Development of a LAMP assay for rapid detection of different intimin variants of attaching and effacing microbial pathogens

2013 ◽  
Vol 62 (11) ◽  
pp. 1665-1672 ◽  
Author(s):  
Zhang Xue-han ◽  
Ye Qing ◽  
Liu Ya-dong ◽  
Li Bin ◽  
Ivanek Renata ◽  
...  
Keyword(s):  
Lamp Assay ◽  
Microbial Pathogens ◽  
Conventional Pcr ◽  
Cell Adherence ◽  
E Coli ◽  
Faecal Samples ◽  
Specialized Equipment ◽  
Field Setting ◽  
Online Software

Intimin harboured by pathogenic Escherichia coli (E. coli) strains is a key virulence factor involved in host cell adherence and colonization. Twenty-seven intimin-encoding E. coli attaching and effacing (eae) gene variants have been reported according to their 3′ binding domain sequences. In our study, we developed a specific and sensitive loop-mediated isothermal amplification (LAMP) assay to detect all known intimin variants. Four primers specific for six regions of eae genes were designed using online software. The eae-LAMP assay was highly specific and detected all 27 tested eae variants; no cross-reactions were observed with genes from enterotoxigenic E. coli (ETEC), E. coli BL21, Salmonella, Shigella, Listeria monocytogenes, or Streptococcus suis type 2 (SS2). With the lowest detection limit of approximately 10 copies per reaction the eae-LAMP assay was 100 times more sensitive than conventional PCR. These results, and the results of tests involving food and faecal samples artificially contaminated with E. coli O157 : H7 (eaeγ+), show that the eae-LAMP assay is a simple, rapid, sensitive and specific tool for detecting intimin variants from pathogenic strains of E. coli. The eae-LAMP assay has great potential for wider applications, not only in the laboratory but also in the field setting, as it does not require specialized equipment.

Development of Isothermal LAMP Assay for Detection of Intimin Gene (eae) in E. coli Associated with Piglet Diarrhea

2020 ◽  
Author(s):  
Sanjeev Kumar ◽  
Jagan Mohanarao Gali ◽  
T.K. Dutta ◽  
P. Roychoudhury ◽  
P.K. Subudhi
Keyword(s):  
Bacterial Species ◽  
False Negative ◽  
Acute Diarrhoea ◽  
Lamp Assay ◽  
Dna Amplification ◽  
Analytical Sensitivity ◽  
Conventional Pcr ◽  
Isolation And Identification ◽  
Reliable Technique ◽  
E Coli

Background: Diarroeagenic Escherichia coli (DEC) including enteropathogenic E. coli (EPEC) and enterohaemorrhagic E. coli (EHEC) is associated with acute diarrhoea in children and young animals. The virulence is associated with attaching and effacing lesions encoded by eaeA gene is considered as marker for EPEC and EHEC. Laboratory diagnosis of such infections is carried out by traditional bacteriological techniques and by conventional PCR assays. Those techniques often provide false negative result and at the same time are costly as well as difficult to perform in the field level. The loop-mediated isothermal amplification (LAMP) is a new generation DNA amplification assay is developed for detection of eaeA gene in E. coli isolated from diarrhoeic piglets.Methods: Samples were collected from diarrhoeic piglets for isolation and identification of E. coli. eaeA gene was detected by conventional PCR using specific primers in all the isolates. LAMP assay was standardized for detection of eaeA gene. Analytical sensitivity of LAMP was evaluated using 10 fold serially diluted E. coli genomic DNA. The specificity of the LAMP assay was determined by evaluating the cross reactivity with 19 other enteric and non-enteric bacterial species. Standardized LAMP was applied for detection of eaeA gene in the field isolates.Result: A total of 37 (24.67%) isolates were recorded as positive for eaeA gene by conventional PCR, while 49 (32.67%) isolates were recorded as positive for eaeA gene by LAMP assay. The LAMP assay was 10 times more sensitive than conventional PCR. LAMP assay was found to be more sensitive, specific, cost effective, user friendly and reliable technique over conventional PCR, which can be applied for screening of the clinical isolates for confirmation of EPEC and/or EHEC.

scholarly journals The importance of P and type 1 fimbriae for the persistence ofEscherichia coliin the human gut

1992 ◽  
Vol 108 (3) ◽  
pp. 415-421 ◽  
Author(s):  
K. Tullus ◽  
I. Kühn ◽  
I. Ørskov ◽  
F. Ørskov ◽  
R. Möllby
Keyword(s):  
Escherichia Coli ◽  
Hela Cell ◽  
Hela Cells ◽  
Human Intestine ◽  
Cell Adherence ◽  
Human Gut ◽  
E Coli ◽  
Type 1 Fimbriae ◽  
Faecal Samples

SUMMARYThe faecalEscherichia coliflora was studied in 89 infants. Each infant was followed with a mean of 12 faecal samples (range 5–21) between 0 and 18 months of age. All isolates were assayed for P fimbriae and biochemically phenotyped and the persistence of each strain (phenotype) in the infant's gut was determined. In a subset of strains the occurrence of type 1 fimbriae and adherence to HeLa cells was studied. Thirty-one per cent of isolates belonging to strains colonizing for longer than 6 months expressed P fimbriae compared to 19% of the isolates from strains colonizing 1–6 months or transient strains colonizing less than 1 month. Type 1 fimbriae and adherence to HeLa cells occurred similarly often in all groups of strains. We conclude that P fimbriae, but not type 1 fimbriae or HeLa cell adherence seemed to contribute to the ability of theE. colistrain to colonize the human intestine.

Loss of an Intimin-Like Protein Encoded on a Uropathogenic E. coli Pathogenicity Island Reduces Inflammation and Affects Interactions with the Urothelium

2021 ◽  
Author(s):  
Allyson E. Shea ◽  
Jolie A. Stocki ◽  
Stephanie D. Himpsl ◽  
Sara N. Smith ◽  
Harry L. T. Mobley
Keyword(s):  
Ex Vivo ◽  
Cell Adherence ◽  
Upec Strain ◽  
E Coli ◽  
Bladder Cell ◽  
Bladder Cells ◽  
Strain Cft073 ◽  
The Individual ◽  
Tract Infections

Uropathogenic Escherichia coli (UPEC) causes the majority of uncomplicated urinary tract infections (UTI), which affect nearly half of women worldwide. Many UPEC strains encode an annotated intimin-like adhesin ( ila ) locus in their genome related to a well-characterized virulence factor in diarrheagenic E. coli pathotypes. Its role in UPEC uropathogenesis, however, remains unknown. In prototype UPEC strain CFT073, there is an ila locus that encodes three predicted intimin-like genes sinH , sinI , and ratA . We used in silico approaches to determine the phylogeny and genomic distribution of this locus among uropathogens. We found that the currently annotated intimin-encoding proteins in CFT073 are more closely related to invasin proteins found in Salmonella . Deletion of the individual sinH , sinI , and ratA genes did not result in measurable effects on growth, biofilm formation, or motility in vitro . On average, sinH was more highly expressed in clinical strains during active human UTI than in human urine ex vivo . Unexpectedly, we found that strains lacking this ila locus had increased adherence to bladder cells in vitro , coupled with a decrease in bladder cell invasion and death. The sinH mutant displayed a significant fitness defect in the murine model of ascending UTI including reduced inflammation in the bladder. These data confirmed an inhibitory role in bladder cell adherence to facilitate invasion and inflammation; therefore, the ila locus should be termed invasin-like, rather than intimin-like. Collectively, our data suggest that loss of this locus mediates measurable interactions with bladder cells in vitro and contributes to fitness during UTI.

Effectiveness of ZnO Nano Particles against the Foodborne Microbial Pathogens E. coli and S. aureus

2020 ◽  
Vol 15 (2) ◽  
pp. 87-94
Keyword(s):  
Antimicrobial Agents ◽  
Inhibitory Concentration ◽  
Antimicrobial Effect ◽  
Nano Particles ◽  
Microbial Pathogens ◽  
Gram Positive ◽  
Gram Negative ◽  
E Coli ◽  
Antimicrobial Susceptibility Tests ◽  
Susceptibility Tests

In this work, various concentrations of ZnO nano particles, prepared by the coprecipitation method with a size range of 47-68 nm, have been investigated as antimicrobial agents. Dilution antimicrobial susceptibility tests were carried out on two kinds of microbes (Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli) according to the standard method recommended by Clinical and Laboratory Standards Institute, CLSI-2015-M07-A10. The results showed that the antimicrobial effect is larger, the higher the concentration of ZnO nano particles in solution. It was also found that Gram-positive microbes are more sensitive to ZnO nano particles when compared with the Gram-negative ones. The minimum inhibitory concentration (MIC) for E. coli was found to be 50 mg/mL while that for S. aureus was 25 mg/mL. The minimum bactericidal concentration (MBC) was 1600 mg/mL for E. coli and 800 mg/mL for S. aureus.

Temperature Dependent On Activated Carbon For Their Biological Activity And Photocatalytical Degradation of Methylene Blue: A Novel Approach To Green Synthesis

2021 ◽  
Author(s):  
Amalanathan.M ◽  
Aravind.M ◽  
Sony Michael Mary.M ◽  
Razan A. Alshgari ◽  
Asma A. Alothman ◽  
...  
Keyword(s):  
Methylene Blue ◽  
Antibacterial Activity ◽  
Activated Carbon ◽  
Hydrothermal Carbonization ◽  
Microbial Pathogens ◽  
X Ray Diffraction ◽  
E Coli ◽  
Novel Approach ◽  
Transmission Electron ◽  
Ft Ir

Abstract In this work, jasmine flower derived activated carbon were successfully synthesized by hydrothermal carbonization process at the different annealing temperature. The Crystallinity, phase, structural, morphological and optical properties of activated carbon were investigated using X-ray diffraction (XRD), Fourier Transform Infrared spectroscopy (FT-IR), Scanning electron microscopy (SEM), Transmission electron microscope (TEM), and UV-visible spectroscopy analysis. The graphitic phase of carbon was obtained from the XRD pattern. Surface morphology reveals irregular-shaped nanoparticles. The photodegradation of methylene blue (MB) was carried out under the visible light irradiation technique to study its photocatalytic activity. The activated carbon obtained at 400oC, 500oC and 600oC shows a photocatalytic degradation efficiency of 86%, 90%, and 94%, respectively. Antibacterial activity of activated carbon was examined against S. Aureus (MTCC-737) and E-Coli (MTCC- 443) microbial pathogens, and their potent antibacterial activity was examined from the zone of inhibition layer.

Characterization of novel ybjG and dacC variants in Escherichia coli

2013 ◽  
Vol 62 (11) ◽  
pp. 1728-1734 ◽  
Author(s):  
Dongguo Wang ◽  
Enping Hu ◽  
Jiayu Chen ◽  
Xiulin Tao ◽  
Katelyn Gutierrez ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Multiple Drug Resistance ◽  
Multiple Drug ◽  
The Novel ◽  
Conventional Pcr ◽  
E Coli ◽  
Novel Variants ◽  
Restriction Sites ◽  
Genetic Structures

A total of 69 strains of Escherichia coli from patients in the Taizhou Municipal Hospital, China, were isolated, and 11 strains were identified that were resistant to bacitracin, chloramphenicol, tetracycline and erythromycin. These strains were PCR positive for at least two out of three genes, ybjG, dacC and mdfA, by gene mapping with conventional PCR detection. Conjugation experiments demonstrated that these genes existed in plasmids that conferred resistance. Novel ybjG and dacC variants were isolated from E. coli strains EC2163 and EC2347, which were obtained from the sputum of intensive care unit patients. Genetic mapping showed that the genes were located on 8200 kb plasmid regions flanked by EcoRI restriction sites. Three distinct genetic structures were identified among the 11 PCR-positive strains of E. coli, and two contained the novel ybjG and dacC variants. The putative amino acid differences in the ybjG and dacC gene variants were characterized. These results provide evidence for novel variants of ybjG and dacC, and suggest that multiple drug resistance in hospital strains of E. coli depends on the synergistic function of ybjG, dacC and mdfA within three distinct genetic structures in conjugative plasmids.

scholarly journals Genetic characterisation and local genotypes of canine parvovirus strains collected from pet dogs in central and eastern China during 2018–2019

2020 ◽  
Vol 64 (4) ◽  
pp. 477-486
Author(s):  
Wen Hu ◽  
Liangyan Zheng ◽  
Xin Xu ◽  
Qiang Liu ◽  
Jun Ji ◽  
...  
Keyword(s):  
Eastern China ◽  
Canine Parvovirus ◽  
Zhejiang Province ◽  
Central China ◽  
High Morbidity ◽  
Faecal Samples ◽  
Amino Acid Mutations ◽  
Canine Parvovirus Type 2 ◽  
Major Amino Acid

AbstractIntroductionCanine parvovirus type-2 (CPV-2) causes acute infectious diseases in puppies, which show high morbidity and mortality. Better effect of vaccination against these diseases could be achieved with deeper knowledge of CPV-2 genotype dissemination and mutation history. This study investigated CPV-2–positive samples collected recently over a wide region of China.Material and MethodsA total of 118 faecal samples from dogs identified as CPV-positive were collected from veterinary clinics in central and eastern China. Overall, 16 strains collected from Anhui, 29 from Henan, and 16 from Zhejiang Province were sequenced to determine the genotypic composition of CPV-2 and mutational complexity of CPV-VP2.ResultsThe CPV-2a, CPV-2b, and CPV-2c genotypes were detected in Anhui and Henan Provinces, while CPV-2c alone was detected in Zhejiang Province. Sequence analysis of all strains showed 98.5%–99.8%, 98.3%–99.9%, and 98.7%–99.8% identity among the 16 Anhui, 29 Henan, and 16 Zhejiang strains, respectively. Strains collected from Anhui and Henan Provinces showed lower identity (97.0%), suggesting greater genetic divergence in central China. The mutation rates of Henan and Anhui strains were lower than that of Zhejiang strains. Major amino acid mutations occurred at sites 5, 370, 426, and 440. Epitope and entropy analyses implied these sites’ likely conformance to the principles of mutation tendency, complexity, and diversity.ConclusionThe findings for the evolutionary structure of CPV-2 strains collected from three provinces in central and eastern China advance trend monitoring of the genetic variation in canine parvovirus and point to its implications in the development of novel vaccines.

scholarly journals A novel report of colistin-resistant Escherichia coli carrying mcr-1 gene from animal and human feacal samples in Nigeria

2018 ◽  
Vol 1 (1) ◽  
pp. 7-10 ◽  
Author(s):  
Olugbenga A. Olowe ◽  
Rita A. Olowe ◽  
Adeolu S. Oluremi ◽  
Olusolabomi J. Adefioye
Keyword(s):  
Escherichia Coli ◽  
Animal Husbandry ◽  
Multiple Drug ◽  
Colistin Resistance ◽  
Pcr Assay ◽  
Southwestern Nigeria ◽  
E Coli ◽  
Multiple Drug Resistant ◽  
Microbiological Methods ◽  
Faecal Samples

Background: The mobilized colistin resistance (m cr)-1 gene confers transferable colistin resistance. Reports of mcr-1-positive Escherichia coli (MCRPE) have attracted substantial attention. However, in Nigeria, there is no report of mcr-1 gene resistance. Since colistin is a last resort for multiple drug-resistant isolates, this study therefore report the prevalence of mcr-1 gene among E. coli isolated from human and animal sources. Methods: Out of a total of 280 samples collected from animal and hum an faecal samples from selected farms in Oyo and Osun States, Southwestern Nigeria between July 2015 and June 2016, 60 E. coli were identified using standard microbiological methods. The mcr-1 gene was detected in the isolates by conventional PCR assay. Results: The m cr-1 gene was low and not statistically significant (p≥0.05). It was detected in 5 (8.3%) of 60 E. coli isolates (4= animals; 1= human) Conclusion: This study is the first report of mcr -1 gene from E. coli from human and animal sources in Nigeria. This calls for urgent caution in the use of colistin in animal husbandry.

scholarly journals Synergy in Polymicrobial Infections in a Mouse Model of Type 2 Diabetes

2005 ◽  
Vol 73 (9) ◽  
pp. 6055-6063 ◽  
Author(s):  
Matthew D. Mastropaolo ◽  
Nicholas P. Evans ◽  
Meghan K. Byrnes ◽  
Ann M. Stevens ◽  
John L. Robertson ◽  
...  
Keyword(s):  
Type 2 Diabetes ◽  
White Blood Cells ◽  
In Vivo Model ◽  
Diabetic Patients ◽  
Diabetic Mice ◽  
E Coli ◽  
Polymicrobial Infections ◽  
Young Mice

ABSTRACT Human diabetics frequently suffer delayed wound healing, increased susceptibility to localized and systemic infections, and limb amputations as a consequence of the disease. Lower-limb infections in diabetic patients are most often polymicrobial, involving mixtures of aerobic, facultative anaerobic, and anaerobic bacteria. The purpose of this study is to determine if these organisms contribute to synergy in polymicrobial infections by using diabetic mice as an in vivo model. The model was the obese diabetic mouse strain BKS.Cg-m +/+ Lepr db /J, a model of human type 2 diabetes. Young (5- to 6-week-old) prediabetic mice and aged (23- to 24-week-old) diabetic mice were compared. The mice were injected subcutaneously with mixed cultures containing Escherichia coli, Bacteroides fragilis, and Clostridium perfringens. Progression of the infection (usually abscess formation) was monitored by examining mice for bacterial populations and numbers of white blood cells at 1, 8, and 22 days postinfection. Synergy in the mixed infections was defined as a statistically significant increase in the number of bacteria at the site of injection when coinfected with a second bacterium, compared to when the bacterium was inoculated alone. E. coli provided strong synergy to B. fragilis but not to C. perfringens. C. perfringens and B. fragilis provided moderate synergy to each other but only in young mice. B. fragilis was anergistic (antagonistic) to E. coli in coinfections in young mice at 22 days postinfection. When age-matched nondiabetic mice (C57BLKS/J) were used as controls, the diabetic mice exhibited 5 to 35 times the number of CFU as did the nondiabetic mice, indicating that diabetes was a significant factor in the severity of the polymicrobial infections.

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