from women) and 85 isolates from male patients (23% of E. coli isolated from men). High percentage of ESBL isolates was detected in the infant age group under one year (36.7%) and in the age group over 60 years (28.8%). All ESBL isolates were susceptible to imipenem and resistant to ampicillin, piperacillin, cefazolin, cefotaxime, ceftazidime and cefepime. There was a significant resistance to amikacin (79.1%), gentamicin (76.8%), amoxicillin/clavulanate (54.8%) and trimethoprim/sulphamethoxazole (45.8%). Resistance to nutrofurantoin was 13.6%. CONCLUSION: This study has demonstrated the presence of ESBL producing E. coli urinary isolates in outpatients, and their extensive susceptibility to imipenem and nitrofurantoin.