Commenced in January 2007
Frequency: Monthly
Edition: International
Paper Count: 534

Search results for: E. coli

534 Visual Detection of Escherichia coli (E. coli) through Formation of Beads Aggregation in Capillary Tube by Rolling Circle Amplification

Authors: Bo Ram Choi, Ji Su Kim, Juyeon Cho, Hyukjin Lee

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Food contaminated by bacteria (E.coli), causes food poisoning, which occurs to many patients worldwide annually. We have introduced an application of rolling circle amplification (RCA) as a versatile biosensor and developed a diagnostic platform composed of capillary tube and microbeads for rapid and easy detection of Escherichia coli (E. coli). When specific mRNA of E.coli is extracted from cell lysis, rolling circle amplification (RCA) of DNA template can be achieved and can be visualized by beads aggregation in capillary tube. In contrast, if there is no bacterial pathogen in sample, no beads aggregation can be seen. This assay is possible to detect visually target gene without specific equipment. It is likely to the development of a genetic kit for point of care testing (POCT) that can detect target gene using microbeads.

Keywords: rolling circle amplification (RCA), Escherichia coli (E. coli), point of care testing (POCT), beads aggregation, capillary tube

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533 Production of Human BMP-7 with Recombinant E. coli and B. subtilis

Authors: Jong Il Rhee

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The polypeptide representing the mature part of human BMP-7 was cloned and efficiently expressed in Escherichia coli and Bacillus subtilis, which had a clear band for hBMP-7, a homodimeric protein with an apparent molecular weight of 15.4 kDa. Recombinant E.coli produced 111 pg hBMP-7/mg of protein hBMP-7 through IPTG induction. Recombinant B. subtilis also produced 350 pg hBMP-7/ml of culture medium. The hBMP-7 was purified in 2 steps using an FPLC system with an ion exchange column and a gel filtration column. The hBMP-7 produced in this work also stimulated the alkaline phosphatase (ALP) activity in a dose-dependent manner, i.e. 2.5- and 8.9-fold at 100 and 300 ng hBMP-7/ml, respectively, and showed intact biological activity.

Keywords: B. subtilis, E. coli, fermentation, hBMP-7

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532 Riparian Buffer Strips’ Capability of E. coli Removal in New York Streams

Authors: Helen Sanders, Joshua Cousins

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The purpose of this study is to ascertain whether riparian buffer strips could be used to reduce Escherichia Coli (E. coli) runoff into streams in Central New York. Mainstream methods currently utilized to reduce E. coli runoff include fencing and staggered fertilizing plans for agriculture. These methods still do not significantly limit E. coli and thus, pose a serious health risk to individuals who swim in contaminated waters or consume contaminated produce. One additional method still in research development involves the planting of vegetated riparian buffers along waterways. Currently, riparian buffer strips are primarily used for filtration of nitrate and phosphate runoff to slow erosion, regulate pH and, improve biodiversity within waterways. For my research, four different stream sites were selected for the study, in which rainwater runoff was collected at both the riparian buffer and the E. coli sourced runoff upstream. Preliminary results indicate that there is an average 70% decrease in E. coli content in streams at the riparian buffer strips compared to upstream runoff. This research could be utilized to include vegetated buffer planting as a method to decrease manure runoff into essential waterways.

Keywords: Escherichia coli, riparian buffer strips, vegetated riparian buffers, runoff, filtration

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531 Cytolethal Distending Toxins in Intestinal and Extraintestinal E. coli

Authors: Katarína Čurová, Leonard Siegfried, Radka Vargová, Marta Kmeťová, Vladimír Hrabovský

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Introduction: Cytolethal distending toxins (CDTs) represent intracellular acting proteins which interfere with cell cycle of eukaryotic cells. They are produced by Gram-negative bacteria with afinity to mucocutaneous surfaces and could play a role in the pathogenesis of various diseases. CDTs induce DNA damage probably through DNAse activity, which causes cell cycle arrest and leads to further changes (cell distension and death, apoptosis) depending on the cell type. Five subtypes of CDT (I to V) were reported in E. coli. Methods: We examined 252 E. coli strains belonging to four different groups. Of these strains, 57 were isolated from patients with diarrhea, 65 from patients with urinary tract infections (UTI), 65 from patients with sepsis and 65 from patients with other extraintestinal infections (mostly surgical wounds, decubitus ulcers and respiratory tract infections). Identification of these strains was performed by MALDI-TOF analysis and detection of genes encoding CDTs and determination of the phylogenetic group was performed by PCR. Results: In this study, we detected presence of cdt genes in 11 of 252 E. coli strains tested (4,4 %). Four cdt positive E. coli strains were confirmed in group of UTI (6,15 %), three cdt positive E. coli strains in groups of diarrhea (5,3 %) and other extraintestinal infections (4,6 %). The lowest incidence, one cdt positive E. coli strain, was observed in group of sepsis (1,5 %). All cdt positive E. coli strains belonged to phylogenetic group B2. Conclusion: CDT-producing E. coli are isolated in a low percentage from patients with intestinal and extraintestinal infections, including sepsis and our results correspond with these studies. A weak prevalence of cdt genes suggests that CDTs are not major virulence factors but in combination with other virulence factors may increase virulence potential of E. coli. We suppose that all 11 cdt positive E. coli strains represent real pathogens because they belong to the phylogenetic group B2 which is pathogenic lineage for bacteria E. coli.

Keywords: cytolethal distending toxin, E. coli, phylogenetic group, extraintestinal infection, diarrhea

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530 Molecular Detection and Characterization of Shiga Toxogenic Escherichia coli Associated with Dairy Product

Authors: Mohamed Al-Hazmi, Abdullah Al-Arfaj, Moussa Ihab

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Raw, unpasteurized milk can carry dangerous bacteria such as Salmonella, E. coli, and Listeria, which are responsible for causing numerous foodborne illnesses. The objective of this study was molecular characterization of shiga toxogenic E. coli in raw milk collected from different Egyptian governorates by multiplex PCR. During the period of 25th May to 25th October 2012, a total of 320 bulk-tank milk samples were collected from 10 cow farms located in different Egyptian governorates. Bacteriological examination of milk samples revealed the presence of E. coli organisms in 65 samples (20.3%), serotyping of the E. coli isolates revealed, 35 strains (10.94%) O111, 15 strains (4.69%) O157: H7, 10 strains (3.13%) O128 and 5 strains (1.56%) O119. Multiplex PCR for detection of shiga toxin type 2 and intimin genes revealed positive amplification of 255 bp fragment of shiga toxin type 2 gene and 384 bp fragment of intimin gene from all E. coli serovar O157: H7, while from serovar O111 were 25 (71.43%), 20 (57.14%) and from serovar O128 were 6 (60%), 8 (80%), respectively. The results of multiplex PCR assay are useful for identification of STEC possessing the eaeA and stx2 genes.

Keywords: raw milk, E. coli, multiplex PCR, Shiga toxin type 2, intimin gene

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529 Prevalence and Risk Factors of Faecal Carriage Fluoroquinolone-Resistant Escherichia coli among Hospitalized Patients in Ado-Ekiti, Nigeria

Authors: C. A. Ologunde

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Escherichia coli have been a major microorganisms associated with, and isolated from feacal samples either in adult or children all over the world. Strains of these organisms are resistant to cephalosporins and fluoroquinolone (FQ) antimicrobial agents among hospitalized patients and FQs are the most frequently prescribed antimicrobial class in hospitals, and the level of resistant of E. coli to these antimicrobial agents is a risk factor that should be assessed. Hence, this study was conducted to determine the prevalence and risk factors for colonization with fluoroquinolone (FQ)-resistant E. coli in hospitalized patients in Ado-Ekiti. Rectal swabs were obtained from patients in hospitals in the study area and FQ-resistant E. coli were isolated and identified by means of Nalidixic acid multi-disk and a 1-step screening procedure. Species identification and FQ resistance were confirmed by automated testing (Vitek, bioMerieux, USA). Individual colonies were subjected to pulse-field gel electrophoresis (PAGE) to determine macro-restriction polymorphism after digestion of chromosomal DNA. FQ-resistant E. coli was detected in the stool sample of 37(62%) hospitalized patient. With multivariable analyses, the use of FQ before hospitalization was the only independent risk factor for FQ-resistant E. coli carriage and was consistent for FQ exposures for the 3-12 months of study. Pulsed-field gel electrophoresis of FQ-resistant E. coli identified conal spread of 1(one) strain among 18 patients. Loss (9 patients) or acquisition (10 residents) of FQ-resistant E. coli was documented and was associated with de novo colonization with genetically distinct strains. It was concluded that FQ-resistant E. coli carriage was associated with clonal spread. The differential effects of individual fluoroquinolone on antimicrobial drug resistance are an important area for future study, as hospitals manipulate their formularies with regard to use of individual fluoroquinolone, often for economic reasons.

Keywords: E. coli, fluoroquinolone, risk factors, feacal carriage, hospitalized patients, Ado-Ekiti

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528 Cell Surface Display of Xylanase on Escherichia coli by TibA Autotransporter

Authors: Yeng Min Yi, Rosli Md Illias, Salehhuddin Hamdan

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Industrial biocatalysis is mainly based on the use of cell free or intracellular enzyme systems. However, the expensive cost and relatively lower operational stability of free enzymes limit practical use in industries. Cell surface display system can be used as a cost-efficient alternative to overcome the laborious purification and substrate transport limitation. In this research, TibA autotransporter from E. coli was used to display Aspergillus fumigatus xylanase (xyn). The amplified xyn was fused in between N-terminal signal peptide and C-terminal β-barrel of TibA. The cloned was transformed and expressed in E. coli BL21 (DE3). Outer membrane localization of TibA-xyn fusion protein was confirmed by SDS PAGE and western blot with expected size of 62.5 kDa. Functional display of xyn was examined by activity assay. Cell surface displayed xyn exhibited the highest activity at 37 °c, 0.3 mM IPTG. As a summary, TibA displaying system has the potential for further industrial applications. Moreover, this is the first report of the display of xylanase using TibA on the surface of E. coli.

Keywords: biocatalysis, cell surface display, Escherichia coli, TibA autotransporter

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527 Purification, Extraction and Visualization of Lipopolysaccharide of Escherichia coli from Urine Samples of Patients with Urinary Tract Infection

Authors: Fariha Akhter Chowdhury, Mohammad Nurul Islam, Anamika Saha, Sabrina Mahboob, Abu Syed Md. Mosaddek, Md. Omar Faruque, Most. Fahmida Begum, Rajib Bhattacharjee

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Urinary tract infection (UTI) is one of the most common infectious diseases in Bangladesh where Escherichia coli is the prevalent organism and responsible for most of the infections. Lipopolysaccharide (LPS) is known to act as a major virulence factor of E. coli. The present study aimed to purify, extract and visualize LPS of E. coli clinical isolates from urine samples of patients with UTI. The E. coli strain was isolated from the urine samples of 10 patients with UTI and then the antibiotic sensitivity pattern of the isolates was determined. The purification of LPS was carried out using the hot aqueous-phenol method and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, which was directly stained using the modified silver staining method and Coomassie blue. The silver-stained gel demonstrated both smooth and rough type LPS by showing trail-like band patterns with the presence and lacking O-antigen region, respectively. Coomassie blue staining showed no band assuring the absence of any contaminating protein. Our successful extraction of purified LPS from E. coli isolates of UTI patients’ urine samples can be an important step to understand the UTI disease conditions.

Keywords: Escherichia coli, electrophoresis, polyacrylamide gel, silver staining, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

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526 The Effect of Cinnamaldehyde on Escherichia coli Survival during Low Temperature Long Time Cooking

Authors: Fuji Astuti, Helen Onyeaka

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The aim of the study was to investigate the combine effects of cinnamaldehyde (0.25 and 0.45% v/v) on thermal resistance of pathogenic Escherichia coli during low temperature long time (LT-LT) cooking below 60℃. Three different static temperatures (48, 53 and 50℃) were performed, and the number of viable cells was studied. The starting concentrations of cells were 10⁸ CFU/ml. In this experiment, heat treatment efficiency for safe reduction indicated by decimal logarithm reduction of viable recovered cells, which was monitored for heating over 6 hours. Thermal inactivation was measured by means of establishing the death curves between the mean log surviving cells (log₁₀ CFU/ml) and designated time points (minutes) for each temperature test. The findings depicted that addition of cinnamaldehyde exhibited to elevate the thermal sensitivity of E. coli. However, the injured cells found to be well-adapted to all temperature tests after certain time point of cooking, in which they grew to more than 10⁵ CFU/ml.

Keywords: cinnamaldehyde, decimal logarithm reduction, Escherichia coli, LT-LT cooking

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525 Open Reading Frame Marker-Based Capacitive DNA Sensor for Ultrasensitive Detection of Escherichia coli O157:H7 in Potable Water

Authors: Rehan Deshmukh, Sunil Bhand, Utpal Roy

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We report the label-free electrochemical detection of Escherichia coli O157:H7 (ATCC 43895) in potable water using a DNA probe as a sensing molecule targeting the open reading frame marker. Indium tin oxide (ITO) surface was modified with organosilane and, glutaraldehyde was applied as a linker to fabricate the DNA sensor chip. Non-Faradic electrochemical impedance spectroscopy (EIS) behavior was investigated at each step of sensor fabrication using cyclic voltammetry, impedance, phase, relative permittivity, capacitance, and admittance. Atomic force microscopy (AFM) and scanning electron microscopy (SEM) revealed significant changes in surface topographies of DNA sensor chip fabrication. The decrease in the percentage of pinholes from 2.05 (Bare ITO) to 1.46 (after DNA hybridization) suggested the capacitive behavior of the DNA sensor chip. The results of non-Faradic EIS studies of DNA sensor chip showed a systematic declining trend of the capacitance as well as the relative permittivity upon DNA hybridization. DNA sensor chip exhibited linearity in 0.5 to 25 pg/10mL for E. coli O157:H7 (ATCC 43895). The limit of detection (LOD) at 95% confidence estimated by logistic regression was 0.1 pg DNA/10mL of E. coli O157:H7 (equivalent to 13.67 CFU/10mL) with a p-value of 0.0237. Moreover, the fabricated DNA sensor chip used for detection of E. coli O157:H7 showed no significant cross-reactivity with closely and distantly related bacteria such as Escherichia coli MTCC 3221, Escherichia coli O78:H11 MTCC 723 and Bacillus subtilis MTCC 736. Consequently, the results obtained in our study demonstrated the possible application of developed DNA sensor chips for E. coli O157:H7 ATCC 43895 in real water samples as well.

Keywords: capacitance, DNA sensor, Escherichia coli O157:H7, open reading frame marker

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524 Prevalence, Antimicrobial Susceptibility Pattern and Associated Risk Factors for Salmonella Species and Escherichia Coli from Raw Meat at Butchery Houses in Mekelle, Tigray, Northern Ethiopia

Authors: Haftay Abraha Tadesse, Dawit Gebreegziabiher Hagos, Atsebaha Gebrekidan Kahsay, Mahumd Abdulkader

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Background: Salmonella species and Escherichia coli (E. coli) are important foodborne pathogens affecting humans and animals. They are among the most important causes of infection that are associated with the consumption of contaminated food. This study was aimed to determine the prevalence, antimicrobial susceptibility patterns and associated risk factors for Salmonella species and E. coli in raw meat from butchery houses of Mekelle, Northern Ethiopia. Method: A cross-sectional study was conducted from January to December 2019. Socio-demographic data and risk factors were collected using a predesigned questionnaire. Meat samples were collected aseptically from the butchery houses and transported using icebox to Mekelle University, College of Veterinary Sciences for the isolation and identification of Salmonella species and E. coli. Antimicrobial susceptibility patterns were determined using Kirby disc diffusion method. Data obtained were cleaned and entered into Statistical Package for the Social Sciences version 22 and logistic regression models with odds ratio were calculated. P-value < 0.05 was considered as statistically significant. Results: A total of 153 out of 384 (39.8%) of the meat specimens were found to be contaminated. The contamination of Salmonella species and E. coli were 15.6% (n=60) and 20.8%) (n=80), respectively. Mixed contamination (Salmonella species and E. coli) was observed in 13 (3.4 %) of the analyzed. Poor washing hands regularly (AOR = 8.37; 95% CI: 2.75-25.50) and not using gloves during meat handling (AOR=11. 28; 95% CI:(4.69 27.10) were associated with overall bacterial contamination. About 100% of the tested isolates were sensitive to ciprofloxacin, gentamicin, Co trimoxazole , sulphamethoxazole, ceftriaxone, and trimethoprim and ciprofloxacin, gentamicin, and norfloxacine of E. coli and Salmonella species, respectively, while the resistance of amoxyclav_amoxicillin and erythromycin were both isolated bacteria species. The overall multidrug resistance pattern for Salmonella and E. coli were 51.4% (n=19) and 31.8% (14), respectively. Conclusion: Of the 153 (153/384) contaminated raw meat, 60 (15.6%) and 80 (20.8%) were contaminated by Salmonella species and E. coli, respectively. Poor handwashing practice and not using glove during meat handling showed a significant association with bacterial contamination. Multidrug-resistant showed in Salmonella species, and E. coli were 19 (51.4%) and 14 (31.8%), respectively.

Keywords: antimicrobial susceptibility test, butchery houses, E. coli, raw meat, salmonella species

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523 Prediction and Identification of a Permissive Epitope Insertion Site for St Toxoid in cfaB from Enterotoxigenic Escherichia coli

Authors: N. Zeinalzadeh, Mahdi Sadeghi

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Enterotoxigenic Escherichia coli (ETEC) is the most common cause of non-inflammatory diarrhea in the developing countries, resulting in approximately 20% of all diarrheal episodes in children in these areas. ST is one of the most important virulence factors and CFA/I is one of the frequent colonization factors that help to process of ETEC infection. ST and CfaB (CFA/I subunit) are among vaccine candidates against ETEC. So, ST because of its small size is not a good immunogenic in the natural form. However to increase its immunogenic potential, here we explored candidate positions for ST insertion in CfaB sequence. After bioinformatics analysis, one of the candidate positions was selected and the chimeric gene (cfaB*st) sequence was synthesized and expressed in E. coli BL21 (DE3). The chimeric recombinant protein was purified with Ni-NTA columns and characterized with western blot analysis. The residue 74-75 of CfaB sequence could be a good candidate position for ST and other epitopes insertion.

Keywords: bioinformatics, CFA/I, enterotoxigenic E. coli, ST toxoid

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522 Antimicrobial Resistance Patterns of Campylobacter from Pig and Cattle Carcasses in Poland

Authors: Renata Szewczyk, Beata Lachtara, Kinga Wieczorek, Jacek Osek

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Campylobacter is recognized as the main cause of bacterial gastrointestinal infections in Europe. A main source of the pathogen is poultry and poultry meat; however, other animals like pigs and cattle can also be reservoirs of the bacteria. Human Campylobacter infections are often self-limiting but in some cases, macrolide and fluoroquinolones have to be used. The aim of this study was to determine antimicrobial resistance patterns (AMR) of Campylobacter isolated from pig and cattle carcasses. Between July 2009 and December 2015, 735 swabs from pig (n = 457) and cattle (n = 278) carcasses were collected at Polish slaughterhouses. All samples were tested for the presence of Campylobacter by ISO 10272-1 and confirmed to species level using PCR. The antimicrobial susceptibility of Campylobacter isolates was determined by a microbroth dilution method with six antimicrobials: gentamicin (GEN), streptomycin (STR), erythromycin (ERY), nalidixic acid (NAL), ciprofloxacin (CIP) and tetracycline (TET). It was found that 167 of 735 samples (22.7%) were contaminated with Campylobacter. The vast majority of them were of pig origin (134; 80.2%), whereas for cattle carcasses Campylobacter was less prevalent (33; 19.8%). Among positive samples C. coli was predominant species (123; 73.7%) and it was isolated mainly from pig carcasses. The remaining isolates were identified as C. jejuni (44; 26.3%). Antimicrobial susceptibility indicated that 22 out of 167 Campylobacter (13.2%) were sensitive to all antimicrobials used. Fourteen of them were C. jejuni (63.6%; pig, n = 6; cattle, n = 8) and 8 was C. coli (36.4%; pig, n = 4; cattle, n = 4). Most of the Campylobacter isolates (145; 86.8%) were resistant to one or more antimicrobials (C. coli, n = 115; C. jejuni, n = 30). Comparing the AMR for Campylobacter species it was found that the most common pattern for C. jejuni was CIP-NAL-TET (9; 30.0%), whereas CIP-NAL-STR-TET was predominant among C. coli (47; 40.9%). Multiresistance, defined as resistance to three or more classes of antimicrobials, was found in 57 C. coli strains, mostly obtained from pig (52 isolates). On the other hand, only one C. jejuni strain, isolated from cattle, showed multiresistance with pattern CIP-NAL-STR-TET. Moreover, CIP-NAL-STR-TET was characteristic for most of multiresistant C. coli isolates (47; 82.5%). For the remaining C. coli the resistance patterns were CIP-ERY-NAL-TET (7 strains; 12.3%) and for one strain of each patterns: ERY-STR-TET, CIP-STR-TET, CIP-NAL-GEN-STR-TET. According to the present findings resistance to erythromycin was observed only in 11 C. coli (pig, n = 10; cattle, n = 1). In conclusion, the results of this study showed that pig carcasses may be a serious public health concern because of contamination with C. coli that might features multiresistance to antimicrobials.

Keywords: antimicrobial resistance, Campylobacter, carcasses, multi resistance

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521 The Incidence of Prostate Cancer in Previous Infected E. Coli Population

Authors: Andreea Molnar, Amalia Ardeljan, Lexi Frankel, Marissa Dallara, Brittany Nagel, Omar Rashid

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Background: Escherichia coli is a gram-negative, facultative anaerobic bacteria that belongs to the family Enterobacteriaceae and resides in the intestinal tracts of individuals. E.Coli has numerous strains grouped into serogroups and serotypes based on differences in antigens in their cell walls (somatic, or “O” antigens) and flagella (“H” antigens). More than 700 serotypes of E. coli have been identified. Although most strains of E. coli are harmless, a few strains, such as E. coli O157:H7 which produces Shiga toxin, can cause intestinal infection with symptoms of severe abdominal cramps, bloody diarrhea, and vomiting. Infection with E. Coli can lead to the development of systemic inflammation as the toxin exerts its effects. Chronic inflammation is now known to contribute to cancer development in several organs, including the prostate. The purpose of this study was to evaluate the correlation between E. Coli and the incidence of prostate cancer. Methods: Data collected in this cohort study was provided by a Health Insurance Portability and Accountability Act (HIPAA) compliant national database to evaluate patients infected with E.Coli infection and prostate cancer using the International Classification of Disease (ICD-10 and ICD-9 codes). Permission to use the database was granted by Holy Cross Health, Fort Lauderdale for the purpose of academic research. Data analysis was conducted through the use of standard statistical methods. Results: Between January 2010 and December 2019, the query was analyzed and resulted in 81, 037 patients after matching in both infected and control groups, respectively. The two groups were matched by Age Range and CCI score. The incidence of prostate cancer was 2.07% and 1,680 patients in the E. Coli group compared to 5.19% and 4,206 patients in the control group. The difference was statistically significant by a p-value p<2.2x10-16 with an Odds Ratio of 0.53 and a 95% CI. Based on the specific treatment for E.Coli, the infected group vs control group were matched again with a result of 31,696 patients in each group. 827 out of 31,696 (2.60%) patients with a prior E.coli infection and treated with antibiotics were compared to 1634 out of 31,696 (5.15%) patients with no history of E.coli infection (control) and received antibiotic treatment. Both populations subsequently developed prostate carcinoma. Results remained statistically significant (p<2.2x10-16), Odds Ratio=0.55 (95% CI 0.51-0.59). Conclusion: This retrospective study shows a statistically significant correlation between E.Coli infection and a decreased incidence of prostate cancer. Further evaluation is needed in order to identify the impact of E.Coli infection and prostate cancer development.

Keywords: E. Coli, prostate cancer, protective, microbiology

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520 Nanobiosensor System for Aptamer Based Pathogen Detection in Environmental Waters

Authors: Nimet Yildirim Tirgil, Ahmed Busnaina, April Z. Gu

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Environmental waters are monitored worldwide to protect people from infectious diseases primarily caused by enteric pathogens. All long, Escherichia coli (E. coli) is a good indicator for potential enteric pathogens in waters. Thus, a rapid and simple detection method for E. coli is very important to predict the pathogen contamination. In this study, to the best of our knowledge, as the first time we developed a rapid, direct and reusable SWCNTs (single walled carbon nanotubes) based biosensor system for sensitive and selective E. coli detection in water samples. We use a novel and newly developed flexible biosensor device which was fabricated by high-rate nanoscale offset printing process using directed assembly and transfer of SWCNTs. By simple directed assembly and non-covalent functionalization, aptamer (biorecognition element that specifically distinguish the E. coli O157:H7 strain from other pathogens) based SWCNTs biosensor system was designed and was further evaluated for environmental applications with simple and cost-effective steps. The two gold electrode terminals and SWCNTs-bridge between them allow continuous resistance response monitoring for the E. coli detection. The detection procedure is based on competitive mode detection. A known concentration of aptamer and E. coli cells were mixed and after a certain time filtered. The rest of free aptamers injected to the system. With hybridization of the free aptamers and their SWCNTs surface immobilized probe DNA (complementary-DNA for E. coli aptamer), we can monitor the resistance difference which is proportional to the amount of the E. coli. Thus, we can detect the E. coli without injecting it directly onto the sensing surface, and we could protect the electrode surface from the aggregation of target bacteria or other pollutants that may come from real wastewater samples. After optimization experiments, the linear detection range was determined from 2 cfu/ml to 10⁵ cfu/ml with higher than 0.98 R² value. The system was regenerated successfully with 5 % SDS solution over 100 times without any significant deterioration of the sensor performance. The developed system had high specificity towards E. coli (less than 20 % signal with other pathogens), and it could be applied to real water samples with 86 to 101 % recovery and 3 to 18 % cv values (n=3).

Keywords: aptamer, E. coli, environmental detection, nanobiosensor, SWCTs

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519 Pathogenic Escherichia Coli Strains and Their Antibiotic Susceptibility Profiles in Cases of Child Diarrhea at Addis Ababa University, College of Health Sciences, Tikur Anbessa Specialized Hospital, Addis Ababa, Ethiopia

Authors: Benyam Zenebe, Tesfaye Sisay, Gurja Belay, Workabeba Abebe

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Background: The prevalence and antibiogram of pathogenic E. coli strains, which cause diarrhea vary from region to region, and even within countries in the same geographical area. In Ethiopia, diagnostic approaches to E. coli induced diarrhea in children less than five years of age are not standardized. The aim of this study was to determine the involvement of pathogenic E. coli strains in child diarrhea and determine the antibiograms of the isolates in children less than 5 years of age with diarrhea at Addis Ababa University College of Health Sciences TikurAnbessa Specialized Hospital, Addis Ababa, Ethiopia. Methods: A purposive study that included 98 diarrheic children less than five years of age was conducted at Addis Ababa University College of Health Sciences, TikurAnbessa Specialized Hospital, Addis Ababa, Ethiopia to detect pathogenic E. coli biotypes. Stool culture was used to identify presumptive E. coliisolates. Presumptive isolates were confirmed by biochemical tests, and antimicrobial susceptibility tests were performed on confirmed E. coli isolates by the disk diffusion method. DNA was extracted from confirmed isolates by a heating method and subjected to Polymerase Chain Reaction or the presence of virulence genes. Amplified PCR products were analyzed by agarose gel electrophoresis. Data were collected on child demographics and clinical conditions using administered questionnaires. The prevalence of E. coli strains from the total diarrheic children, and the prevalence of pathogenic strains from total E. coli isolates along with their susceptibility profiles; the distribution of pathogenic E.coli biotypes among different age groups and between the sexes were determined by using descriptive statistics. Result: Out of 98 stool specimens collected from diarrheic children less than 5 years of age, 75 presumptive E. coli isolates were identified by culture; further confirmation by biochemical tests showed that only 56 of the isolates were E. coli; 29 of the isolates were found in male children and 27 of them in female children. Out of the 58 isolates of E. coli, 25 pathotypes belonging to different classes of pathogenic strains: STEC, EPEC, EHEC, EAEC were detected by using the PCR technique. Pathogenic E. coli exhibited high rates of antibiotic resistance to many of the antibiotics tested. Moreover, they exhibited multiple drug resistance. Conclusion: This study found that the isolation rate of E. coli and the involvement of antibiotic-resistant pathogenic E. coli in diarrheic children is prominent, and hence focus should be given on the diagnosis and antimicrobial sensitivity testing of pathogenic E. coli at Addis Ababa University College of Health Sciences TikurAnbessa Specialized Hospital. Among antibiotics tested, Cefotitan could be a drug of choice to treat E. coli.

Keywords: antibiotic susceptibility profile, children, diarrhea, E. coli, pathogenic

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518 Assessment of Escherichia coli along Nakibiso Stream in Mbale Municipality, Uganda

Authors: Abdul Walusansa

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The aim of this study was to assess the level of microbial pollution along Nakibiso stream. The study was carried out in polluted waters of Nakibiso stream, originating from Mbale municipality and running through ADRA Estates to Namatala Wetlands in Eastern Uganda. Four sites along the stream were selected basing on the activities of their vicinity. A total of 120 samples were collected in sterile bottles from the four sampling locations of the stream during the wet and dry seasons of the year 2011. The samples were taken to the National water and Sewerage Cooperation Laboratory for Analysis. Membrane filter technique was used to test for Erischerichia coli. Nitrogen, Phosphorus, pH, dissolved oxygen, electrical conductivity, total suspended solids, turbidity and temperature were also measured. Results for Nitrogen and Phosphorus for sites; 1, 2, 3 and 4 were 1.8, 8.8, 7.7 and 13.8 NH4-N mg/L; and 1.8, 2.1, 1.8 and 2.3 PO4-P mg/L respectively. Basing on these results, it was estimated that farmers use 115 and 24 Kg/acre of Nitrogen and Phosphorus respectively per month. Taking results for Nitrogen, the same amount of Nutrients in artificial fertilizers would cost $ 88. This shows that reuse of wastewater has a potential in terms of nutrients. The results for E. coli for sites 1, 2, 3 and 4 were 1.1 X 107, 9.1 X 105, 7.4 X 105, and 3.4 X 105 respectively. E. coli hence decreased downstream with statistically significant variations between sites 1 and 4. Site 1 had the highest mean E.coli counts. The bacterial contamination was significantly higher during the dry season when more water was needed for irrigation. Although the water had the potential for reuse in farming, bacterial contamination during both seasons was higher than 103 FC/100ml recommended by WHO for unrestricted Agriculture.

Keywords: E. coli, nitrogen, phosphorus, water reuse, waste water

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517 Molecular Detection of Naegleria fowleri and Fecal Indicator Bacteria in Brackish Water of Lake Pontchartrain, Louisiana

Authors: Jia Xue, Frederica G. Lamar, Siyu Lin, Jennifer G. Lamori, Samendra Sherchan

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Brackish water samples from Lake Pontchartrain in Louisiana were assessed for the presence of pathogenic amoeba Naegleria fowleri, which causes primary amoebic meningoencephalitis (PAM). In our study, quantitative polymerase chain reaction (qPCR) methods were used to determine N. fowleri, E. coli, and Enterococcus in water collected from Lake Pontchartrain. A total of 158 water samples were analyzed over the 10-month sampling period. Statistically significant positive correlation between water temperature and N. fowleri concentration was observed. N. fowleri target sequence was detected at 35.4% (56/158) of the water samples from ten sites around the Lake ranged from 11.6 GC/100 ml water to 457.8 GC/100 ml water. A single factor (ANOVA) analysis shows the average concentration of N. fowleri in summer (119.8 GC/100 ml) was significantly higher than in winter (58.6 GC/100 ml) (p < 0.01). Statistically significant positive correlations were found between N. fowleri and qPCR E. coli results and N. fowleri and colilert E. coli (culture method), respectively. A weak positive correlation between E. coli and Enterococcus was observed from both qPCR (r = 0.27, p < 0.05) and culture based method (r = 0.52, p < 0.05). Meanwhile, significant positive correlation between qPCR and culture based methods for E. coli (r = 0.30, p < 0.05) and Enterococcus concentration was observed (r = 0.26, p < 0.05), respectively. Future research is needed to determine whether sediment is a source of N. fowleri found in the water column.

Keywords: brackish water, Escherichia coli, Enterococcus, Naegleria fowleri, primary amoebic meningoencephalitis (PAM), qPCR

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516 Production of Recombinant Human Serum Albumin in Escherichia coli: A Crucial Biomolecule for Biotechnological and Healthcare Applications

Authors: Ashima Sharma, Tapan K. Chaudhuri

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Human Serum Albumin (HSA) is one of the most demanded therapeutic protein with immense biotechnological applications. The current source of HSA is human blood plasma. Blood is a limited and an unsafe source as it possesses the risk of contamination by various blood derived pathogens. This issue led to exploitation of various hosts with the aim to obtain an alternative source for the production of the rHSA. But, till now no host has been proven to be effective commercially for rHSA production because of their respective limitations. Thus, there exists an indispensable need to promote non-animal derived rHSA production. Of all the host systems, Escherichia coli is one of the most convenient hosts which has contributed in the production of more than 30% of the FDA approved recombinant pharmaceuticals. E. coli grows rapidly and its culture reaches high cell density using inexpensive and simple substrates. The fermentation batch turnaround number for E. coli culture is 300 per year, which is far greater than any of the host systems available. Therefore, E. coli derived recombinant products have more economical potential as fermentation processes are cheaper compared to the other expression hosts available. Despite of all the mentioned advantages, E. coli had not been successfully adopted as a host for rHSA production. The major bottleneck in exploiting E. coli as a host for rHSA production was aggregation i.e. majority of the expressed recombinant protein was forming inclusion bodies (more than 90% of the total expressed rHSA) in the E. coli cytosol. Recovery of functional rHSA form inclusion body is not preferred because it is tedious, time consuming, laborious and expensive. Because of this limitation, E. coli host system was neglected for rHSA production for last few decades. Considering the advantages of E. coli as a host, the present work has targeted E. coli as an alternate host for rHSA production through resolving the major issue of inclusion body formation associated with it. In the present study, we have developed a novel and innovative method for enhanced soluble and functional production of rHSA in E.coli (~60% of the total expressed rHSA in the soluble fraction) through modulation of the cellular growth, folding and environmental parameters, thereby leading to significantly improved and enhanced -expression levels as well as the functional and soluble proportion of the total expressed rHSA in the cytosolic fraction of the host. Therefore, in the present case we have filled in the gap in the literature, by exploiting the most well studied host system Escherichia coli which is of low cost, fast growing, scalable and ‘yet neglected’, for the enhancement of functional production of HSA- one of the most crucial biomolecule for clinical and biotechnological applications.

Keywords: enhanced functional production of rHSA in E. coli, recombinant human serum albumin, recombinant protein expression, recombinant protein processing

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515 Effect of Oxidative Stress on Glutathione Reductase Activity of Escherichia coli Clinical Isolates from Patients with Urinary Tract Infection

Authors: Fariha Akhter Chowdhury, Sabrina Mahboob, Anamika Saha, Afrin Jahan, Mohammad Nurul Islam

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Urinary tract infection (UTI) is frequently experienced by the female population where the prevalence increases with aging. Escherichia coli, one of the most common UTI causing organisms, retains glutathione defense mechanism that aids the organism to withstand the harsh physiological environment of urinary tract, host oxidative immune response and even to affect antibiotic-mediated cell death and the emergence of resistance. In this study, we aimed to investigate the glutathione reductase activity of uropathogenic E. coli (UPEC) by observing the reduced glutathione (GSH) level alteration under stressful condition. Urine samples of 58 patients with UTI were collected. Upon isolation and identification, 88% of the samples presented E. coli as UTI causing organism among which randomly selected isolates (n=9), obtained from urine samples of female patients, were considered for this study. E. coli isolates were grown under normal and stressful conditions where H₂O₂ was used as the stress-inducing agent. GSH level estimation of the isolates in both conditions was carried out based on the colorimetric measurement of 5,5'-dithio-bis (2-nitrobenzoic acid) (DTNB) and GSH reaction product using microplate reader assay. The GSH level of isolated E. coli sampled from adult patients decreased under stress compared to normal condition (p = 0.011). On the other hand, GSH production increased markedly in samples that were collected from elderly subjects (p = 0.024). A significant partial correlation between age and change of GSH level was found as well (p = 0.007). This study may help to reveal ways for better understanding of E. coli pathogenesis of UTI prevalence in elderly patients.

Keywords: Escherichia coli, glutathione reductase activity, oxidative stress, reduced glutathione (GSH), urinary tract infection (UTI)

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514 Effects of β-Glucan on the Release of Nitric Oxide by RAW264.7 Cells Stimulated with Escherichia coli Lipopolysaccharide

Authors: Eun Young Choi, So Hui Choe, Jin Yi Hyeon, Ji Young Jin, Bo Ram Keum, Jong Min Lim, Hyung Rae Cho, Kwang Keun Cho, In Soon Choi

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This research analyzed the effect of β-glucan that is expected to alleviate the production of inflammatory mediator in macrophagocyte, which was processed by the lipopolysaccharide (LPS) of Escherichia, a pathogen related to allergy. The incubated layer was used for nitric oxide (NO) analysis. The DNA-binding activation of the small unit of NF-κB was measured using ELISA-based kit. In RAW264.7 cells that were vitalized by E.coli LPS, β-glucan inhibited both the combatant and rendering phases of inducible NO synthase (iNOS)-derived NO. β-glucan increased the expression of heme oxygenase-1 (HO-1) in the cell that was stimulated by E.coli LPS, and HO-1 activation was inhibited by SnPP. This shows that NO production induced by LPS is related to the inhibition effect of β-glucan. The phosphorylation of JNK and p38 induced by LPS were not influenced by β-glucan, and IκB-α decomposition was not influenced either. Instead, β-glucan remarkably inhibited the phosphorylation of STAT1 that was induced by E.coli LPS. Overall, β-glucan inhibited the production of NO in macrophagocyte that was vitalized by E.coli LPS through HO-1 induction and STAT1 pathways inhibition in this research. As the host inflammation reaction control by β-glucan weakens the progress of allergy, β-glucan can be used as an effective treatment method.

Keywords: β-glucan, lipopolysaccharide (LPS), nitric oxide (NO), RAW264.7 cells, STAT1

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513 Inhibitory Mechanism of Ag and Fe Colloidal Nanoparticles on P. aeruginosa and E.coli Growth

Authors: Fatemeh Moradian, Razieh Ghorbani, Poria Biparva

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Growing resistance of microorganisms to potent antibiotics has renewed a great interest towards investigating bactericidal properties of nanoparticles and their Nano composites as an alternative. The use of metal nanoparticles to combat bacterial infections is one of the most wide spread applications of nanotechnology in the field of antibacterial. Nanomaterials have unique properties compared to their bulk counterparts. In this report, we demonstrate the antimicrobial activity of zerovalent Iron(ZVI) and Ag(silver) nanoparticles against Gram-negative bacteria E.coli(DH5α) and Pseudomonas aeruginosa. At first ZVI and Ag nanoparticles were synthesized by chemical reduction method and using scanning electron microscopy (SEM) the nanoparticle size determined. Different concentrations of Ag and ZVI nanoparticles were added to bacteria on nutrient agar medium. Minimum inhibitory concentration (MIC) of Ag and Fe nanoparticles for P. aeruginosa were 5µM and 1µg as well as for E.coli were 6µM. and 10 µg, respectively. Among the two nanoparticles, ZVI showed that the greatest antimicrobial activity against E.coli and Ag nanoparticle on P.aeruginosa. Results suggested that the bactericidal effect of metal nanoparticles has been attributed to their small size as well as high surface to volume ratio and NPs could be used as an effective antibacterial material.

Keywords: bactericidal properties, MIC, nanoparticle, SEM

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512 Effect of Hydrogen Peroxide Concentration Produced by Cold Atmospheric Plasma on Inactivation of Escherichia Coli in Water

Authors: Zohreh Rashmei

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Introduction: Plasma inactivation is one of the emerging technologies in biomedical field and has been applied to the inactivation of microorganisms in water. The inactivation effect has been attributed to the presence of active plasma species, i.e. OH, O, O3, H2O2, UV and electric fields, generated by the discharge of plasma. Material and Method: To evaluate germicidal effects of plasma, the electric spark discharge device was used. After the effect of the plasma samples were collected for culture medium agar plate count. In addition to biological experiments, the concentration of hydrogen peroxide was also measured. Results: The results showed that Plasma is able to inactivate a high concentration of E. coli. After a short period of plasma radiation on the surface of water, the amount log8 reduced the microbial load. Starting plasma radiation on the surface of the water, the measurements show of production and increasing the amount of hydrogen peroxide in water. So that at the end of the experiment, the concentration of hydrogen peroxide to about 100 mg / l increased. Conclusion: Increasing the concentration of hydrogen peroxide is directly related to the reduction of microbial load. The results of E. coli culture in media containing certain concentrations of H2O2 showed that E. coli can not to grow in a medium containing more than 2/5 mg/l of H2O2. Surely we can say that the main cause of killing bacteria is a molecule of H2O2.

Keywords: plasma, hydrogen peroxide, disinfection, E. coli

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511 An Investigation of E. coli Contamination in Fars Province, Iran and Methods of Reducing the Contamination

Authors: Ali Mohagheghzadeh, Samad Vaez Badiegard, Bita Shomali

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Nowadays, with the increase in population, the need for protein sources is increasing. Different bacteria can cause food poisoning while most of the symptoms of food poisoning are similar to those of gastrointestinal infections. As a result, the diagnosis of bacteria and viruses causing food poisoning would not be possible without a stool culture. Cases of food poisoning are often accompanied by gastrointestinal disorders such as diarrhea, vomit, and gastrointestinal stomach cramps. Thus, providing enough food, taking into account health issues has always been a concern of authorities. Since E. coli bacterium is one of the important indicators of food hygiene and quality, producing food without being contaminated by this bacterium is desired in the food industry. This study aimed at assessing the E. coli contamination of poultry meat produced in slaughterhouses. Samples were taken from critical areas of slaughterhouses, namely the feather picking area, viscera and carcass evacuation area the area after cooling chillers. The results showed that 60% of contamination occurs in feather picking area. Among antiseptic and detergent materials, the highest reduction belongs to Epimax.

Keywords: slaughterhouse, E. coli, Epimax, contamination

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510 Intracellular Strategies for Gene Delivery into Mammalian Cells Using Bacteria as a Vector

Authors: Kumaran Narayanan, Andrew N. Osahor

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E. coli has been engineered by our group and by others as a vector to deliver DNA into cultured human and animal cells. However, so far conditions to improve gene delivery using this vector have not been investigated, resulting in a major gap in our understanding of the requirements for this vector to function optimally. Our group recently published novel data showing that simple addition of the DNA transfection reagent Lipofectamine increased the efficiency of the E. coli vector by almost 3-fold, providing the first strong evidence that further optimization of bactofection is possible. This presentation will discuss advances that demonstrate the effects of several intracellular strategies that improve the efficiency of this vector. Conditions that promote endosomal escape of internalized bacteria to evade lysosomal destruction after entry in the cell, a known obstacle limiting this vector, are elucidated. Further, treatments that increase bacterial lysis so that the vector can release its transgene into the mammalian environment for expression will be discussed. These experiments will provide valuable new insight to advance this E. coli system as an important class of vector technology for genetic correction of human disease models in cells and whole animals.

Keywords: DNA, E. coli, gene expression, vector

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509 Functional Cell Surface Display Using Ice Nucleation Protein from Erwina ananas on Escherischia coli

Authors: Mei Yuin Joanne Wee, Rosli Md. Illias

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Cell surface display is the expression of a protein with an anchoring motif on the surface of the cell. This approach offers advantages when used in bioconversion in terms of easier purification steps and more efficient enzymatic reaction. A surface display system using ice nucleation protein (InaA) from Erwina ananas as an anchoring motif has been constructed to display xylanase (xyl) on the surface of Escherischia coli. The InaA was truncated so that it is made up of the N- and C-terminal domain (INPANC-xyl) and it has successfully directed xylanase to the surface of the cell. A study was also done on xylanase fused to two other ice nucleation proteins, InaK (INPKNC-xyl) and InaZ (INPZNC-xyl) from Pseudomonas syringae KCTC 1832 and Pseudomonas syringae S203 respectively. Surface localization of the fusion protein was verified using SDS-PAGE and Western blot on the cell fractions and all anchoring motifs were successfully displayed on the outer membrane of E. coli. Upon comparison, whole-cell activity of INPANC-xyl was more than six and five times higher than INPKNC-xyl and INPZNC-xyl respectively. Furthermore, the expression of INPANC-xyl on the surface of E. coli did not inhibit the growth of the cell. This is the first report of surface display system using ice nucleation protein, InaA from E. ananas. From this study, this anchoring motif offers an attractive alternative to the current surface display systems.

Keywords: cell surface display, Escherischia coli, ice nucleation protein, xylanase

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508 Identification of Associated-Virulence Genes in Quinolone-Resistant Escherichia coli Strains Recovered from an Urban Wastewater Treatment Plant

Authors: Alouache Souhila, Messai Yamina, Torres Carmen, Bakour Rabah

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Objective: It has often been reported an association between antibiotic resistance and virulence. However, resistance to quinolones seems to be an exception, it tends instead to be associated with an attenuation of virulence, particularly in clinical strains. The purpose of this study was to evaluate the potential virulence of 28 quinolone-resistant E. coli strains recovered from water at the inflow (n=16) and outflow (n=12) of an urban wastewater treatment plant (WWTP). Methods: E. coli isolates were selected on Tergitol-7 agar supplemented with 2µg/ml of ciprofloxacin, they were screened by PCR for 11 virulence genes related to Extraintestinal pathogenic E. coli (ExPEC): papC, papG, afa/draBC, sfa/foc, kpsMTII, iutA, iroN, hlyF, ompT, iss and traT. The phylogenetic groups were determined by PCR and clonal relationship was evaluated by ERIC-PCR. Results: Genotyping by ERIC-PCR showed 7 and 12 DNA profiles among strains of wastewater (inflow) and treated water (outflow), respectively. Strains were assigned to the following phylogenetic groups: B2 (n = 1, 3.5%), D (n = 3, 10.7%), B1 (n = 10, 35.7%.) and A (n = 14, 50%). A total of 8 virulence-associated genes were detected, traT (n=19, 67.8%), iroN (n= 16, 57 .1%), hlyF (n=15, 53 .5%), ompT (n=15, 53 .5%), iss (n=14, 50%), iutA (n=9, 32.1%) , sfa/foc (n=7, 25%) and kpsMTII (n=2, 7.1%). Combination of virulence factors allowed to define 16 virulence profiles. The pathotype APEC was observed in 17.8% (D=1, B1=4) and human ExPEC in 7% (B2=1, D=1) of strains. Conclusion: The study showed that quinolone-resistant E. coli strains isolated from wastewater and treated water in WWTP harbored virulence genes with the presence of APEC and human ExPEC strains.

Keywords: E. coli, quinolone-resistance, virulence, WWTP

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507 Wide Dissemination of CTX-M-Type Extended-Spectrum β-Lactamases in Korean Swine Farms

Authors: Young Ah Kim, Hyunsoo Kim, Eun-Jeong Yoon, Young Hee Seo, Kyungwon Lee

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Extended-spectrum β-lactamase (ESBL)-producing Escherichia coli from food animals are considered as a reservoir for transmission of ESBL genes to human. The aim of this study is to assess the prevalence and molecular epidemiology of ESBL-producing E. coli colonization in pigs, farm workers, and farm environments to elucidate the transmission of multidrug-resistant clones from animal to human. Nineteen pig farms were enrolled across the country in Korea from August to December 2017. ESBL-producing E. coli isolates were detected in 190 pigs, 38 farm workers, and 112 sites of farm environments using ChromID ESBL (bioMerieux, Marcy l'Etoile, France), directly (stool or perirectal swab) or after enrichment (sewage). Antimicrobial susceptibility tests were done with disk diffusion methods and blaTEM, blaSHV, and blaCTX-M were detected with PCR and sequencing. The genomes of the four CTX-M-55-producing E. coli isolates from various sources in one farm were entirely sequenced to assess the relatedness of the strains. Whole genome sequencing (WGS) was performed with PacBio RS II system (Pacific Biosciences, Menlo Park, CA, USA). ESBL genotypes were 85 CTX-M-1 group (one CTX-M-3, 23 CTX-M-15, one CTX-M-28, 59 CTX-M-55, one CTX-M-69) and 60 CTX-M-9 group (41 CTX-M-14, one CTX-M-17, one CTX-M-27, 13 CTX-M-65, 4 CTX-M-102) in total 145 isolates. The rectal colonization rates were 53.2% (101/190) in pigs and 39.5% (15/38) in farm workers. In WGS, sequence types (STs) were determined as ST69 (E. coli PJFH115 isolate from a human carrier), ST457 (two E. coli isolates PJFE101 recovered from a fence and PJFA1104 from a pig) and ST5899 (E. coli PJFA173 isolate from the other pig). The four plasmids encoding CTX-M-55 (88,456 to 149, 674 base pair), whether it belonged to IncFIB or IncFIC-IncFIB type, shared IncF backbone furnishing the conjugal elements, suggesting of genes originated from same ancestor. In conclusion, the prevalence of ESBL-producing E. coli in swine farms was surprisingly high, and many of them shared common ESBL genotypes of clinical isolates such as CTX-M-14, 15, and 55 in Korea. It could spread by horizontal transfer between isolates from different reservoirs (human-animal-environment).

Keywords: Escherichia coli, extended-spectrum β-lactamase, prevalence, whole genome sequencing

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506 High Prevalence of Multi-drug Resistant Diarrheagenic Escherichia coli among Hospitalised Diarrheal Patients in Kolkata, India

Authors: Debjani Ghosh, Goutam Chowdhury, Prosenjit Samanta, Asish Kumar Mukhopadhyay

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Acute diarrhoea caused by diarrheagenic Escherichia coli (DEC) is one of the major public health problem in developing countries, mainly in Asia and Africa. DEC consists of six pathogroups, but the majority of the cases were associated with the three pathogropus, enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), and enteropathogenic E. coli (EPEC). Hence, we studied the prevalence and antimicrobial resistance of these three major DEC pathogroups in hospitalized diarrheal patients in Kolkata, India, during 2012-2019 with a large sample size. 8,891 stool samples were processed, and 7.8% of them was identified as DEC infection screened by multiplex PCR, in which ETEC was most common (47.7%) followed by EAEC (38.4%) and EPEC (13.9%). Clinical patient history suggested that children <5 years of age were mostly affected with ETEC and EAEC, whereas people within >5-14 years of age were significantly associated with EPEC and ETEC infections. Antibiogram profile showed a high prevalence of multidrug resistant (MDR) isolates among DEC (56.9%), in which 9% were resistant to antibiotics of six different antimicrobial classes. Screening of the antibiotic resistance conferring genes in DEC showed the presence of blaCTX-M (30.2%) in highest number followed by blaTEM (27.5%), tetB (18%), sul2 (12.6%), strA (11.8%), aadA1 (9.8%), blaOXA-1 (9%), dfrA1 (1.6%) and blaSHV (1.2%) which indicates the existence of mobile genetic elements in those isolates. Therefore, the presence of MDR DEC strains in higher number alarms the public health authorities to take preventive measures before the upsurge of the DEC caused diarrhea cases in near future.

Keywords: diarrheagenic escherichia coli, ETEC, EAEC, EPEC

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505 Understanding the Diversity of Antimicrobial Resistance among Wild Animals, Livestock and Associated Environment in a Rural Ecosystem in Sri Lanka

Authors: B. M. Y. I. Basnayake, G. G. T. Nisansala, P. I. J. B. Wijewickrama, U. S. Weerathunga, K. W. M. Y. D. Gunasekara, N. K. Jayasekera, A. W. Kalupahana, R. S. Kalupahana, A. Silva- Fletcher, K. S. A. Kottawatta

Abstract:

Antimicrobial resistance (AMR) has attracted significant attention worldwide as an emerging threat to public health. Understanding the role of livestock and wildlife with the shared environment in the maintenance and transmission of AMR is of utmost importance due to its interactions with humans for combating the issue in one health approach. This study aims to investigate the extent of AMR distribution among wild animals, livestock, and environment cohabiting in a rural ecosystem in Sri Lanka: Hambegamuwa. One square km area at Hambegamuwa was mapped using GPS as the sampling area. The study was conducted for a period of five months from November 2020. Voided fecal samples were collected from 130 wild animals, 123 livestock: buffalo, cattle, chicken, and turkey, with 36 soil and 30 water samples associated with livestock and wildlife. From the samples, Escherichia coli (E. coli) was isolated, and their AMR profiles were investigated for 12 antimicrobials using the disk diffusion method following the CLSI standard. Seventy percent (91/130) of wild animals, 93% (115/123) of livestock, 89% (32/36) of soil, and 63% (19/30) of water samples were positive for E. coli. Maximum of two E. coli from each sample to a total of 467 were tested for the sensitivity of which 157, 208, 62, and 40 were from wild animals, livestock, soil, and water, respectively. The highest resistance in E. coli from livestock (13.9%) and wild animals (13.3%) was for ampicillin, followed by streptomycin. Apart from that, E. coli from livestock and wild animals revealed resistance mainly against tetracycline, cefotaxime, trimethoprim/ sulfamethoxazole, and nalidixic acid at levels less than 10%. Ten cefotaxime resistant E. coli were reported from wild animals, including four elephants, two land monitors, a pigeon, a spotted dove, and a monkey which was a significant finding. E. coli from soil samples reflected resistance primarily against ampicillin, streptomycin, and tetracycline at levels less than in livestock/wildlife. Two water samples had cefotaxime resistant E. coli as the only resistant isolates out of 30 water samples tested. Of the total E. coli isolates, 6.4% (30/467) was multi-drug resistant (MDR) which included 18, 9, and 3 isolates from livestock, wild animals, and soil, respectively. Among 18 livestock MDRs, the highest (13/ 18) was from poultry. Nine wild animal MDRs were from spotted dove, pigeon, land monitor, and elephant. Based on CLSI standard criteria, 60 E. coli isolates, of which 40, 16, and 4 from livestock, wild animal, and environment, respectively, were screened for Extended Spectrum β-Lactamase (ESBL) producers. Despite being a rural ecosystem, AMR and MDR are prevalent even at low levels. E. coli from livestock, wild animals, and the environment reflected a similar spectrum of AMR where ampicillin, streptomycin, tetracycline, and cefotaxime being the predominant antimicrobials of resistance. Wild animals may have acquired AMR via direct contact with livestock or via the environment, as antimicrobials are rarely used in wild animals. A source attribution study including the effects of the natural environment to study AMR can be proposed as this less contaminated rural ecosystem alarms the presence of AMR.

Keywords: AMR, Escherichia coli, livestock, wildlife

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