scholarly journals Intestinal Enteroid Monolayers Model the Human Intestinal Environment for Escherichia coli Infection

2021 ◽  
Author(s):  
Jason Small ◽  
Alison Weiss
Keyword(s):  
Escherichia Coli ◽  
Bacterial Growth ◽  
Culture Media ◽  
Enterohemorrhagic Escherichia Coli ◽  
Suitable Model ◽  
Apical Surface ◽  
Human Intestine ◽  
Multipotent Stem Cells ◽  
Epithelial Damage ◽  
E Coli

Enterohemorrhagic Escherichia coli O157:H7 is an enteric pathogen responsible for bloody diarrhea, hemolytic uremic syndrome, and in severe cases even death. The study of O157:H7 is difficult due to the high specificity of the bacteria for the human intestine, along with our lack of sufficiently complex human cell culture models. The recent development of human intestinal enteroids derived from intestinal crypt multipotent stem cells has allowed us to construct 2-dimensional differentiated epithelial monolayers grown in transwells that mimic the human intestine. Unlike previous studies, saline was added to the apical surface, while maintaining culture media in the basolateral well. The monolayers continued to grow and differentiate with apical saline. Apical infection with O157:H7 or commensal E. coli resulted in robust bacterial growth from 105 to over 108 over 24 hours. Despite this robust bacterial growth, commensal E. coli neither adhered to nor damaged the epithelial barrier over 30 hours. However, O157:H7 was almost fully adhered (>90%) by 18 hours with epithelial damage observed by 30 hours. O157:H7 contains the locus of enterocyte effacement (LEE) pathogenicity island responsible for attachment and damage to the intestinal epithelium. Previous studies report the ability of nutrients such as biotin, D-serine, and L-fucose to downregulate LEE gene expression. O157:H7 treated with biotin or L-fucose, but not D-serine displayed both decreased attachment and reduced epithelial damage over 36 hours. These data illustrate enteroid monolayers can serve as a suitable model for the study of O157:H7 pathogenesis, and identification of potential therapeutics.

Extracts of Haematoxylon brasiletto Inhibit Growth, Verotoxin Production, and Adhesion of Enterohemorrhagic Escherichia coli O157:H7 to HeLa Cells

2005 ◽  
Vol 68 (7) ◽  
pp. 1346-1351 ◽  
Author(s):  
NORMA HEREDIA ◽  
MARCO ESCOBAR ◽  
CRISTINA RODRÍGUEZ-PADILLA ◽  
SANTOS GARCÍA
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Activity ◽  
Bacterial Growth ◽  
Hela Cells ◽  
Enterohemorrhagic Escherichia Coli ◽  
Partial Purification ◽  
Escherichia Coli O157 ◽  
Active Fraction ◽  
E Coli ◽  
Ethanolic Extracts

The extracts of 33 plants were evaluated for their effects on growth of Escherichia coli O157:H7 (EHEC). The extract of Haematoxylon brasiletto was the only one that effectively inhibited bacterial growth. The effects of ethanolic extracts of this plant on growth, verotoxin production, and adhesion of E. coli O157:H7 to HeLa cells were determined. The MBC for growth was 4 mg/ml. No verotoxin formation was detected at 1, 2, or 3 mg/ml. Preexposing bacteria and HeLa cells to various concentrations of extracts affected the adhesion between non-EHEC and HeLa cells. Partial purification of the active fraction suggested that polyphenols might play a role in the antimicrobial activity exhibited by H. brasiletto extracts.

The Influence of Glycosylation on the Catalytic and Fibrinolytic Properties of Pro-Urokinase

1992 ◽  
Vol 68 (05) ◽  
pp. 539-544 ◽  
Author(s):  
Catherine Lenich ◽  
Ralph Pannell ◽  
Jack Henkin ◽  
Victor Gurewich
Keyword(s):  
Escherichia Coli ◽  
Mammalian Cell ◽  
Human Gene ◽  
Culture Media ◽  
Mammalian Cell Culture ◽  
Glycosylation Site ◽  
Clot Lysis ◽  
Cell Culture Media ◽  
E Coli ◽  
The Uk

SummaryWe previously found that human pro-UK expressed in Escherichia coli is more active in fibrinolysis than recombinant human pro-UK obtained from mammalian cell culture media. To determine whether this difference is related to the lack of glycosylation of the E. coli product, we compared the activity of E. coli-derived pro-UK [(-)pro-UK] with that of a glycosylated pro-UK [(+)pro-UK] and of a mutant of pro-UK missing the glycosylation site at Asn-302 [(-) (302) pro-UK]. The latter two pro-UKs were obtained by expression of the human gene in a mammalian cell. The nonglycosylated pro-UKs were activated by plasmin more efficiently (≈2-fold) and were more active in clot lysis (1.5-fold) than the (+)pro-UK. Similarly, the nonglycosylated two-chain derivatives (UKs) were more active against plasminogen and were more rapidly inactivated by plasma inhibitors than the (+)UK.These findings indicate that glycosylation at Asn-302 influences the activity of pro-UK/UK and could be the major factor responsible for the enhanced activity of E. coli-derived pro-UK.

Polyester Cloth–Based Hybridization Array System for Identification of Enterohemorrhagic Escherichia coli Serogroups O26, O45, O103, O111, O121, O145, and O157

2012 ◽  
Vol 75 (9) ◽  
pp. 1691-1697 ◽  
Author(s):  
BURTON W. BLAIS ◽  
MARTINE GAUTHIER ◽  
MYLÈNE DESCHÊNES ◽  
GEORGE HUSZCZYNSKI
Keyword(s):  
Escherichia Coli ◽  
Marker Gene ◽  
Enterohemorrhagic Escherichia Coli ◽  
Oligonucleotide Probes ◽  
E Coli ◽  
Polyester Cloth ◽  
Gene Profiles ◽  
Pcr Products ◽  
Different Strains ◽  
Immunoenzymatic Assay

A cloth-based hybridization array system (CHAS) was developed for the identification of foodborne colony isolates of seven priority enterohemorrhagic Escherichia coli (EHEC-7) serogroups targeted by U.S. food inspection programs. Gene sequences associated with intimin; Shiga-like toxins 1 and 2; and the antigenic markers O26, O45, O103, O111, O121, O145, and O157 were amplified in a multiplex PCR incorporating a digoxigenin label, and detected by hybridization of the PCR products with an array of specific oligonucleotide probes immobilized on a polyester cloth support, with subsequent immunoenzymatic assay of the captured amplicons. The EHEC-7 CHAS exhibited 100% inclusivity and 100% exclusivity characteristics with respect to detection of the various markers among 89 different E. coli strains, with various marker gene profiles and 15 different strains of non–E. coli bacteria.

scholarly journals Quorum Sensing Is a Global Regulatory Mechanism in Enterohemorrhagic Escherichia coli O157:H7

2001 ◽  
Vol 183 (17) ◽  
pp. 5187-5197 ◽  
Author(s):  
Vanessa Sperandio ◽  
Alfredo G. Torres ◽  
Jorge A. Girón ◽  
James B. Kaper
Keyword(s):  
Escherichia Coli ◽  
Quorum Sensing ◽  
Type Strain ◽  
Wild Type Strain ◽  
Regulatory Mechanism ◽  
Sos Response ◽  
Enterohemorrhagic Escherichia Coli ◽  
Trna Genes ◽  
Wild Type ◽  
E Coli

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is responsible for outbreaks of bloody diarrhea and hemolytic-uremic syndrome in many countries. EHEC virulence mechanisms include the production of Shiga toxins (Stx) and formation of attaching and effacing (AE) lesions on intestinal epithelial cells. We recently reported that genes involved in the formation of the AE lesion were regulated by quorum sensing through autoinducer-2, which is synthesized by the product of the luxS gene. In this study we hybridized an E. coli gene array with cDNA synthesized from RNA that was extracted from EHEC strain 86-24 and its isogenicluxS mutant. We observed that 404 genes were regulated by luxS at least fivefold, which comprises approximately 10% of the array genes; 235 of these genes were up-regulated and 169 were down-regulated in the wild-type strain compared to in theluxS mutant. Down-regulated genes included several involved in cell division, as well as ribosomal and tRNA genes. Consistent with this pattern of gene expression, theluxS mutant grows faster than the wild-type strain (generation times of 37.5 and 60 min, respectively, in Dulbecco modified Eagle medium). Up-regulated genes included several involved in the expression and assembly of flagella, motility, and chemotaxis. Using operon::lacZ fusions to class I, II, and III flagellar genes, we were able to confirm this transcriptional regulation. We also observed fewer flagella by Western blotting and electron microscopy and decreased motility halos in semisolid agar in the luxS mutant. The average swimming speeds for the wild-type strain and the luxS mutant are 12.5 and 6.6 μm/s, respectively. We also observed an increase in the production of Stx due to quorum sensing. Genes encoding Stx, which are transcribed along with λ-like phage genes, are induced by an SOS response, and genes involved in the SOS response were also regulated by quorum sensing. These results indicate that quorum sensing is a global regulatory mechanism for basic physiological functions of E. coli as well as for virulence factors.

scholarly journals Colonization of Arabidopsis thaliana with Salmonella enterica and Enterohemorrhagic Escherichia coli O157:H7 and Competition by Enterobacter asburiae

2003 ◽  
Vol 69 (8) ◽  
pp. 4915-4926 ◽  
Author(s):  
Michael B. Cooley ◽  
William G. Miller ◽  
Robert E. Mandrell
Keyword(s):  
Escherichia Coli ◽  
Arabidopsis Thaliana ◽  
Salmonella Enterica ◽  
Fluorescent Protein ◽  
Enterohemorrhagic Escherichia Coli ◽  
Plant Responses ◽  
Human Pathogens ◽  
E Coli ◽  
Green Fluorescent ◽  
Enterobacter Asburiae

ABSTRACT Enteric pathogens, such as Salmonella enterica and Escherichia coli O157:H7, have been shown to contaminate fresh produce. Under appropriate conditions, these bacteria will grow on and invade the plant tissue. We have developed Arabidopsis thaliana (thale cress) as a model system with the intention of studying plant responses to human pathogens. Under sterile conditions and at 100% humidity, S. enterica serovar Newport and E. coli O157:H7 grew to 109 CFU g−1 on A. thaliana roots and to 2 × 107 CFU g−1 on shoots. Furthermore, root inoculation led to contamination of the entire plant, indicating that the pathogens are capable of moving on or within the plant in the absence of competition. Inoculation with green fluorescent protein-labeled S. enterica and E. coli O157:H7 showed invasion of the roots at lateral root junctions. Movement was eliminated and invasion decreased when nonmotile mutants of S. enterica were used. Survival of S. enterica serovar Newport and E. coli O157:H7 on soil-grown plants declined as the plants matured, but both pathogens were detectable for at least 21 days. Survival of the pathogen was reduced in unautoclaved soil and amended soil, suggesting competition from indigenous epiphytes from the soil. Enterobacter asburiae was isolated from soil-grown A. thaliana and shown to be effective at suppressing epiphytic growth of both pathogens under gnotobiotic conditions. Seed and chaff harvested from contaminated plants were occasionally contaminated. The rate of recovery of S. enterica and E. coli O157:H7 from seed varied from undetectable to 19% of the seed pools tested, depending on the method of inoculation. Seed contamination by these pathogens was undetectable in the presence of the competitor, Enterobacter asburiae. Sampling of 74 pools of chaff indicated a strong correlation between contamination of the chaff and seed (P = 0.025). This suggested that contamination of the seed occurred directly from contaminated chaff or by invasion of the flower or silique. However, contaminated seeds were not sanitized by extensive washing and chlorine treatment, indicating that some of the bacteria reside in a protected niche on the seed surface or under the seed coat.

Enterohemorrhagic Escherichia coli Colony Check Assay for the Identification of Serogroups O26, O45, O103, O111, O121, O145, and O157 Colonies Isolated on Plating Media

2014 ◽  
Vol 77 (7) ◽  
pp. 1212-1218 ◽  
Author(s):  
BURTON BLAIS ◽  
MYLÈNE DESCHÊNES ◽  
GEORGE HUSZCZYNSKI ◽  
MARTINE GAUTHIER
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
High Throughput Screening ◽  
Enterohemorrhagic Escherichia Coli ◽  
Test Results ◽  
Bacterial Strains ◽  
Enrichment Broth ◽  
Substrate Solution ◽  
E Coli ◽  
Assay Performance

A simple immunoenzymatic enterohemorrhagic Escherichia coli (EHEC) colony check (ECC) assay was developed for the presumptive identification of priority EHEC colonies isolated on plating media from enrichment broth cultures of foods. With this approach, lipopolysaccharide extracted from a colony is spotted on the grid of a polymyxin-coated polyester cloth strip, and bound E. coli serogroup O26, O45, O103, O111, O121, O145, and O157 antigens are subsequently detected by sequential reactions with a pool of commercially available peroxidase-conjugated goat antibodies and tetramethylbenzidine substrate solution. Each strip can accommodate up to 15 colonies, and test results are available within 30 min. Assay performance was verified using colonies from a total of 73 target EHEC isolates covering the range of designated priority serogroups (all of which were reactive), 41 nontarget E. coli isolates including several nontarget Shiga toxin–producing E. coli serogroups (all unreactive), and 33 non–E. coli strains (all unreactive except two bacterial strains possessing O-antigenic structures in common with those of the priority EHEC). The ECC assay was reactive with target colonies grown on several types of selective and nonselective plating media designed for their cultivation. These results support the use of the ECC assay for high-throughput screening of colonies isolated on plating media for detecting priority EHEC strains in foods.

Bioluminescence: A Rapid Indicator of Escherichia Coli O157:H7 in Selected Yogurt and Cheese Varieties

1997 ◽  
Vol 60 (8) ◽  
pp. 891-897 ◽  
Author(s):  
L. M. HUDSON ◽  
J. CHEN ◽  
A. R. HILL ◽  
M. W. GRIFFITHS
Keyword(s):  
Escherichia Coli ◽  
Enterohemorrhagic Escherichia Coli ◽  
Cottage Cheese ◽  
Plate Count ◽  
Escherichia Coli O157 ◽  
Potential Hazard ◽  
Growth And Survival ◽  
E Coli ◽  
Standard Plate Count ◽  
Standard Plate

Outbreaks of enterohemorrhagic Escherichia coli O157:H7 have been commonly associated with products derived from ground beef, but recently the organism has been implicated as the causative agent in outbreaks involving yogurt and cheese. This finding has raised concern about the potential for its growth and survival in fermented dairy products. A bioluminescent strain of E. coli O157:H7 was used to determine postprocessing survival in yogurt with live cultures at pH 4.17, 4.39, and 4.47 stored at 4 and 10°C. In addition, survival of E. coli O157:H7 was monitored during the manufacture of Cottage, Colby, Romano, and Feta cheeses. Results indicated survival for 8 and 5 days at 4 and 10°C respectively in yogurt at pH 4.17, 17 and 15 days at 4 and 10°C respectively in yogurt at pH 4.39, and 17days at both 4 and 10°C in yogurt at pH 4.47. E. coli O157:H7 did not survive cooking procedures at 56°C in Cottage cheese. However, the pathogen survived for 27, 30, and 27 days in Colby, Romano, and Feta cheeses respectively. A high correlation of r2 > 0.89 was obtained between counts of bioluminescenct colonies and standard plate count for all yogurt and cheese varieties, indicating that bioluminescence was a sensitive and rapid indicator of cellular viability for E. coli O157:H7. Survival of the pathogen, as indicated by this method, is possible in highly acidic environments even at refrigeration temperatures. This poses a potential hazard should postprocessing contamination occur.

scholarly journals Impact of Different Carbon Sources on the in vitro Growth and Viability of Escherichia coli (SUBE01) and Salmonella spp. (SUBS01) Cells

2016 ◽  
pp. 39-44
Author(s):  
Ifra Tun Nur ◽  
Jannatun Tahera ◽  
Md Sakil Munna ◽  
M Majibur Rahman ◽  
Rashed Noor
Keyword(s):  
Escherichia Coli ◽  
Bacterial Growth ◽  
Culture Media ◽  
Bacterial Species ◽  
Carbon Sources ◽  
Growth Dynamics ◽  
Luria Bertani ◽  
Tween 20 ◽  
Salmonella Spp

With a previous observation of Escherichia coli growth cessation along with temperature variation within three different bacteriological culture media (nutrient agar, Luria-Bertani agar and minimal agar), current investigation further depicted on the possible growth dynamics of Escherichia coli (SUBE01) and Salmonella (SUBS01) growth and viability upon supplementation of different carbon sources (dextrose, sucrose, lactose, glycerol and tween 20) at 37°C under the aeration of 100 rpm. Viability of the tested bacterial species was assessed through the enumeration of the colony forming unit (cfu) appeared upon prescribed incubation for 12-24 hours on different agar plates consisting of the above mentioned carbon sources. Besides, to inspect the cellular phenotypic changes, morphological observations were conducted under the light microscope. Variations in bacterial growth (either growth acceleration or cessation) were further noticed through the spot tests on the agar plates. Considerable shortfalls in the culturable cells of E. coli and Salmonella spp. were noted in the minimal media separately consisting of sucrose, lactose, glycerol or tween 20 while an opposite impact of accelerated growth was noticed in the media supplied with dextrose. The data revealed a hierarchy of consequence of carbon sources as nutrient generators whereby the favourable bacterial growth and survival order of the carbon sources was estimated as dextrose > glycerol > lactose > tween 20 > sucrose.Bangladesh J Microbiol, Volume 32, Number 1-2,June-Dec 2015, pp 39-44

scholarly journals Antibiotic-Mediated Modulations of Outer Membrane Vesicles in Enterohemorrhagic Escherichia coli O104:H4 and O157:H7

2017 ◽  
Vol 61 (9) ◽  
Author(s):  
Andreas Bauwens ◽  
Lisa Kunsmann ◽  
Helge Karch ◽  
Alexander Mellmann ◽  
Martina Bielaszewska
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Shiga Toxin ◽  
Membrane Vesicles ◽  
Polymyxin B ◽  
Outer Membrane Vesicles ◽  
Enterohemorrhagic Escherichia Coli ◽  
Content Type ◽  
E Coli ◽  
Major Virulence Factor

ABSTRACT Ciprofloxacin, meropenem, fosfomycin, and polymyxin B strongly increase production of outer membrane vesicles (OMVs) in Escherichia coli O104:H4 and O157:H7. Ciprofloxacin also upregulates OMV-associated Shiga toxin 2a, the major virulence factor of these pathogens, whereas the other antibiotics increase OMV production without the toxin. These two effects might worsen the clinical outcome of infections caused by Shiga toxin-producing E. coli. Our data support the existing recommendations to avoid antibiotics for treatment of these infections.

Survival of Enterohemorrhagic Escherichia coli O157:H7 in Retail Mustard

2001 ◽  
Vol 64 (6) ◽  
pp. 783-787 ◽  
Author(s):  
CAROLYN M. MAYERHAUSER
Keyword(s):  
Escherichia Coli ◽  
Foodborne Illness ◽  
Enterohemorrhagic Escherichia Coli ◽  
Test Strain ◽  
Escherichia Coli O157 ◽  
Time Intervals ◽  
Fermented Sausage ◽  
Apple Cider ◽  
E Coli ◽  
Predetermined Time

Escherichia coli O157:H7 survival in acid foods such as unpasteurized apple cider and fermented sausage is well documented. Researchers have determined that E. coli O157:H7 can survive in refrigerated acid foods for weeks. The potential of acid foods to serve as a vector of E. coli O157:H7 foodborne illness prompted this study to determine the fate of this organism in retail mustard containing acetic acid when stored at room and refrigerated temperatures. Various retail brands of dijon, yellow, and deli style mustard, pH ranging from 3.17 to 3.63, were inoculated individually with three test strains of E. coli O157:H7. Samples were inoculated with approximately 1.0 × 106 CFU/g, incubated at room (25 ± 2.5°C) and refrigerated (5 ± 3°C) temperatures, and assayed for surviving test strains at predetermined time intervals. An aliquot was appropriately diluted and plated using sorbitol MacConkey agar (SMAC). When the test strain was not recoverable by direct plating, the sample was assayed by enrichment in modified tryptic soy broth and recovered using SMAC. Growth of E. coli O157:H7 test strains was inhibited in all retail mustard styles. E. coli O157:H7 was not detected in dijon style mustard beyond 3 h at room and 2 days at refrigerated temperatures. Survival in yellow and deli style mustard was not detected beyond 1 h. Overall, test strain survival was greater at refrigerated than room temperature. Retail mustard demonstrated the ability to eliminate effectively any chance contamination by this organism within hours to days, suggesting that these products are not a likely factor in E. coli O157:H7 foodborne illness.

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