Development of an immunomagnetic separation method for efficient enrichment of Escherichia coli O157:H7

Food Control 37 (2014) 41e45

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Development of an immunomagnetic separation method for efficient enrichment of Escherichia coli O157:H7 Qirong Xiong a, b, Xi Cui a, Jasdeep K. Saini c, Daofeng Liu a, Shan Shan a, Yong Jin b, Weihua Lai a, * a b c

State Key Laboratory of Food Science and Technology, Nanchang University, 235 Nanjing East Road, Nanchang 330047, China Institute of Food Safety, Chinese Academy of Inspection and Quarantine, Beijing, 100123, China Department of Animal Sciences and Industry, Kansas State University, KS 66506, USA

a r t i c l e i n f o

a b s t r a c t

Article history: Received 14 April 2013 Received in revised form 24 July 2013 Accepted 20 August 2013

Immunomagnetic separation uses antibody-coated paramagnetic particles to selectively bind and purify the target organisms from a comprehensive range of complex food matrices. Aim of this study was to develop and optimize an immunomagnetic separation method based on monoclonal antibody for efficient isolation of Escherichia coli O157:H7 in food samples. The key parameters for preparing the immunomagnetic beads; the coupling rate between monoclonal antibody and magnetic beads, additive amount of immunomagnetic beads, and magnetic separation times were optimized with different concentrations of E. coli O157:H7. Under optimized conditions, the capture efficiency (CE) was greater than 98% against 101e106 cells of E. coli O157:H7 with 0.05 mg immunomagnetic beads. The immunomagnetic beads exhibited high specific binding with E. coli O157:H7 strains (CE > 98%), and low binding with non-target bacteria (CE < 2%) except for S. aureus (CE ¼ 23.6%). The capture efficiency of immunomagnetic beads against E. coli O157:H7 in ground beef and milk samples were 94.4% and 99.8%, respectively. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: Immunomagnetic separation Escherichia coli O157:H7 Monoclonal antibody

1. Introduction Escherichia coli O157:H7 is an important serotype of Enterohemorrhagic E. coli (EHEC) that was first identified as a human pathogen in 1982 (Riley et al., 1983). The pathogen is known to cause several serious diseases that include hemorrhagic colitis, hemolytic uremic syndrome, and thrombotic thrombocytopenic purpura (Mead & Griffin, 1998). Cattle are considered to be the primary reservoirs for E. coli O157:H7. Infected cattle can carry the organism in their gastrointestinal tract without showing any symptoms of disease (Hancock et al., 1998). Outbreaks of the foodborne disease caused by E. coli O157:H7 have been reported throughout the world, most frequently in the Europe, the United States, Canada, and Japan. Based on a 1999 estimate, 73,000 cases of infection occur in the United States each year due to E. coli O157:H7 (Mead et al., 1999). Standard methods for detection of E. coli O157:H7 in food require 5e7 days. Initial cultural enrichment is required to achieve a critical threshold concentration needed for efficient detection; * Corresponding author. Tel.: þ86 791 83969526; fax: þ86 791 88157619. E-mail addresses: [email protected], [email protected] (W. Lai). 0956-7135/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodcont.2013.08.033

followed by isolation from a selective agar and a series of biochemical tests that can isolate and distinguish E. coli O157:H7 from other commensal bacteria (Doyle, 1991). Subsequent serological tests are done with anti-O157 and anti-H7 antibodies to confirm the O157:H7 serotype. This is a time-consuming and laborintensive process before definitive results are obtained. Several novel rapid methods that combine diverse capture and/or detection technologies to improve total assay time and further amplification of signals are being developed to replace the existing traditional techniques. Immunomagnetic separation (IMS) is one such a method that has been suggested to reduce the total time of analysis and improve the sensitivity of detection of pathogenic microorganisms (Weagant & Bound, 2001; Wright, Chapman, & Siddons, 1994). Superparamagnetic particles are coated with antibodies against the target pathogen, forming immunomagnetic beads (IMBs). The IMBs can bind to the target bacteria, forming a beadebacteria complex that is easily separated from food matrix and background bacteria and then concentrated into a smaller volume by exposure to a magnetic field. It can significantly shorten detection time, improve sensitivity, and eliminate interference from fat, protein, and viscosity of food matrices; thus improving foodborne pathogen

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detection strategies (Islam, Heuvelink, Talukder, Zwietering, & de Boer, 2006; Sarimehmetoglu et al., 2009; Wang, Li, Wang, & Slavik, 2011; Zhu et al., 2011). The most common magnetic carriers are Dynabeads (Dynal, Oslo, Norway), which are polystyrene-based microbeads ranging from 1 to 10 mm. However, in recent years, some reports have shown that magnetic nanobeads (30e150 nm diameter) could improve the capture efficiency (CE) of bacterial pathogens in food samples. In comparison with magnetic microbeads, magnetic nanobeads are more mobile in complex samples and have faster reaction kinetics, lower mass, higher surface-to-volume ratio, less blocking effect on optical measurement, and lower cost (Varshney, Yang, Su, & Li, 2005; Wang et al., 2011). However, smaller nanobeads contain less magnetic materials in their cores which makes it difficult to separate target bacteria rapidly from food samples (Shields et al., 2012). For this study, approximately 180 nm magnetic nanobeads were chosen to prepare the IMBs. The CE of IMBs mainly depends on the quality of the antibodies that should have high affinity with E. coli O157:H7 and no crossreactivity with other bacteria. The monoclonal antibodies (MAbs) have higher stability and specificity than polyclonal antibodies (PAbs), and the MAbs used as capture antibodies in Bacillus anthracis spores, Vibrio vulnificus, and Listeria monocytogenes separation have been reported (Jadeja, Janes, & Simonson, 2010; Shields et al., 2012; Shim et al., 2008). In this study, the MAbs specific for E. coli O157:H7 were used for capturing target bacteria by IMS. At the same time, the effect of IMBs volumes, immunoreaction time, and magnetic separation time on the immunomagnetic separation of E. coli O157:H7 were studied. 2. Materials and methods 2.1. Bacteria and culture conditions E. coli O157:H7 (American type culture collection, ATCC 43888), E. coli O157:H7 (China Medical Culture Collection, CMCC 44828), E. coli O157:H7 (National Collection of Type Cultures, NCTC 12900), E. coli O157:H7 (XY0540 from Jiang Xi Province Center for Disease Control and Prevention, China), vulgaris E. coli CMCC 44102, E. coli ATCC 25922, Enteropathogenic E. coli CMCC 44496, Enteroinvasive E. coli CMCC 44350, Salmonella typhimurium ATCC 13311, and Staphylococcus aureus CMCC 26003 were cultured in LuriaeBertani medium (LB, Oxoid, Basingstoke, UK) at 37  C for 20 h before use, L. monocytogenes CMCC 54001 were cultivated with brain heart infusion (BHI; Becton, Dickinson and Company, Sparks, MD) at 37  C for 24 h. To determine the number of viable cells, serial dilutions of cultures in phosphatebuffered saline (PBS, Sigma Chemical Company, St. Louis, MO, 0.01 M, pH 7.4) were made and plated onto trypticase soy agar (TSA; Becton, Dickinson and Company, Sparks, MD). The plates were then incubated at 37  C for 24 h. 2.2. The magnetic beads and monoclonal antibody Carboxylated magnetic beads (mean diameter of 181.4 nm, 10 mg/ml) were obtained from allrunnano technology co. Ltd. (Shanghai, China). The particles sizes distributions were determined by Nicomp 380 ZLS Particles Size Analyzer (Particle Sizing Systems, Santa Barbara, California, USA). The MAbs 10C5eH3eB6 which has high specificity and affinity against E. coli O157:H7 was prepared in our laboratory. The MAbs was produced by immunizing mice at 2-week intervals by injection of 50 mg whole-cell antigen of E. coli O157:H7 ATCC 43888. It was determined to react with the LPS of E. coli O157:H7 by Western blotting. The concentration of MAbs 10C5eH3eB6 was 6.25 mg/ml, determined

by Bicinchoninic acid (BCA, Thermo Scientific, Rockford, IL) protein assay. A 1:10 dilution of the antibodies was prepared in PBS (0.01 M, pH 7.4) before use. 2.3. Preparation of immunomagnetic beads Carboxylated magnetic beads were resuspended in 500 ml of freshly made ethyl (dimethylaminopropyl) carbodiimide (EDC)e N-hydroxysuccinimide sodium salt (NHSS) (Sigma Chemical Company, St. Louis, USA) solution (20 mg/ml EDC and 20 mg/ml NHSS in 0.01 M 2-Morpholinoethanesulfonic acid buffer, pH 6.0) and placed on a variable speed rotator (Ningbo Scientz Biotechnology company, Ningbo, China) for 15 min at 15 rpm. After incubation, EDC and NHSS solutions were removed and the magnetic beads were resuspended with 1 ml of 0.01 M borate saline buffer (pH 8.5). Purified MAbs was immediately added into the solution with thorough mixing and placed on the rotator 15 rpm at room temperature for 4 h. Antibody-conjugated magnetic beads were washed with BST (BS containing 0.05% Tween 20) three times. The Antibody-conjugated magnetic beads were blocked in 1 ml BS containing 1% BSA at room temperature for 1 h and washed four times with BST, resuspended at a final concentration of 10 mg/ml in 0.01 M PBS (pH 7.4), and stored at 4  C. 2.4. Procedure of IMS E. coli O157:H7 (ATCC 43888) was grown in LB at 37  C for 12 h and serially diluted to 104e105 CFU/ml in PBS containing 1% BSA and then mixed with immunomagnetic beads. The mixture was incubated on a rotator at 15 rpm and separated by magnet. An aliquot of 0.1 ml from supernatant was spread on sorbitol MacConkey (SMAC; Beijing Land Bridge Technology Co. Ltd., Beijing, China) agar for bacterial enumeration after appropriate dilution. The beads were washed three times with 1 ml PBST (0.01 M PBS containing 0.05% Tween 20, pH 7.4). An aliquot of 0.1 ml washing solution was spread on SMAC agar after appropriate dilution. Triplets of each sample were plated. All the SMAC agar plates were incubated at 37  C for 18e20 h for bacterial enumeration. 2.5. CE calculations CE defined as the percentage fraction of the total bacteria retained on the surface of the IMBs, was calculated using a method that is based on the cells unbound to IMBs or left in the supernatant. The following equation was used for calculating CE:

CEð%Þ ¼ ð1  B=AÞ  100%

(1)

where A is the total number of cells presents in the sample (CFU/ml) and B is the number of cells unbound to IMBs (CFU/ml, in supernatant and washed solution). 2.6. Key parameters of the immunomagnetic separation The capture kinetics and capacity of the IMBs against E. coli O157:H7 were studied for five coupling rates between MAbs and magnetic beads (10, 25, 50, 75, and 100 mg/mg), six volumes of IMBs (0.005, 0.01, 0.02, 0.05, 0.1, and 0.2 mg), five immunoreaction times (5, 15, 30, 45, and 60 min), five magnetic separation times (0.5, 1, 2, 3, and 5 min) and a series of concentrations (101e108 CFU/ml). CE was equal to the number of E. coli O157:H7 isolated divided by the number of E. coli O157:H7 present in the sample.

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2.7. Specificity test Four different E. coli O157:H7 strains (CMCC 44828, ATCC 43888, NCTC 12900, XY 0540) along with 7 nontarget bacteriadE. coli (CMCC 44102, ATCC 25922), Enteropathogenic E. coli (CMCC 44496), Enteroinvasive E. coli (CMCC 44350), S. Typhimurium (ATCC 13311), L. monocytogenes (CMCC 54001), S. aureus (CMCC 26003) were cultured in LB at 37  C for 12 h, and serially diluted to approx. 106 CFU/ml in PBS containing 1% BSA. Binding of IMBs against target and non-target bacteria was tested by the surface plating method as described above. E. coli O157:H7 was plated onto SMAC agar plates, other bacteria were plated onto TSA plates. 2.8. Food sample preparation and enrichment Ground beef and whole milk (3% milk fat) were purchased from a local grocery store and used for the food spiking experiments E. coli O157:H7 cultures were serially diluted to 102 CFU/ml in PBS containing 1% BSA. Twenty five gram of ground beef was mixed with 225 ml of modified EC broth (EC broth contain 0.002% novobiocin) was stomached for (Seward 400 Stomacher, Norfolk, UK) for 2 min. Twenty five milliliter of whole milk was mixed with 225 ml of modified EC broth in a flask. Both food samples were inoculated with decimally diluted cultures of E. coli O157:H7 to final inoculation concentrations of 100e101 CFU/ml, followed by enrichment of 6 h at 37  C, with shaking at 160 rpm. After enrichment, 1 ml of the sample was obtained and mixed with 0.05 mg IMBs and captured according to the method described above. Bacterial recovery was evaluated by plating captured beads onto SMAC agar supplemented with cefixime (0.05 mg/l) and tellurite (2.5 mg/l) (CT-SMAC; Beijing Land Bridge Technology Co. Limited, Beijing, China). The postenrichment blank was plated onto both TSA plates and CT-SMAC agar plates to enumerate background microflora and ensure no indigenous E. coli O157:H7 in the ground beef. The commercial Dynabeads anti-E. coli O157 (Invitrogen Dynal AS, Oslo, Norway) were also used in this study according to the manufacturer’s instructions. Three replications of the study were performed in triplicate.

Fig. 1. Effect of immunoreaction time on the capture efficiency against 8.04  104 CFU/ ml E. coli O157:H7 at room temperature.

CE for E. coli O157:H7 was 99.4% when cell concentrations were approx. 105 CFU/ml. Higher MAb concentrations did not alter the bacterial capture efficiencies of IMBs. 3.2. Optimization of IMS method Table 1 showed the effect of volumes of IMBs on capture efficiencies. The CE was 99.2% when 0.05 mg of IMBs was added to 1 ml of bacterial suspensions (1  105 CFU/ml in PBS). For overall economics and efficiency, 0.05 mg of IMBs was selected as the optimal volume in the IMS system. The effect of immunoreaction time on the magnetic separation of E. coli O157:H7 at room temperature is shown in Fig. 1. The CE increased from 62.7% to 98.7% with the immunoreaction time from 5 min to 30 min. The magnetic separation time of E. coli O157:H7 was examined from 8.33  104 CFU/ml samples at room temperature. The CE arrived at 98.1% as magnetic separation time was 2 min (Fig. 2).

2.9. Statistical analysis 3.3. Characterization of IMS system The statistical analysis of the capture efficiency was performed with the Statistical Analytical Software (Systat SigmaPlot 10, Systat Software, Inc., CA). 3. Results and discussion 3.1. Preparation of IMBs Optimal coupling rate between monoclonal antibody and magnetic beads was determined by conducting some preliminary studies in which IMBs were prepared with different concentrations of MAb from 10 to 100 mg per 1 mg MBs. The IMBs prepared by 50 mg IgG per 1 mg magnetic beads were optimum. It showed that

Fig. 3 showed the relationship between the bacterial concentration and the CE. When the concentration of E. coli O157:H7 was lower than 106 CFU/ml, the CE was greater than 98%. The CE decreased from 97.3% to 46.7% as the concentration of E. coli O157:H7 increased from 8.93  106 CFU/ml to 8.13  107 CFU/ml. It indicated that there were a finite number of cells that could be captured by the IMBs. For evaluating the specificity, 4 strains of E. coli O157:H7 and 7 nontarget bacteria were tested. As shown in Fig. 4, all E. coli O157 (approx. 105 CFU/ml) could be recovered by the IMS technique (CE > 98%), while the CE of non-target bacteria (approx. 106 CFU/ ml) was lower than 2% except for S. aureus (23.6%). The specificity of

Table 1 Effect of different volume of immunomagnetic beads on the capture efficiency of the IMBs against E. coli O157:H7. Volumes of IMBs (mg) 0.005 0.01 0.02 0.05 0.1 0.2

E. coli O157:H7concentration(CFU/ml) (8.70 (8.70 (8.70 (8.70 (8.70 (8.70

     

1.21) 1.21) 1.21) 1.21) 1.21) 1.21)

     

4

10 104 104 104 104 104

Immunoreaction times (min)

Magnetic separation times (min)

CE(%), n ¼ 3

45 45 45 45 45 45

3 3 3 3 3 3

42.15 86.74 94.94 99.17 99.40 99.13

     

6.74 1.27 1.60 0.21 0.52 0.43

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Fig. 2. Effect of magnetic separation times on the capture efficiency against 8.33  104 CFU/m E. coli O157:H7.

IMS system mainly depends on the antibody coated on the magnetic beads. The high binding with S. aureus should be explained by the fact that the cell wall of S. aureus contains a covalently bound protein called protein A that binds the Fc region of IgG of most mammalian species with high affinity. In our experiments, E. coli O157:H7 inoculated at 1001 CFU/ml reached approx. 106 CFU/ml after a 6 h enrichment step in ground beef and whole milk. The CE of IMBs for E. coli O157:H7 in ground beef and milk samples were 94.4% and 99.8%, respectively, and the CE of Dynabeads anti-O157 in the same samples were 72.8% and 95.8%. Many researchers have suggested that composition of the food matrix has a great influence on the IMS (Fu, Rogelj, & Kieft, 2005). A previous study reported a decrease in the capture of E. coli O157:H7 in beef samples due to the presence of fat, tissue, and other organic materials in the samples (Varshney et al., 2005). However, in our study, the CE of the IMBs coated with MAb 10C5eH3eB6 did not significantly reduce in the ground beef (94.4%) and whole milk samples (99.8%). This can be explained by the high affinity of MAb

Fig. 4. The specificity of IMBs with selected bacterial strains.

10C5eH3eB6 for E. coli O157:H7. The higher CE of the IMBs coated with MAb 10C5eH3eB6 than Dynabeads anti-O157 may be due to the stronger mobility in complex samples and faster reaction kinetics of the IMBs (180 nm) in comparison to Dynabeads anti-O157 (2.8 mm). 4. Conclusion IMBs coated with MAb 10C5eH3eB6 could be used for the rapid separation of E. coli O157:H7 in a wide range of concentrations with high specificity. Different conditions, such as volumes of IMBs, immunoreaction times and magnetic separation times, were optimized in order to increasing CE of IMBs. In comparison to the Dynabeads anti-O157, the IMBs studied in our laboratory showed higher CE in ground beef and whole milk samples. Acknowledgments We are grateful to the Research Program of State Key Laboratory of Food Science and Technology, Nanchang University (Project No. SKLF-ZZB-201307) and the Nanchang Technological Program (2012-CYH-DW-SP-001) for financial support. References

Fig. 3. The capture efficiency of the 0.05 mg IMBs against 1 ml 5.7  101, 5.6  102, 3.5  103, 7.6  104, 9.3  105, 8.9  106, 8.1  107 and 6.6  108 CFU/ml of E. coli O157:H7 for 30 min of immunoreaction time.

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