Strategy for Accurate Detection of Escherichia coli O157:H7 in Ground Pork Using a Lateral Flow Immunoassay

Escherichia coli O157:H7 is known to cause serious diseases including hemorrhagic colitis and hemolytic uremic syndrome. A gold nanoparticle lateral flow immunoassay (Au-LFIA) was used to detect Escherichia coli O157:H7 in ground pork samples. False-positive results were detected using Au-LFIA; a Citrobacter freundii strain was isolated from the ground pork samples and identified by using CHROmagarTM plates, API 20E, and 16S RNA sequencing. Since C. freundii showed cross-reactivity with E. coli O157:H7 when Au-LFIA test strips were used, a novel method combining modified enrichment with a lateral flow immunoassay for accurate and convenient detection of E. coli O157:H7 in ground pork was developed in this study to minimize these false positives. MacConkey broth was optimized for E. coli O157:H7 enrichment and C. freundii inhibition by the addition of 5 mg/L potassium tellurite and 0.10 mg/L cefixime. Using the proposed modified enrichment procedure, the false-positive rate of ground pork samples spiked with 100 CFU/g C. freundii decreased to 5%.


Introduction
Escherichia coli O157:H7 is a dangerous foodborne pathogen because of its low infectious dose (minimum 10 cells) and high pathogenicity (diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome) [1]. The "gold standard" for detecting E. coli O157:H7 in food samples is a traditional separation-identification method which is time-consuming (at least five days) and laborious (involves pre-enrichment, selective enrichment, culture isolation, and identification). Other methods, including the polymerase chain reaction (PCR) [2,3] and enzyme-linked immunosorbent assay (ELISA) [4,5], also may require laborious procedures and expensive instruments.
Lateral flow immunoassay (LFIA), which is widely used in the field of food safety, presents a number of advantages, including ease of use, rapidity, and sensitivity [6]. However, LFIA-based methods have the disadvantage that the antibody used in the immunoassay can show cross-reactivity with bacteria other than the target [7,8].
A gold nanoparticle LFIA (Au-LFIA) method was previously optimized for detecting Escherichia coli O157:H7 with high sensitivity [9][10][11][12][13][14][15]. Detection of E. coli O157:H7 in uninoculated ground pork samples using Au-LFIA test strips prepared in our laboratory in combination with the enrichment in modified E.Coli (EC) broth yielded positive results. In this study, the reasons behind the false-positive results obtained when Au-LFIA test strips are used to detect E. coli O157:H7 in ground pork were determined. samples using Au-LFIA test strips prepared in our laboratory in combination with the enrichment in modified E.Coli (EC) broth yielded positive results. In this study, the reasons behind the false-positive results obtained when Au-LFIA test strips are used to detect E. coli O157:H7 in ground pork were determined. A novel method combining modified enrichment with Au-LFIA for accurate detection of E. coli O157:H7 in ground pork was developed to minimize false positives ( Figure 1).

Figure 1.
Overall process of the immunochromatographic assay for detecting E. coli O157:H7.

Preparation of Au-LFIA Test Strips
Au-LFIA test strips to detect E. coli O157:H7 were prepared in laboratory as described previously [14]. The test strip consisted of sample pad, conjugate pad, nitrocellulose membrane (NC), and absorbent pad. Anti-E. coli O157:H7 polyclonal antibody was applied to the nitrocellulose membrane as the test line. Goat anti-mouse antibody was applied to the nitrocellulose membrane as the control lines. Anti-E. coli O157:H7 monoclonal antibody-AuNPs complex was applied to a conjugate pad.

Preparation of Au-LFIA Test Strips
Au-LFIA test strips to detect E. coli O157:H7 were prepared in laboratory as described previously [14]. The test strip consisted of sample pad, conjugate pad, nitrocellulose membrane (NC), and absorbent pad. Anti-E. coli O157:H7 polyclonal antibody was applied to the nitrocellulose membrane as the test line. Goat anti-mouse antibody was applied to the nitrocellulose membrane as the control lines. Anti-E. coli O157:H7 monoclonal antibody-AuNPs complex was applied to a conjugate pad.

Pretreatment of Ground Pork Samples
Twenty ground pork samples were tested negative for E. coli O157:H7. Then 25 g of these ground pork samples was transferred into a stomacher bag as negative sample. Another 25 g of these ground pork samples was transferred into a stomacher bag and inoculated 2 CFU/g E. coli O157:H7 as positive samples.

Enrichment with Modified EC Broth and Evaluation by Using Au-LFIA Test Strip
Twenty negative samples and 20 positive samples (25 g) were mixed with 225 mL of modified EC broth containing 20 mg/L of novobiocin respectively, and stomached (Seward 400 Stomacher, Norfolk, UK) for 2 min. All of the samples were incubated at 37 • C with shaking at 160 r/min for 12 h. One hundred microliters of the 40 enriched broths of the ground pork samples was respectively added to the sample pad of Au-LFIA test strips for detection. When the target analyte (E. coli O157:H7) was added onto the sample pad of the test strip, it flowed laterally through the test strip. The E. coli O157:H7 interacted with the antibody-AuNPs complex in the conjugate pad, and aggregated subsequently at the T line because of the specific interaction between the E. coli O157:H7 and the antibody, which leads to a red color of the T line. Two visual bands (control line and test line) indicated a positive result. One visual band (control line) indicated a negative test result. The colored gold-antibody conjugate should bind to the control line and form a red-colored band regardless of the presence of E. coli O157:H7. The signal intensity of the test line can be detected by colloidal gold test strip reader. When the sample is positive, the signal intensity of the test line is equal or greater than 30.

Identification of the False-Positive Bacterium
CHROmagar TM O157 agar without potassium tellurite and cefixime was used after ground pork sample enrichment. The enrichment broth was diluted 1:100,000 with sterile phosphate buffer, and the diluted broth (100 µL) was streaked onto CHROmagar TM O157 agar. After incubation at 37 • C for 24 h, bacteria with different colors and colony morphology were inoculated into LB medium for enrichment and identified through API 20E and 16S RNA sequencing.

Cross Activity of C. freundii with Au-LFIA Strip
The isolated C. freundii strain was identified, inoculated into 250 mL of modified EC broth (10 CFU/mL), and incubated at 37 • C with shaking at 160 r/min for 12 h. The sample of the enrichment broth (100 µL) was added to the sample pad of Au-LFIA test strips. Three experiments were repeated.

Optimization of Enrichment Conditions
CT-SMAC culture medium with potassium tellurite and cefixime was optimized by adjusting the concentrations of potassium tellurite (2.50 mg/L, 3.75 mg/L, and 5.00 mg/L) and cefixime (0.05 mg/L, 0.075 mg/L, and 0.10 mg/L). Two portions of the ground pork samples that had been tested to be negative for E. coli O157:H7 and C. freundii were spiked with E. coli O157:H7 (2 CFU/g) and C. freundii which was identified in 2.6 (100 CFU/g). Two hundred and twenty-five milliliters of CT-SMAC with potassium tellurite and cefixime was mixed with 25 g of the ground pork samples and stomached for 2 min. All of the test samples were incubated at 37 • C with shaking at 160 r/min for 12 h.

Evaluation of the Optimized Enrichment by Using Au-LFIA Test Strip
Twenty negative ground pork samples were spiked with 100 CFU/g C. freundii as negative samples, while another 20 negative ground pork samples were spiked with 2 CFU/g E. coli O157:H7 as positive samples. Twenty-five grams of these 40 samples were respectively mixed with 225 mL of the optimized CT-SMAC culture medium and stomached for 2 min. All of the samples were incubated at 37 • C with shaking at 160 r/min for 12 h. One hundred microliters of the 40 enrichment broth of the ground pork samples were then respectively added to the sample pad of Au-LFIA test strips for detection.

Specificity of the Au-LFIA
The results showed that the five strains of E. coli O157:H7 were detected successfully, and no cross-reaction with the other 40 strains was observed (Table 1).  Table 2).

False-Positive Result of Au-LFIA Test Strip
The results obtained from Au-LFIA test strips indicated that some bacterial species in the ground pork samples exhibit cross-reactivity with E. coli O157:H7 ( Figure S1). All 20 negative samples from the slaughterhouse showed false positives, whereas only eight of the 20 negative samples from the supermarket revealed false positives. Table 2. Results of ground pork samples detected using Au-LFIA test strips.

Samples
Negative

Isolation and Identification of C. freundii from Incubated Broth
The diluted modified EC broth (100 µL) was streaked onto CHROmagar TM O157 agar. After incubation at 37 • C for 24 h, colonies with different colors and colony morphologies were produced. E. coli O157:H7 appeared mauve in CHROmagar TM plates ( Figure S2A), while some non-O157 bacteria showed other colors. The bacteria in 14 negative samples with false-positive Au-LFIA results were isolated using the CHROmagar TM plate. These bacteria exhibited cross-reactivity with E. coli O157:H7 when the Au-LFIA test strips were used for detection and appeared blue on the CHROmagar TM plate ( Figure S2B). The bacteria producing false positives were identified by API 20E (Figure S3), and one bacterium, namely C. freundii, was identified through 16S RNA sequencing (Supplementary Material 4). The nucleotide sequence of C. freundii was identified with 99.9% accuracy by NCBI Blast, and the results obtained agreed with the API 20E findings.

Cross-Reactivity of C. freundii with Au-LFIA Strip
The positive results indicated that the Au-LFIA test strips for detecting E. coli O157:H7 exhibited cross-reactivity with C. freundii.

Optimization of the Modified Culture Medium for C. freundii Inhibition
A series of various potassium tellurite and cefixime concentrations in CT-SMAC were studied to demonstrate the inhibition of C. freundii (Figure 2). When the potassium tellurite and cefixime concentrations in CT-SMAC were 5 and 0.10 mg/L, respectively, C. freundii did not multiply and E. coli O157:H7 was enriched. One hundred microliters of the enrichment broth of the ground pork samples was added to the sample pad of Au-LFIA test strips prepared in our laboratory. The signal intensities of the test lines of the strips with ground pork broth samples spiked with E. coli O157:H7 and C. freundii were 175 and 0, respectively.
The positive results indicated that the Au-LFIA test strips for detecting E. coli O157:H7 exhibited cross-reactivity with C. freundii.

Optimization of the Modified Culture Medium for C. freundii Inhibition
A series of various potassium tellurite and cefixime concentrations in CT-SMAC were studied to demonstrate the inhibition of C. freundii (Figure 2). When the potassium tellurite and cefixime concentrations in CT-SMAC were 5 and 0.10 mg/L, respectively, C. freundii did not multiply and E. coli O157:H7 was enriched. One hundred microliters of the enrichment broth of the ground pork samples was added to the sample pad of Au-LFIA test strips prepared in our laboratory. The signal intensities of the test lines of the strips with ground pork broth samples spiked with E. coli O157:H7 and C. freundii were 175 and 0, respectively.

Evaluation of Modified CT-SMAC in Ground Pork Test
Twenty ground pork samples that were negative for E. coli O157:H7 and C. freundii were spiked with 100 CFU/g C. freundii as negative controls and 2 CFU/g E. coli O157:H7 as positive controls. After enrichment by the developed culture medium, 100 µL of the enrichment broth of the ground pork samples was detected using Au-LFIA test strips, as well as CHROmagar TM plates. Results (Table 3) indicated that the modified CT-SMAC was suitable for E. coli O157:H7 enrichment in ground pork samples. Using this enrichment procedure, ground pork samples spiked with 2 CFU/g E. coli O157:H7 showed 100% positive results and only a 5% false-positive result from C. freundii.
Previous studies had shown that some strains of C. freundii presented cross-reactivity with anti-O157 sera [16,17]. With the developed enrichment procedure, Au-LFIA had good specificity. Bennett and Zadik have also obtained a good specificity result with potassium tellurite and cefixime [18,19]. Some scholars also acquired sensitive and specific results based on lectin recognition of E. coli O157:H7 [20]. Table 3. Evaluation of modified CT-SMAC in ground pork samples.

Conclusions
In this study, E. coli O157:H7 in ground pork samples was detected by using Au-LFIA test strips. A large number of false-positive results were obtained. The C. freundii strain was isolated and identified from the ground pork samples and determined to induce these false positives. A modified enrichment procedure by the addition of 5 mg/L potassium tellurite and 0.10 mg/L cefixime was evaluated for the enrichment of E. coli O157:H7 and the inhibition of C. freundii. Combining the modified enrichment