• 1

    Okeke IN, Nataro JP, 2001. Enteroaggregative Escherichia coli. Lancet Infect Dis 1 :304–313.

  • 2

    Nataro JP, Kaper JB, Robins-Browne R, Prado V, Vial P, Levine MM, 1987. Patterns of adherence of diarrheagenic Escherichia coli to HEp-2 cells. Pediatr Infect Dis J 6 :829–831.

    • Search Google Scholar
    • Export Citation
  • 3

    Cerna JF, Nataro JP, Estrada-Garcia T, 2003. Multiplex PCR for detection of three plasmid-borne genes of enteroaggregative Escherichia coli strains. J Clin Microbiol 41 :2138–2140.

    • Search Google Scholar
    • Export Citation
  • 4

    Miqdady MS, Jiang ZD, Nataro JP, DuPont HL, 2002. Detection of enteroaggregative Escherichia coli with formalin-preserved HEp-2 cells. J Clin Microbiol 40 :3066–3067.

    • Search Google Scholar
    • Export Citation
  • 5

    Baudry B, Savarino SJ, Vial P, Kaper JB, Levine MM, 1990. A sensitive and specific DNA probe to identify enteroaggregative Escherichia coli, a recently discovered diarrheal pathogen. J Infect Dis 161 :1249–1251.

    • Search Google Scholar
    • Export Citation
  • 6

    Cobeljic M, Miljkovic-Selimovic B, Paunovic-Todosijevic D, Velickovic Z, Lepsanovic Z, Zec N, Savic D, Ilic R, Konstantinovic S, Jovanovic B, Kostic V, 1996. Enteroaggregative Escherichia coli associated with an outbreak of diarrhoea in a neonatal nursery ward. Epidemiol Infect 117 :11–16.

    • Search Google Scholar
    • Export Citation
  • 7

    Itoh Y, Nagano I, Kunishima M, Ezaki T, 1997. Laboratory investigation of enteroaggregative Escherichia coli O untype-able:H10 associated with a massive outbreak of gastrointestinal illness. J Clin Microbiol 35 :2546–2550.

    • Search Google Scholar
    • Export Citation
  • 8

    Schmidt H, Knop C, Franke S, Aleksic S, Heesemann J, Karch H, 1995. Development of PCR for screening of enteroaggregative Escherichia coli. J Clin Microbiol 33 :701–705.

    • Search Google Scholar
    • Export Citation
  • 9

    Tsukamoto T, 1996. PCR methods for detection of enteropathogenic Escherichia coli (localized adherence) and enteroaggregative Escherichia coli. Kansenshogaku Zasshi 70 :569–573.

    • Search Google Scholar
    • Export Citation
  • 10

    Czeczulin JR, Whittam TS, Henderson IR, Navarro-Garcia F, Nataro JP, 1999. Phylogenetic analysis of enteroaggregative and diffusely adherent Escherichia coli. Infect Immun 67 :2692–2699.

    • Search Google Scholar
    • Export Citation
  • 11

    Nataro JP, Kaper JB, 1998. Diarrheagenic Escherichia coli. Clin Microbiol Rev 11 :142–201.

  • 12

    Sheikh J, Hicks S, Dall’Agnol M, Phillips AD, Nataro JP, 2001. Roles for Fis and YafK in biofilm formation by enteroaggregative Escherichia coli. Mol Microbiol 41 :983–997.

    • Search Google Scholar
    • Export Citation
  • 13

    Nishi J, Sheikh J, Mizuguchi K, Luisi B, Burland V, Boutin A, Rose DJ, Blattner FR, Nataro JP, 2003. The export of coat protein from enteroaggregative Escherichia coli by a specific ATP-binding cassette transporter system. J Biol Chem 278 :45680–45689.

    • Search Google Scholar
    • Export Citation
  • 14

    Sarantuya J, Nishi J, Wakimoto N, Erdene S, Nataro JP, Sheikh J, Iwashita M, Manago K, Tokuda K, Yoshinaga M, Miyata K, Kawano Y, 2004. Typical enteroaggregative Escherichia coli is the most prevalent pathotype among E. coli strains causing diarrhea in Mongolian children. J Clin Microbiol 42 :133–139.

    • Search Google Scholar
    • Export Citation
  • 15

    Iwanaga M, Song T, Higa N, Kakinohana S, Toma C, Nakasone N, 2002. Enteroaggregative Escherichia coli: incidence in Japan and usefulness of the clump-formation test. J Infect Chemother 8 :345–348.

    • Search Google Scholar
    • Export Citation
  • 16

    Albert MJ, Qadri F, Haque A, Bhuiyan NA, 1993. Bacterial clump formation at the surface of liquid culture as a rapid test for identification of enteroaggregative Escherichia coli. J Clin Microbiol 31 :1397–1399.

    • Search Google Scholar
    • Export Citation
  • 17

    Nataro JP, 2004. Enteroaggregative Escherichia coli. Scheld WM, Murray BE, Hughes JM, eds. Emerging Infections 6. Washington, DC: American Society for Microbiology Press, 101–110.

 

 

 

 

QUANTITATIVE BIOFILM ASSAY USING A MICROTITER PLATE TO SCREEN FOR ENTEROAGGREGATIVE ESCHERICHIA COLI

View More View Less
  • 1 Department of Pediatrics, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan; Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland

The gold standard for identification of Enteroaggregative Escherichia coli (EAEC) remains the HEp-2 cell adherence test, which is time-consuming and requires specialized facilities. We evaluated the usefulness of a quantitative biofilm assay to screen for EAEC from a total of 1,042 E. coli strains from children with diarrhea. Bacteria were incubated overnight in high-glucose Dulbecco’s modified Eagle’s medium using a polystyrene microtiter plate. The plate was stained with crystal violet after washing, and the biofilm was quantified using an enzyme-linked immunosorbent assay plate reader. The aggR gene was evaluated by a polymerase chain reaction. Forty-eight (77.4%) of 62 strains with an optical density at 570 nm (OD570) > 0.2 were identified as EAEC by the HEp-2 adherence test, while no EAEC was found in strains with an OD570 ≤ 0.2. Twenty-one aggR+ and 27 aggR - EAEC strains could be screened by an OD570 > 0.2 using this assay. Although confirmation by a HEp-2 cell adherence test is needed, this biofilm assay is convenient and useful in screening for EAEC.

Save