Confirmation of viable E. coli O157:H7 by enrichment and PCR after rapid biosensor detection

Journal of Microbiological Methods 55 (2003) 141 – 147 www.elsevier.com/locate/jmicmeth

Confirmation of viable E. coli O157:H7 by enrichment and PCR after rapid biosensor detection T. Bryan Tims, Daniel V. Lim * Department of Biology, University of South Florida, 4202 East Fowler Ave, SCA 110, Tampa, FL 33620-5200, USA Received 6 January 2003; received in revised form 28 February 2003; accepted 7 April 2003

Abstract Many rapid tests have been developed for the detection of Escherichia coli O157:H7 from complex matrices such as food and water. However, many of these methods rely on traditional culture steps for confirmation, which can take an extra 24 – 48 h. The fiber optic biosensor has been used to rapidly detect pathogens from complex matrices. In this paper, we demonstrate a method using a rapid biosensor assay, recovery through a short enrichment, and PCR to detect and confirm the presence of at least 103 CFU/ml of E. coli O157:H7 in a sample in less than 10 h. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Detection; Biosensor; PCR; E. coli O157:H7; Immunoassay

1. Introduction Escherichia coli O157:H7 is a facultative gramnegative bacillus that has been implicated in outbreaks of illness due to ingestion of meats (Chapman et al., 2000), water, and uncooked fruits and vegetables (Pebody et al., 1999). This pathogen is known to cause diarrhea and hemolytic uremic syndrome (HUS) in humans (DeCludt et al., 2000). It is the most common strain of Shiga toxin-producing E. coli (STEC) in the United States, Canada, and the United Kingdom (Kaper, 1998). Many detection methods have been employed to rapidly detect low levels of organisms in complex matrices such as food. Current techniques include traditional enrichment and plating * Corresponding author. Tel.: +1-813-974-1618; fax: +1-813974-3263. E-mail address: [email protected] (D.V. Lim).

methods with selective media such as Sorbitol MacConkey agar and Rainbow agar (Manafi and Kremsmaier, 2001), immunomagnetic separation of the organism from the sample matrix followed by PCR identification (Chapman et al., 2001; Uyttendaele et al., 1999), and immunological techniques such as ELISA and fiber optic biosensors (DeMarco and Lim, 2001; DeMarco et al., 1999; DeMarco and Lim, 2002). Each of these methods has its own unique set of drawbacks. Enrichment and plating often take 24 – 48 h to identify the organism. Immunological techniques cannot differentiate viable and nonviable cells and often require enrichments. PCR is more rapid than plating techniques, but it can be inhibited by components of the sample matrix and cannot be used to determine viability. PCR also requires DNA purification from sample matrices. The solution to these problems lies in a combination of all of these techniques.

0167-7012/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0167-7012(03)00133-7

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We have previously reported a rapid fiber optic biosensor assay that can detect low levels of E. coli O157:H7 in ground beef and apple cider in 30 min (DeMarco and Lim, 2001; DeMarco et al., 1999; DeMarco and Lim, 2002). We have also been able to recover isolated colonies of the target organism directly from the biosensor through enrichment after completion of the fiber optic biosensor assay from a complex matrix like ground beef (Kramer et al., 2002). In this study, we describe a 10 h procedure for selective enrichment of low levels of E. coli O157:H7 recovered from fiber optic waveguides followed by selective enrichment and plating to confirm viability and molecular identification by PCR.

2. Materials and methods 2.1. Bacterial strains and culture conditions E. coli O157:H7 was obtained from Dr. Harvey George (Massachusetts Department of Public State Laboratory Institute, Jamaica Plains, MA). The strain chosen was isolated from hamburger meat used at a taco stand implicated in a county fair outbreak. All cultures were maintained on tryptic soy agar (TSA, Remel, Lenexa, KS) plates at 4 jC. Cultures for assays and viable counts were grown on TSA for 18 h in a 37 jC incubator and serially diluted in sterile 0.01 M phosphate-buffered saline, 0.85% NaCl, pH 7.4 (PBS) for use in experiments. 2.2. PCR amplification PCR was performed with primers specific to the 3V end of the E. coli O157:H7 eaeA gene (Louie et al., 1994). Each 50 Al reaction volume contained the following: eaeA primer set as defined by Louie et al. (1994) at a concentration of 0.4 AM, 30.25 Al sterile water, 200 AM each deoxynucleoside triphosphate, 1  iTaq buffer (20 mM Tris –HCl, 50 mM KCl, pH 8.4), 1.5 mM MgCl2, 10 Al sample, and 1.25U of iTaq DNA polymerase (iTaq DNA polymerase, Bio-Rad Laboratories, Hercules, CA). Samples were run on a Bio-Rad iCycler with the following conditions: 95 jC for 3 min; 40 cycles of 94 jC for 30 s, 59 jC for 30 s, and 72 jC for 45 s; and extension at 72 jC for 10 min. PCR products were run on a 1% agarose gel at 80 V,

stained with ethidium bromide, destained with water, and viewed under a UV light. Samples were considered positive if a band at approximately 450 bp was visible with the naked eye. 2.3. Antibodies Lyophilized affinity-purified biotin-labeled antibody to E. coli O157:H7 was purchased from Kirkegaard and Perry Laboratories (KPL, Gaithersburg, MD). This antibody was rehydrated using 0.1 M phosphate buffer, pH 7.4. 2.4. Instrument and waveguide preparation The Analyte 2000 is a portable, fiber optic wave biosensor manufactured by Research International (Woodinville, WA). Tapered polystyrene waveguides produced by Research International were used with the Analyte 2000. These waveguides were prepared as described by DeMarco and Lim (2001) and coated with the biotinylated antibodies from KPL. 2.5. Biosensor sample incubation and preparation After waveguides were coated with biotinylated antibody, 1 ml of blocking buffer was added and incubated for 20 min at 25 jC (PBS, 2 mg/ml casein, 2 mg/ml BSA). Waveguides were then rinsed with 1 ml PBS with 0.01% Tween 20 (PBST). One milliliter of sample of E. coli O157:H7 at various concentrations was added and incubated for 10 min. Waveguides were then rinsed with 1 ml PBST. After rinsing, waveguides were either cut into approximately 1-mm sections and added directly to the PCR reaction or incubated for 10 min with 0.5 M glycine, 0.5 M NaCl, pH 3.2. When the waveguides were treated with glycine, the glycine-treated material was used directly as the PCR sample for non-enriched samples, or the cells on the waveguide and in the glycine buffer were enriched in 5 ml modified Luria – Bertani broth with acriflavin (mLB) as described by Kramer et al. (2002). For those experiments where the cells from the waveguides were enriched in mLB, samples were cultured for 6 h at 42 jC and centrifuged at 450  g for 20 min in a IEC clinical centrifuge (International Equipment Company, Needham Hts., MA). The supernatant fluid was then decanted,

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Fig. 1. Procedure for confirming results after rapid biosensor assay.

and the sample was resuspended in 100 Al of sterile water. Ten microliters of the resuspended cells were used as the sample for PCR. For the growth studies, 100 Al of the unconcentrated mLB enrichment broth were serially diluted and plated on TSA every hour to determine the number of viable cells at each time point. Fig. 1 illustrates the final method for recovering a sample containing E. coli O157:H7 from the waveguide, confirming its presence by PCR, and testing cell viability by plating.

results illustrate the PCR sensitivity in an ideal sample of sterile water. 3.2. PCR directly from waveguides We attempted to remove organisms directly to allow a more direct and rapid preparation of cells for PCR confirmation. PCR was initially attempted

3. Results 3.1. PCR sensitivity in water Sterile water was seeded with concentrations of E. coli O157:H7 and a 10 Al sample was used for the PCR reaction as described. The lowest number of cells that could be visualized after the PCR from water seeded with E. coli O157:H7 was 6.9  104 CFU/ml, which was equivalent to 6.9  102 total cells per PCR reaction mixture (Fig. 2). Strong bands were observed with a positive control of 10 ng total purified E. coli O157:H7 DNA. This experiment allowed comparison between ideal PCR conditions and those conditions present after capture, and after enrichment. These

Fig. 2. PCR sensitivity in water. Lane 1, 100 bp marker; lane 2, 6.9  104 CFU per reaction; lane 3, 6.9  103 CFU per reaction; lane 4, 6.9  102 CFU per reaction; lane 5, 69 CFU per reaction; lane 6, 6.9 CFU per reaction; lane 7, positive control, 10 ng purified DNA per reaction; lane 8, negative control, no DNA template.

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directly from the waveguide as a more rapid method of recovery. Small pieces of an unused waveguide were placed directly into the PCR mixture containing 10 ng total purified E. coli O157:H7 DNA. Amplified product was present in the samples that contained DNA template only in the reaction mixture, but not in the samples containing DNA template and waveguide. Other attempts to recover organisms from the waveguide such as boiling were also unsuccessful (data not shown). The PCR was inhibited when the sterile water used for the positive control samples was boiled for 10 min with an unused waveguide (data not shown). It appears that heating of the waveguide releases substances that are inhibitory to PCR. Glycine buffer (0.5 M glycine, 0.5 M NaCl, pH 3.2) was also used in an attempt to directly recover organism for PCR. Glycine buffer at a low pH has previously been used to dissociate antigen/antibody interactions (Suzuki et al., 2002). After cells were captured on the biosensor waveguides by immobilized antibodies, glycine buffer was added to dissociate the cells. Viable and direct counts were performed on the buffer suspension to measure the removal of cells from the waveguide. Recovery of E. coli O157:H7 from the waveguide with glycine buffers of varying molarities of glycine and NaCl and varying pH was tested. Approximately 0.02% of cells injected into the biosensor were recovered on TSA for each different buffer formulation. Only slightly more cells were observed when cells were enumerated by direct count. In order to determine the inhibition of PCR by the different glycine buffer components, cells were resuspended in glycine buffer, and PCR was run using PCR buffer with no KCl. Samples were also resuspended in glycine buffer without 0.5 M NaCl, and PCR was run with unchanged PCR buffer. Fig. 3 shows the inhibition in both cases, suggesting that samples were inhibited in these experiments by the presence of the glycine or the low pH of the buffer. This inhibition, coupled with the poor recovery rates of glycinedissociated cells, led to enrichment as the preferred method of recovery. 3.3. PCR amplification of E. coli O157:H7 in mLB broth In order to determine if the enrichment media would inhibit PCR, mLB broth with acriflavin was spiked

Fig. 3. Inhibition of PCR with glycine buffer. Lane 1, 100 bp marker; lane 2, 1.7  104 CFU per reaction; lane 3, 1.7  103 CFU per reaction; lane 4, 1.7  102 CFU per reaction; lane 5, positive control; lane 6, 1.7  104 CFU per reaction; lane 7, 1.7  103 CFU per reaction; lane 8, 1.7  102 CFU per reaction; lane 9, positive control; lane 10, 1.7  104 CFU per reaction; lane 11, 1.7  103 CFU per reaction; lane 12, 1.7  102 CFU per reaction. Lanes 2 – 4 are in glycine buffer minus 0.5 M NaCl; lanes 6 – 8 are in glycine buffer with 1  PCR buffer minus KCl; lanes 10 – 12 are run in water.

with various concentrations of E. coli O157:H7, and the lower limit of detection of PCR was determined. Five milliliters of mLB were seeded with various concentrations of E. coli O157:H7 and centrifuged for 20 min at 450  g. The pellet was resuspended in 100 Al of sterile water, and 10 Al were removed and used as the PCR template. Detection was achieved when the mLB was seeded with 2.9  103 CFU/ml, which was 1.5  104 CFU per PCR reaction mixture (Fig. 4). Therefore, the mLB with acriflavin did not totally inhibit the PCR reaction, and reasonable sensitivity was obtained after the centrifugation and resuspension step that removed inhibitors such as acriflavin (Scheu et al., 1998). Centrifugation also concentrated the cells so that more cells could be included in the smaller volume required for the PCR reaction. 3.4. Enrichment times from waveguides For enrichment studies, one ml of E. coli O157:H7 at a concentration of at least 103 CFU/ml was captured with the biosensor waveguide, treated with glycine buffer, and added to the enrichment broth. A detection step was not performed prior to recovery and enrichment as detection has been documented to have no

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Fig. 4. PCR sensitivity in enrichment media after centrifugation and resuspension. Lane 1, 100 bp marker; lane 2, 1.5  102 CFU per reaction; lane 3, 1.5  103 CFU per reaction; lane 4, 1.5  104 CFU per reaction; lane 5, 1.5  105 CFU per reaction; lane 6, 1.5  106 CFU per reaction; lane 7, positive control, 10 ng purified DNA per reaction; lane 8, negative control, no DNA template.

effect on recovery (Kramer et al., 2002). Viable counts were performed at 1-h intervals. In order to determine the enrichment time needed to reach the lower limit of at least 2.9  103 CFU/ml for PCR detection from enriched cultures, it was necessary to enrich the bacteria in mLB broth at 42 jC for 5 – 6 h after capture on the biosensor waveguide. Concentrations of E. coli O157:H7 after enrichment at various times and with different concentrations injected into the biosensor are shown in Table 1. 3.5. Final results of capture, enrichment, and PCR from different starting concentrations Finally, all steps were performed in sequence to confirm the starting concentration and enrichment Table 1 Concentration of E. coli O157:H7 in enrichment culture after recovery from biosensor waveguides Biosensor capture 3

4.1  10 CFU/ml 4.1  104 CFU/ml

Concentration after enrichment (CFU/ml) 1h

2h

3h

4h

5h 2

6h 2

0

0

0

4.0  10

7.3  10

1.4  104

0

0

60

1.3  103

1.1  104

1.2  105

Biosensor capture is the concentration of bacteria injected into the biosensor.

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Fig. 5. PCR after recovery and enrichment. Lane 1, 100 bp marker; lane 2, negative control PBS; lane 3, 6.5  102 CFU/ml injected into biosensor; lane 4, 6.5  103 CFU/ml injected into biosensor; lane 5, 6.5  104 CFU/ml injected into biosensor; lane 6, positive control, 10 ng purified DNA added to sample from lane 2.

time required for detection of E. coli O157:H7 by PCR. Samples of PBS containing varying starting concentrations of E. coli O157:H7 were injected and captured on the biosensor waveguides. These waveguides were then treated with glycine buffer, enriched in mLB, and confirmed with PCR. Fig. 5 illustrates a representative experiment in which 1 ml samples of E. coli O157:H7 were resuspended in PBS at concentrations of 0 CFU/ml (PBS buffer), 6.5  10 2 , 6.5  103, or 6.5  104 CFU/ml and were injected into the biosensor. Positive samples were confirmed by PCR at the 6.5  103 and 6.5  104 CFU/ml concentrations. The inability to confirm a positive sample at the 6.5  102 CFU/ml concentration is likely due to the inability to recover viable organisms at this concentration and is consistent with our previously reported data (Kramer et al., 2002).

4. Discussion E. coli O157:H7 is an important food pathogen that can cause serious human illness. Rapid detection and identification of the pathogen allows physicians to begin appropriate treatment in a more timely fashion and prevents costly recalls and potential illness from food products. However, new rapid methods such as real-time PCR do not allow for rapid follow-up confirmatory tests. In fact, some diagnostic tools

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allow for no confirmation at all without splitting or diluting the sample. For example, after real-time PCR, there is no whole cell left for either culture confirmation or immunological testing. The biosensor has been shown to be an efficient method to detect low levels of pathogens from complex matrices (DeMarco and Lim, 2001; DeMarco et al., 1999; DeMarco and Lim, 2002). However, even the most reliable tests require some type of confirmation. Therefore, we have developed culture techniques for recovery of live organisms for traditional confirmational tests (Kramer et al., 2002). Because the biosensor relies on immunological techniques, the potential for cross-reaction with epitopes from other organisms exists. In fact, immunological cross reaction between E. coli O157:H7 and some strains of Citrobacter freundii and Escherichia hermanii, among others, has been documented with other immunological procedures (Bettelheim et al., 1993; Perry et al., 1986). PCR detection relies on a specific DNA sequence, and primers to the eaeA gene are specific for E. coli O157:H7 and O55:H7 (Louie et al., 1994). Therefore, there is only a small chance of cross-reactivity in any PCR-based test. Thus, by using two rapid tests (immunological and PCR) that are based on different biological interactions, confirmation of the specific organism is assured. Because both the detection step and the confirmatory steps are rapid, the organism can be tested and identification can be confirmed in one working day. Many PCR assays depend on immunomagnetic separation (IMS) to remove inhibitory substances that are present in complex matrices. The biosensor assay can be used in a similar manner as a solid-phase immunoseparation instrument to capture target organisms from complex matrices. However, unlike IMS, the biosensor assay adds a rapid detection step as well. Because organisms can be recovered after the assay with inhibitory substances removed, PCR can subsequently be run on recovered captured microorganisms. Thus, without much additional time, the biosensor assay can perform the same function as IMS but with an added preliminary detection step. We found that recovery of E. coli O157:H7 from the biosensor waveguide through culture enrichment may be somewhat slower than the other methods that were tested, but it provided the greatest sensitivity. Recovery rates for the more direct recovery methods

of boiling or glycine treatment were not as high as for enrichment. These methods were also found to inhibit the PCR reaction. In addition, whereas neither the biosensor nor PCR alone can determine viability, viability can be determined with the enrichment culture step. We found that the PCR is not sensitive enough to detect E. coli O157:H7 directly from the waveguide when present at low concentrations. Therefore, at these low initial concentrations, the PCR product will be present only when viable cells that can be enriched from the waveguide are in the sample. Culture also allows for confirmation of viability by traditional plating methods as previously demonstrated (Kramer et al., 2002). In addition, it provides a viable culture for performance of other tests such as antimicrobial susceptibility or for epidemiological and criminal investigations. The importance of a viable culture, even in the context of real time PCR, has been documented in the literature (Bell et al., 2002). PCR amplification of recovered cells in this study indicates the ability to perform additional molecular techniques after a rapid biosensor detection and recovery. In these experiments, PCR was used as a molecular confirmatory step. However, other techniques such as ribotyping, pulsed field gel electrophoresis, or sequencing could be performed to compare the sample strain to a known database to help determine the source of the organism. Thus, the results of this study indicate that it is possible to detect and identify a target microorganism such as E. coli O157:H7, recover the captured microorganism and culture it in enrichment broth, and confirm the identity of the microbe by PCR, all within 10 h.

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