Search results for: enteropathogenic Eschericia coli O157

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

Abstract:

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

Procedia PDF Downloads 253

Authors: Wan-Ling Jiang, Hsin Chi, Jia-Lu Cheng, Ming-Fang Cheng

Abstract:

Background: Investigating the risk factors for the fecal carriage of third-generation cephalosporin-resistant E.coli could contribute to further disease prevention. Previous research on third-generation cephalosporin-resistant prevalence in children in different regions of Taiwan is limited. This project aims to explore the risk factors and regional differences in the prevalence of third-generation cephalosporin-resistant and other antibiotic-resistant E. coli in the northern, southern, and eastern regions of Taiwan. Methods: We collected data from children aged 0 to 18 from community or outpatient clinics from July 2022 to May 2023 in southern, northern, and eastern Taiwan. The questionnaire was designed to survey the characteristics of participants and possible risk factors, such as clinical information, household environment, drinking water, and food habits. After collecting fecal samples and isolating stool culture with E.coli, antibiotic sensitivity tests and MLST typing were performed. Questionnaires were used to analyze the risk factors of third-generation cephalosporin-resistant E. coli in the three different regions of Taiwan. Results: In the total 246 stool samples, third-generation cephalosporin-resistant E.coli accounted for 37.4% (97/246) of all isolates. Among the three different regions of Taiwan, the highest prevalence of fecal carriage with third-generation cephalosporin-resistant E.coli was observed in southern Taiwan (42.7%), followed by northern Taiwan (35.5%) and eastern Taiwan (28.4%). Multi-drug resistant E. coli had prevalence rates of 51.9%, 66.3%, and 37.1% in the northern, southern, and eastern regions, respectively. MLST typing revealed that ST131 was the most prevalent type (11.8%). The prevalence of ST131 in northern, southern, and eastern Taiwan was 10.1%, 12.3%, and 13.2%, respectively. Risk factors analysis identified lower paternal education, overweight status, and non-vegetarian diet as statistical significance risk factors for third-generation cephalosporin-resistant E.coli. Conclusion: The fecal carriage rates of antibiotic-resistant E. coli among Taiwanese children were on the rise. This study found regional disparities in the prevalence of third-generation cephalosporin-resistant and multi-drug-resistant E. coli, with southern Taiwan having the highest prevalence. Lower paternal education, overweight, and non-vegetarian diet were the potential risk factors of third-generation cephalosporin-resistant E. coli in this study.

Keywords: Escherichia coli, fecal carriage, antimicrobial resistance, risk factors, prevalence

Procedia PDF Downloads 21

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

Abstract:

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)

Procedia PDF Downloads 355

Authors: Fuji Astuti, Helen Onyeaka

Abstract:

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

Procedia PDF Downloads 331

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

Abstract:

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

Procedia PDF Downloads 140

Authors: N. Zeinalzadeh, Mahdi Sadeghi

Abstract:

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

Procedia PDF Downloads 419

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

Abstract:

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

Procedia PDF Downloads 292

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

Abstract:

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

Procedia PDF Downloads 197

Authors: Haftay Abraha Tadesse, Atsebaha Gebrekidan Kahsay, Mahumd Abdulkader

Abstract:

Background: Salmonella species and Escherichia 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. Methodology: A cross-sectional study was conducted from January to September 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 an overall bacterial contamination.About 95.5% of the tested isolates were sensitive to chloramphenicol and norfloxacin 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 hand washing practice and not using glove during meat handling showed 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, salmonella species

Procedia PDF Downloads 12

Authors: Ganesh K. Maurya, Reema Chaudhary, Neha Pandey, Hari S. Misra

Abstract:

PprA, a pleiotropic protein participating in radioresistance, has been reported for its roles in DNA replication initiation, genome segregation, cell division and DNA repair in polyextremophile Deinococcus radiodurans. Interestingly, expression of deinococcal PprA in E. coli suppresses its growth by reducing the number of colony forming units and provides better resistance against γ-radiation than control. We employed different biochemical and cell biology studies using PprA and its DNA binding/polymerization mutants (K133E & W183R) in E. coli. Cells expressing wild type PprA or its K133E mutant showed reduction in the amount of genomic DNA as well as chromosome copy number in comparison to W183R mutant of PprA and control cells, which suggests the role of PprA protein in regulation of DNA replication initiation in E. coli. Further, E. coli cells expressing PprA or its mutants exhibited different impact on cell morphology than control. Expression of PprA or K133E mutant displayed a significant increase in cell length upto 5 folds while W183R mutant showed cell length similar to uninduced control cells. We checked the interaction of deinococcal PprA and its mutants with E. coli DnaA using Bacterial two-hybrid system and co-immunoprecipitation. We observed a functional interaction of EcDnaA with PprA and K133E mutant but not with W183R mutant of PprA. Further, PprA or K133E mutant has suppressed the ATPase activity of EcDnaA but W183R mutant of PprA failed to do so. These observations suggested that PprA protein regulates DNA replication initiation and cell morphology of surrogate E. coli.

Keywords: DNA replication, radioresistance, protein-protein interaction, cell morphology, ATPase activity

Procedia PDF Downloads 34

Authors: Ganesh K. Maurya, Reema Chaudhary, Neha Pandey, Hari S. Misra

Abstract:

PprA, a pleiotropic protein participating in radioresistance, has been reported for its roles in DNA replication initiation, genome segregation, cell division and DNA repair in polyextremophile Deinococcus radiodurans. Interestingly, expression of deinococcal PprA in E. coli suppresses its growth by reducing the number of colony forming units and provide better resistance against γ-radiation than control. We employed different biochemical and cell biology studies using PprA and its DNA binding/polymerization mutants (K133E & W183R) in E. coli. Cells expressing wild type PprA or its K133E mutant showed reduction in the amount of genomic DNA as well as chromosome copy number in comparison to W183R mutant of PprA and control cells, which suggests the role of PprA protein in regulation of DNA replication initiation in E. coli. Further, E. coli cells expressing PprA or its mutants exhibited different impact on cell morphology than control. Expression of PprA or K133E mutant displayed a significant increase in cell length upto 5 folds while W183R mutant showed cell length similar to uninduced control cells. We checked the interaction of deinococcal PprA and its mutants with E. coli DnaA using Bacterial two-hybrid system and co-immunoprecipitation. We observed a functional interaction of EcDnaA with PprA and K133E mutant but not with W183R mutant of PprA. Further, PprA or K133E mutant has suppressed the ATPase activity of EcDnaA but W183R mutant of PprA failed to do so. These observations suggested that PprA protein regulates DNA replication initiation and cell morphology of surrogate E. coli.

Keywords: DNA replication, radioresistance, protein-protein interaction, cell morphology, ATPase activity

Procedia PDF Downloads 34

Authors: Abdul Walusansa

Abstract:

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

Procedia PDF Downloads 216

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

Abstract:

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

Procedia PDF Downloads 139

Authors: Ashima Sharma, Tapan K. Chaudhuri

Abstract:

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

Procedia PDF Downloads 308

Authors: Abdullahi Musa, Yakubu Kukure Enebe Ibrahim, Adeshina Gujumbola

Abstract:

The emergence of multidrug resistance in clinical Escherichia coli has been associated with plasmid-mediated genes. DNA transfer among bacteria is critical to the dissemination of resistance. Plasmids have proved to be the ideal vehicles for dissemination of resistance genes. Plasmids coding for antibiotic resistance were long being recognized by many researchers globally. The study aimed at determining the antibiotic susceptibility pattern of ESBL E. coli isolates claimed to be multidrug resistance using disc diffusion method. Antibacterial activity of the test isolates was carried out using disk diffusion methods. The results showed that, majority of the multidrug resistance among clinical isolates of ESBL E. coli was as a result of acquisition of plasmid carrying antibiotic-resistance genes. Production of these ESBL enzymes by these organisms which are normally carried by plasmid and transfer from one bacterium to another has greatly contributed to the rapid spread of antibiotic resistance amongst E. coli isolates, which lead to high economic burden, increase morbidity and mortality rate, complication in therapy and limit treatment options. To curtail these problems, it is of significance to checkmate the rate at which over the counter drugs are sold and antibiotic misused in animal feeds. This will play a very important role in minimizing the spread of resistance bacterial strains in our environment.

Keywords: Escherichia coli, plasmid, multidrug resistance, ESBL, pan drug resistance

Procedia PDF Downloads 24

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

Abstract:

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)

Procedia PDF Downloads 297

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

Abstract:

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

Procedia PDF Downloads 379

Authors: Fatemeh Moradian, Razieh Ghorbani, Poria Biparva

Abstract:

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

Procedia PDF Downloads 565

Authors: Zohreh Rashmei

Abstract:

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

Procedia PDF Downloads 111

Authors: Ali Mohagheghzadeh, Samad Vaez Badiegard, Bita Shomali

Abstract:

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

Procedia PDF Downloads 676

Authors: Kumaran Narayanan, Andrew N. Osahor

Abstract:

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

Procedia PDF Downloads 329

Authors: Mei Yuin Joanne Wee, Rosli Md. Illias

Abstract:

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

Procedia PDF Downloads 364

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

Abstract:

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

Procedia PDF Downloads 435

Authors: Eleni Poulou-Sidiropoulou, Charalampos N. Bompas, Martina Samiotaki, Alexios Vlamis-Gardikas

Abstract:

The glutaredoxin (Grx) and thioredoxin (Trx) systems keep the intracellular environment reduced in almost all organisms. In Escherichia coli (E. coli), the Grx system relies on NADPH+ to reduce GSH reductase (GR), the latter reducing oxidized diglutathione to glutathione (GSH) which in turn reduces cytosolic Grxs, the electron donors for different intracellular substrates. In the Trx system, GR and GSH are replaced by Trx reductase (TrxR). Three of the Grxs of E. coli (Grx1, 2, 3) are reduced by GSH, while Grx4 is likely reduced by TrxR. Trx1 and Grx1 from E. coli may reduce ribonucleotide reductase Ia to ensure a constant supply of deoxyribonucleotides for the synthesis of DNA. The role of the other three Grxs is relatively unknown, especially for Grx2 that may amount up to 1 % of total cellular protein in the stationary phase of growth. The protein is known as a potent antioxidant, but no specific functions have been attributed to it. Herein, affinity chromatography of cellular extracts on immobilized Grx2, followed by MS analysis of the resulting eluates, was employed to identify protein ligands that could provide insights into the biological role of Grx2. Ionic, strong non-covalent, and covalent (disulfide) interactions with relevant proteins were detected. As a means of verification, the identified ligands were subjected to in silico docking with monothiol Grx2. In other experiments, protein extracts from E. coli cells lacking the gene for Grx2 (grxB) were compared to those of wild type. Taken together, the two approaches suggest that Grx2 is involved in protein synthesis, nucleotide metabolism, DNA damage repair, stress responses, and various metabolic processes. Grx2 appears as a versatile protein that may participate in a wide range of biological pathways beyond its known general antioxidant function.

Keywords: Escherichia coli, glutaredoxin 2, interactome, thiol-disulfide oxidoreductase

Procedia PDF Downloads 15

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

Abstract:

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

Procedia PDF Downloads 175

Authors: Mazoun Nasser Rashid Al Kharusi, Nada Al Siyabi

Abstract:

Objectives: The main aim of this study is to Find the rate of susceptibility of ESBL E. coli causing UTI to oral antibiotics. Secondary objectives: Prevalence of ESBL E. coli from community urine samples, identify the best empirical oral antibiotics with the least resistance rate for UTI and identify alternative oral antibiotics for testing and utilization. Methods: This study is a retrospective descriptive study of the last three years in five major hospitals in Oman (Khowla Hospital, AN’Nahdha Hospital, Rustaq Hospital, Nizwa Hospital, and Ibri Hospital) equipped with a microbiologist. Inclusion criteria include all eligible outpatient urine culture isolates, excluding isolates from admitted patients with hospital-acquired urinary tract infections. Data was collected through the MOH database. The MOH hospitals are using different types of testing, automated methods like Vitek2 and manual methods. Vitek2 machine uses the principle of the fluorogenic method for organism identification and a turbidimetric method for susceptibility testing. The manual method is done by double disc diffusion for identifying ESBL and the disc diffusion method is for antibiotic susceptibility. All laboratories follow the clinical laboratory science institute (CLSI) guidelines. Analysis was done by SPSS statistical package. Results: Total urine cultures were (23048). E. coli grew in (11637) 49.6% of the urine, whereas (2199) 18.8% of those were confirmed as ESBL. As expected, the resistance rate to amoxicillin and cefuroxime is 100%. Moreover, the susceptibility of those ESBL-producing E. coli to nitrofurantoin, trimethoprim+sulfamethoxazole, ciprofloxacin and amoxicillin-clavulanate is progressing over the years; however, still low. ESBL E. coli was predominating in the female gender and those aged 66-74 years old throughout all the years. Other oral antibiotic options need to be explored and tested so that we add to the pool of oral antibiotics for ESBL E. coli causing UTI in the community. Conclusion: High rate of ESBL E. coli in urine from the community. The high resistance rates to oral antibiotics highlight the need for alternative treatment options for UTIs caused by these bacteria. Further research is needed to identify new and effective treatments for UTIs caused by ESBL-E. Coli.

Keywords: UTI, ESBL, oral antibiotics, E. coli, susceptibility

Procedia PDF Downloads 54

Authors: Charalampos N. Bompas, Eleni Poulou-Sidiropoulou, Martina Samiotaki, Alexios Vlamis-Gardikas

Abstract:

Ιn all living organisms, antioxidant defenses are orchestrated by the thioredoxin (Trx) and glutaredoxin (Grx) systems. The Trx system of Escherichia coli (E. coli) is comprised of Trx1 and Trx2, both reduced by thioredoxin reductase (TrxR). The Grx system consists of four Grxs (Grx1, Grx2, Grx3, and Grx4), all reduced by glutathione (GSH) except for Grx4, which is reduced by TrxR. Under normal conditions, the GSH reductase of the Grx system keeps GSH at its reduced state. NADPH+ provides the electrons for all reductions in the Trx and Grx systems. Although the role of the E. coli Trx system is widely known, the function of the Grx system reflects the main property of Grx1, which is the reduction of ribonucleotide reductase Ia (RRIa). E. coli Grx3 (encoded by grxC) may also reduce RRIa in vitro but with slow kinetics. The molecule may account for up to 0.4% of total soluble protein and has been the subject of extensive structural studies. Its biological function, however, remains unknown. Herein, affinity chromatography with monothiol Grx3 serving as bait was used to detect the interactions of Grx3 with other proteins. Different types of interactions were identified (covalent, weak, and strong non-covalent) that suggested novel functions for Grx3. In silico approaches were employed to validate selected interactions. In addition, total protein extracts from the null mutant for grxC and the wild-type strain were compared. The overall findings suggest that Grx3 is involved in various metabolic processes, protein synthesis, and stress responses, expanding the recognized functions of Grx3 beyond the possible reduction of RRIa.

Keywords: escherichia coli, glutaredoxin 3, interactome, thiol-disulfide oxidoreductase

Procedia PDF Downloads 17

Authors: Maria Nejjari, Michel Cloutier, Guylaine Talbot, Martin Lanthier

Abstract:

The fluorescence in situ hybridization (FISH) is a staining technique that allows the identification, detection and quantification of microorganisms without prior cultivation by means of epifluorescence and confocal laser scanning microscopy (CLSM). Oligonucleotide probes have been used to detect bacteria and archaea that colonize the cattle and swine digestive systems. These bacterial strains have been obtained from fecal samples issued from cattle manure and swine slurry. The collection of these samples has been done at 3 different pit’s levels A, B and C with same height. Two collection depth levels have been taken in consideration, one collection level just under the pit’s surface and the second one at the bottom of the pit. Cells were fixed and FISH was performed using oligonucleotides of 15 to 25 nucleotides of length associated with a fluorescent molecule Cy3 or Cy5. The double hybridization using Cy3 probe targeting bacteria (Cy3-EUB338-I) along with a Cy5 probe targeting Archaea (Gy5-ARCH915) gave a better signal. The CLSM images show that there are more bacteria than archaea in swine slurry. However, the choice of fluorescent probes is critical for getting the double hybridization and a unique signature for each microorganism. FISH technique is an easy way to detect pathogens like E. coli O157, Listeria, Salmonella that easily contaminate water streams, agricultural soils and, consequently, food products and endanger human health.

Keywords: archaea, bacteria, detection, FISH, fluorescence

Procedia PDF Downloads 358

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

Procedia PDF Downloads 178

Authors: Malalage Mudara Peiris

Abstract:

Objectives of the study are: the biosynthesis of silver nanoparticles (AgNPs) using Escherichia coli, Acinetobacter baumannii and Staphylococcus aureus, characterization of silver nanoparticles and determination of antimicrobial activity against E. coli, P. aeruginosa, S. aureus, MRSA, and C. Albicans. Methods: E. coli (ATCC 25922), A. baumanii (clinical strain), S. aureus (clinical strain) cultured in nutrient broth medium were used for biosynthesis of AgNPs. Culture conditions (AgNO3 concentration, pH, incubation time and temperature) were optimized. Characterization of synthesized NPs was done by UV-Visible spectroscopy. The antimicrobial activity of the synthesized NPs was studied using the good diffusion assay against E. coli, S. aureus, MRSA (Methicillin-resistant Staphylococcus aureus), P. aeruginosa and C. Albicans. Results: All the selected bacteria produced silver nanoparticles at alkaline pH above 0.3 g/L AgNO3 concentration. The optimum reaction temperature was 60oC. According to the UV-Visible spectroscopy, the maximum absorbance was found to be around 420 - 430 nm indicating the presence of AgNPs. According to the good diffusion results, AgNPs produced by S. aureus resulted in the larger zone of inhibition (ZOI) against the selected pathogens, while AgNPs produced by E. coli showed comparatively smaller ZOI. In general, biosynthesized AgNPs were highly effective against gram-negative bacteria compared to gram-positive bacterial and fungal species. Conclusions: Green AgNPs produced by each bacterium show antimicrobial activity against the selected pathogens. AgNPs produced by S. aureus are the most effective NPs among tested AgNPs, while AgNPs produced by E. coli are the least effective. Further characterization of NPs is required to study the physical properties of silver NPs.

Keywords: green nanotechnology, silver nanoparticles, bacteria, antimicrobial activity

Procedia PDF Downloads 176