Detection of water-borne E. coli O157 using the integrating waveguide biosensor

Abstract

Escherichia coli O157:H7, the most common serotype of enterohemorrhagic E. coli (EHEC), is responsible for numerous food-borne and water-borne infections worldwide. An integrating waveguide biosensor is described for the detection of water-borne E. coli O157, based on a fluorescent sandwich immunoassay performed inside a glass capillary waveguide. The genomic DNA of captured E. coli O157 cells was extracted and quantitative real-time PCR subsequently performed to assess biosensor-capture efficiency. In vitro microbial growth in capillary waveguide is also documented. The biosensor allows for quantitative detection of as few as 10 cells per capillary (0.075 ml volume) and can be used in conjunction with cell amplification, PCR and microarray technologies to positively identify a pathogen.

Introduction

Enterohemorrhagic Escherichia coli (e.g., E. coli O157:H7) is a major food-borne and water-borne pathogen that causes diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (Su and Brandt, 1995). Outbreaks have occurred in many developed countries, including Canada, Europe, Australia, and Japan. The Center for Disease Control and Prevention (CDC) estimates that E. coli O157:H7 causes nearly 75,000 human infections in the U.S. each year (Mead et al., 1999).

Many methods have been developed to detect E. coli O157:H7 in food and water matrices, including traditional culturing with selective media (Hammack et al., 1997, March and Ratnam, 1986), serotyping with specific antibodies to O157 and H7 antigens (Chapman et al., 1997, Czajka and Batt, 1996, Shelton and Karns, 2001, Tomoyasu, 1998), amplification of specific genes by PCR (Higgins et al., 2003, Johnson and Stell, 2001, Maurer et al., 1999, Wang et al., 2002) or hybridization of virulence genes by DNA microarrays (Bekal et al., 2003). Each method has limitations with respect to sensitivity, specificity, and quantitation. Consequently, multiple assays are required to detect and quantify small numbers of water-borne E. coli O157:H7 and confirm strain identity.

The integrating waveguide biosensor was originally developed by Ligler et al. (2002) at the Naval Research Laboratory, Washington, DC. The biosensor utilizes a sandwich antibody technique for capture and detection, with the capture antibodies attached to the inner surface of a glass capillary tube. Detection and quantitation are achieved by illuminating the capillary tube (i.e., optical waveguide) at a 90° angle relative to the length of the waveguide and subsequent collection of the emitted fluorescence from the end of the waveguide (Fig. 1). Initial results gave a detection limit of 40 pg ml⁻¹ for mouse IgG and 30 pg ml⁻¹ for staphylococcal enterotoxin B (SEB) in the sandwich assays (Ligler et al., 2002), which is more sensitive than other fiber optic and array biosensors (Anderson et al., 1994, Rowe et al., 1999).

Compared to the existing technologies, the integrating waveguide biosensor provides a platform with multiple advantages for the detection of E. coli O157:H7 and other analytes in low concentration. Although the capillary tube was originally envisioned only as a waveguide, it is readily available, convenient for laboratory experimentation, and compatible with other sample preparation and detection protocols. The capillary tube can serve as incubation vessel for growth of bacterial pathogens after capture, allowing for confirmation of viability, as well as amplification and retrieval for further characterization. The enclosed structure of capillary tubes is of particular benefit when dealing with pathogenic substances. In addition, clean up of the contaminants by washing, followed with in vitro lysis of pathogens, allows for rapid confirmation of strain identity using PCR or microarray technologies. Since the integrating waveguide biosensor was initially described for the detection of protein targets such as mouse IgG and SEB (Ligler et al., 2002), it remained unclear whether the biosensor could be used for the detection of whole bacterial cells or viral particles. Therefore, we conducted these studies to evaluate the use of the biosensor for the detection of water-borne E. coli O157, including assessment of capture efficiency, detection limit, and in vitro growth rates.