Logo of aemPermissionsJournals.ASM.orgJournalAEM ArticleJournal InfoAuthorsReviewers
Appl Environ Microbiol. 1997 Feb; 63(2): 703–709.
PMCID: PMC168360

Detection system for Escherichia coli-specific virulence genes: absence of virulence determinants in B and C strains.

Abstract

We describe a rational approach to simultaneously test Escherichia coli strains for the presence of known virulence genes in a reverse dot blot procedure. Specific segments of virulence genes of E. coli designed to have similar hybridization parameters were subcloned on plasmids and subsequently amplified by PCR as unlabeled probes in amounts sufficient to be bound to nylon membranes. Various pathogenic isolates and laboratory strains of E. coli were probed for the presence of virulence genes by labeling the genomic DNA of these strains with digoxigenin and then hybridizing them to the prepared nylon membranes. These hybridization results demonstrated that besides the E. coli K-12 safety strain derivatives, E. coli B and C strains are also devoid of genes encoding any of the investigated virulence factors. In contrast, pathogenic E. coli control strains, used to evaluate the method, showed typical hybridization patterns. The described probes and their easy application on a single filter were shown to provide a useful tool for the safety assessment of E. coli strains to be used as hosts in biotechnological processes. This approach might also be used for the identification and characterization of clinically significant E. coli isolates from human and animal species.

Full Text

The Full Text of this article is available as a PDF (2.9M).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Abe A, Komase K, Bangtrakulnonth A, Ratchtrachenchat OA, Kawahara K, Danbara H. Trivalent heat-labile- and heat-stable-enterotoxin probe conjugated with horseradish peroxidase for detection of enterotoxigenic Escherichia coli by hybridization. J Clin Microbiol. 1990 Dec;28(12):2616–2620. [PMC free article] [PubMed]
  • Baudry B, Savarino SJ, Vial P, Kaper JB, Levine MM. A sensitive and specific DNA probe to identify enteroaggregative Escherichia coli, a recently discovered diarrheal pathogen. J Infect Dis. 1990 Jun;161(6):1249–1251. [PubMed]
  • Bell BP, Goldoft M, Griffin PM, Davis MA, Gordon DC, Tarr PI, Bartleson CA, Lewis JH, Barrett TJ, Wells JG, et al. A multistate outbreak of Escherichia coli O157:H7-associated bloody diarrhea and hemolytic uremic syndrome from hamburgers. The Washington experience. JAMA. 1994 Nov 2;272(17):1349–1353. [PubMed]
  • Blum G, Falbo V, Caprioli A, Hacker J. Gene clusters encoding the cytotoxic necrotizing factor type 1, Prs-fimbriae and alpha-hemolysin form the pathogenicity island II of the uropathogenic Escherichia coli strain J96. FEMS Microbiol Lett. 1995 Feb 15;126(2):189–195. [PubMed]
  • Blum G, Ott M, Lischewski A, Ritter A, Imrich H, Tschäpe H, Hacker J. Excision of large DNA regions termed pathogenicity islands from tRNA-specific loci in the chromosome of an Escherichia coli wild-type pathogen. Infect Immun. 1994 Feb;62(2):606–614. [PMC free article] [PubMed]
  • Boylan M, Coleman DC, Smyth CJ. Molecular cloning and characterization of the genetic determinant encoding CS3 fimbriae of enterotoxigenic Escherichia coli. Microb Pathog. 1987 Mar;2(3):195–209. [PubMed]
  • Boylan M, Smyth CJ, Scott JR. Nucleotide sequence of the gene encoding the major subunit of CS3 fimbriae of enterotoxigenic Escherichia coli. Infect Immun. 1988 Dec;56(12):3297–3300. [PMC free article] [PubMed]
  • Connell TD, Holmes RK. Characterization of hybrid toxins produced in Escherichia coli by assembly of A and B polypeptides from type I and type II heat-labile enterotoxins. Infect Immun. 1992 Apr;60(4):1653–1661. [PMC free article] [PubMed]
  • Cotter PA, Miller JF. Triggering bacterial virulence. Science. 1996 Aug 30;273(5279):1183–1184. [PubMed]
  • Coulton JW, Mason P, Cameron DR, Carmel G, Jean R, Rode HN. Protein fusions of beta-galactosidase to the ferrichrome-iron receptor of Escherichia coli K-12. J Bacteriol. 1986 Jan;165(1):181–192. [PMC free article] [PubMed]
  • Dallas WS, Gill DM, Falkow S. Cistrons encoding Escherichia coli heat-labile toxin. J Bacteriol. 1979 Sep;139(3):850–858. [PMC free article] [PubMed]
  • Donnenberg MS, Girón JA, Nataro JP, Kaper JB. A plasmid-encoded type IV fimbrial gene of enteropathogenic Escherichia coli associated with localized adherence. Mol Microbiol. 1992 Nov;6(22):3427–3437. [PubMed]
  • Falbo V, Pace T, Picci L, Pizzi E, Caprioli A. Isolation and nucleotide sequence of the gene encoding cytotoxic necrotizing factor 1 of Escherichia coli. Infect Immun. 1993 Nov;61(11):4909–4914. [PMC free article] [PubMed]
  • Gross R, Engelbrecht F, Braun V. Genetic and biochemical characterization of the aerobactin synthesis operon on pColV. Mol Gen Genet. 1984;196(1):74–80. [PubMed]
  • Hartman AB, Venkatesan M, Oaks EV, Buysse JM. Sequence and molecular characterization of a multicopy invasion plasmid antigen gene, ipaH, of Shigella flexneri. J Bacteriol. 1990 Apr;172(4):1905–1915. [PMC free article] [PubMed]
  • Jerse AE, Yu J, Tall BD, Kaper JB. A genetic locus of enteropathogenic Escherichia coli necessary for the production of attaching and effacing lesions on tissue culture cells. Proc Natl Acad Sci U S A. 1990 Oct;87(20):7839–7843. [PMC free article] [PubMed]
  • Karjalainen TK, Evans DG, So M, Lee CH. Molecular cloning and nucleotide sequence of the colonization factor antigen I gene of Escherichia coli. Infect Immun. 1989 Apr;57(4):1126–1130. [PMC free article] [PubMed]
  • Klemm P. The fimA gene encoding the type-1 fimbrial subunit of Escherichia coli. Nucleotide sequence and primary structure of the protein. Eur J Biochem. 1984 Sep 3;143(2):395–399. [PubMed]
  • Kuhnert P, Nicolet J, Frey J. Rapid and accurate identification of Escherichia coli K-12 strains. Appl Environ Microbiol. 1995 Nov;61(11):4135–4139. [PMC free article] [PubMed]
  • Law D. Adhesion and its role in the virulence of enteropathogenic Escherichia coli. Clin Microbiol Rev. 1994 Apr;7(2):152–173. [PMC free article] [PubMed]
  • Levine MM, Xu JG, Kaper JB, Lior H, Prado V, Tall B, Nataro J, Karch H, Wachsmuth K. A DNA probe to identify enterohemorrhagic Escherichia coli of O157:H7 and other serotypes that cause hemorrhagic colitis and hemolytic uremic syndrome. J Infect Dis. 1987 Jul;156(1):175–182. [PubMed]
  • Marklund BI, Tennent JM, Garcia E, Hamers A, Båga M, Lindberg F, Gaastra W, Normark S. Horizontal gene transfer of the Escherichia coli pap and prs pili operons as a mechanism for the development of tissue-specific adhesive properties. Mol Microbiol. 1992 Aug;6(16):2225–2242. [PubMed]
  • Martínez JL, Herrero M, de Lorenzo V. The organization of intercistronic regions of the aerobactin operon of pColV-K30 may account for the differential expression of the iucABCD iutA genes. J Mol Biol. 1994 Apr 29;238(2):288–293. [PubMed]
  • McDaniel TK, Jarvis KG, Donnenberg MS, Kaper JB. A genetic locus of enterocyte effacement conserved among diverse enterobacterial pathogens. Proc Natl Acad Sci U S A. 1995 Feb 28;92(5):1664–1668. [PMC free article] [PubMed]
  • Mühldorfer I, Blum G, Donohue-Rolfe A, Heier H, Olschläger T, Tschäpe H, Wallner U, Hacker J. Characterization of Escherichia coli strains isolated from environmental water habitats and from stool samples of healthy volunteers. Res Microbiol. 1996 Oct;147(8):625–635. [PubMed]
  • Mühldorfer I, Hacker J. Genetic aspects of Escherichia coli virulence. Microb Pathog. 1994 Mar;16(3):171–181. [PubMed]
  • Nataro JP, Baldini MM, Kaper JB, Black RE, Bravo N, Levine MM. Detection of an adherence factor of enteropathogenic Escherichia coli with a DNA probe. J Infect Dis. 1985 Sep;152(3):560–565. [PubMed]
  • Newland JW, Neill RJ. DNA probes for Shiga-like toxins I and II and for toxin-converting bacteriophages. J Clin Microbiol. 1988 Jul;26(7):1292–1297. [PMC free article] [PubMed]
  • Noegel A, Rdest U, Goebel W. Determination of the functions of hemolytic plasmid pHly152 of Escherichia coli. J Bacteriol. 1981 Jan;145(1):233–247. [PMC free article] [PubMed]
  • Paton AW, Paton JC, Goldwater PN, Heuzenroeder MW, Manning PA. Sequence of a variant Shiga-like toxin type-I operon of Escherichia coli O111:H-. Gene. 1993 Jul 15;129(1):87–92. [PubMed]
  • Pickett CL, Weinstein DL, Holmes RK. Genetics of type IIa heat-labile enterotoxin of Escherichia coli: operon fusions, nucleotide sequence, and hybridization studies. J Bacteriol. 1987 Nov;169(11):5180–5187. [PMC free article] [PubMed]
  • Salvi RJ, Ahroon W, Saunders SS, Arnold SA. Evoked potentials: computer-automated threshold-tracking procedure using an objective detection criterion. Ear Hear. 1987 Jun;8(3):151–156. [PubMed]
  • Roberts I, Mountford R, High N, Bitter-Suermann D, Jann K, Timmis K, Boulnois G. Molecular cloning and analysis of genes for production of K5, K7, K12, and K92 capsular polysaccharides in Escherichia coli. J Bacteriol. 1986 Dec;168(3):1228–1233. [PMC free article] [PubMed]
  • Schmidt H, Beutin L, Karch H. Molecular analysis of the plasmid-encoded hemolysin of Escherichia coli O157:H7 strain EDL 933. Infect Immun. 1995 Mar;63(3):1055–1061. [PMC free article] [PubMed]
  • Schmidt H, Knop C, Franke S, Aleksic S, Heesemann J, Karch H. Development of PCR for screening of enteroaggregative Escherichia coli. J Clin Microbiol. 1995 Mar;33(3):701–705. [PMC free article] [PubMed]
  • Schmoll T, Hoschützky H, Morschhäuser J, Lottspeich F, Jann K, Hacker J. Analysis of genes coding for the sialic acid-binding adhesin and two other minor fimbrial subunits of the S-fimbrial adhesin determinant of Escherichia coli. Mol Microbiol. 1989 Dec;3(12):1735–1744. [PubMed]
  • Sears CL, Kaper JB. Enteric bacterial toxins: mechanisms of action and linkage to intestinal secretion. Microbiol Rev. 1996 Mar;60(1):167–215. [PMC free article] [PubMed]
  • Spicer EK, Noble JA. Escherichia coli heat-labile enterotoxin. Nucleotide sequence of the A subunit gene. J Biol Chem. 1982 May 25;257(10):5716–5721. [PubMed]
  • Studier FW, Moffatt BA. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. [PubMed]
  • van Die I, van Geffen B, Hoekstra W, Bergmans H. Type 1C fimbriae of a uropathogenic Escherichia coli strain: cloning and characterization of the genes involved in the expression of the 1C antigen and nucleotide sequence of the subunit gene. Gene. 1985;34(2-3):187–196. [PubMed]
  • Venkatesan MM, Buysse JM, Hartman AB. Sequence variation in two ipaH genes of Shigella flexneri 5 and homology to the LRG-like family of proteins. Mol Microbiol. 1991 Oct;5(10):2435–2445. [PubMed]
  • Weinstein DL, Jackson MP, Samuel JE, Holmes RK, O'Brien AD. Cloning and sequencing of a Shiga-like toxin type II variant from Escherichia coli strain responsible for edema disease of swine. J Bacteriol. 1988 Sep;170(9):4223–4230. [PMC free article] [PubMed]
  • Zapata G, Crowley JM, Vann WF. Sequence and expression of the Escherichia coli K1 neuC gene product. J Bacteriol. 1992 Jan;174(1):315–319. [PMC free article] [PubMed]
  • Zapata G, Vann WF, Aaronson W, Lewis MS, Moos M. Sequence of the cloned Escherichia coli K1 CMP-N-acetylneuraminic acid synthetase gene. J Biol Chem. 1989 Sep 5;264(25):14769–14774. [PubMed]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

  • MedGen
    MedGen
    Related information in MedGen
  • PubMed
    PubMed
    PubMed citations for these articles

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...