In the present study, seven Cronobacter strains were used to produce antibody and to assess the cross-reactivity of developed method, while eight other bacterial strains belonging to different genera were selected to check the cross-reactivity of developed assay due to their partial gene sequence similarity and to confirm the accurate detection of pathogen contaminants such as Salmonella spp., Citrobacter spp., and Bacillus cereus (Iversen et al., 2004; World Health Organization [WHO], 2007; Pinto et al., 2013). In the present study, B. cereus and S. Enteritidis were selected since it was found that the chances of contamination in PIF were highly concerned due to the presence of B. cereus and S. Enteritidis (Giammanco et al., 2011; Pinto et al., 2013). Giammanco et al. (2011) also reported that Salmonella spp. and Cronobacter spp. (formerly E. sakazakii) are the microorganisms of greatest concern in PIF. In addition, regarding the use of Citrobacter freundii, Giammanco et al. (2011) observed that molecular epidemiological survey of C. freundii was misidentified as Cronobacter spp. isolated from PIF. The phylogenetic tree also showed that C. freundii and Cronobacter spp. are very closely related species based on their smaller amounts of mahalanobis distances than other species. Hence, it was suggested that C. freundii may also be an under-reported cause of bacterial infection, especially in high risk neonates, due to misidentification. B. cereus (KCCM 40935), Buttiauxella noackiae (ATCC 51713), C. condimenti (LMG 26250), C. dublinensis (LMG 23823), C. malonaticus (LMG 23826), C. muytjensii (CDC 3523-75), C. turicensis (LMG 23827), C. muytjensii (ATCC 51329), C. sakazakii (ATCC 29544), C. sakazakii (ATCC 29004), C. universalis (LMG 26249), C. freundi (ATCC 8090), Escherichia coli (ATCC 39418), Franconibacter helveticus (LMG 23732), Franconibacter pulveris (LMG 24057), and Salmonella Typhimurium (ATCC 13311) were used in this study. C. muytjensii (CDC 3523–75) was donated by Dr. Carol Iversen from University College Dublin, Ireland. Other strains used in this study were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and Korean Culture Center of Microorganisms (KCCM; Seoul, Korea). Strains indicated with LMG were purchased from the Belgian Coordinated Collections of Microorganisms (BCCM; Gent, Belgium). All strains were cultured in nutrient broth (NB) for 18 h at 37°C in a shaking incubator (150 rpm).

NB, nutrient agar (NA), peptone, and skim milk were purchased from Difco (Franklin Lakes, NJ, USA). Enterobacteriaceae enrichment (EE) broth and violet red bile glucose (VRBG) agar were purchased from MB cell (Seoul, Korea). Sodium carbonate, sodium bicarbonate, sodium azide, potassium phosphate monobasic, potassium phosphate dibasic, sodium chloride, caprylic acid, ammonium sulfate, and alkaline phosphatase yellow liquid substrate p-nitrophenyl phosphate (pNPP) were purchased from Sigma (St. Louis, MO, USA). Phosphatase-labeled goat anti-rabbit IgG was purchased from Kierkegaard & Perry Laboratories, Inc. (Gaithersburg, MD, USA). Lipopolysaccharide (LPS) extraction kit was purchased from Intron Biotechnology (Seongnam, Korea).

Animal use protocol was reviewed by the committee members of Yeungnam University and approved by Korea Food and Drug Administration, Republic of Korea (Animal Ethics License No. 2013-012 and 2012-010).

Three types of immunogens were prepared for the development of a genus-specific antibody against Cronobacter spp. A sonicated cell protein (SCP) mixture of seven Cronobacter spp. was used in this study. The seven Cronobacter spp. were separately cultured in NB at 37°C for 18 h to a concentration of 1 × 10⁹ CFU/mL. Each culture (30 mL) was centrifuged at 3,000 × g at 4°C for 30 min. The pellets were washed three times with 30 mL of 0.01 M phosphate-buffered saline (PBS) and then suspended in 10 mL of 0.01 M PBS for sonication under 20 KHz power for 5 min on ice. Supernatant was collected after centrifugation at 12,000 × g, 4°C for 20 min. Protein concentrations of supernatants were checked with a Bradford kit, after which SCP solutions were adjusted to a concentration of 0.5 mg/L with 0.01 M PBS. SCP solutions were stored at -20°C before use. For immunization, equal volumes of the seven SCP solutions were mixed for use as an immunogen in New Zealand white rabbits.

LPS mixture of the seven Cronobacter spp. was prepared using an LPS extraction kit following the instructions of the manufacturer. Each Cronobacter spp. culture was centrifuged at 10,000 × g at room temperature for 20 min to harvest bacterial cells. Lysis buffer was then added and vortexed vigorously, followed by addition of chloroform, vortexing for 20 s, and incubated at room temperature for 5 min. The mixture was then centrifuged at 10,000 × g for 10 min at 4°C. An aliquot of supernatant was transferred to a new tube, mixed well with purification buffer, and kept at -20°C for 10 min. The solution was then centrifuged at 10,000 × g for 15 min at 4°C, after which the upper layer was removed to obtain an LPS pellet. The pellet was washed three times with 1 mL of ethanol, centrifuged for 3 min at 10,000 × g at 4°C, and then collected and dried at room temperature. For the LPS solution, 50 μL of Tris-HCl buffer (10 mM, pH 8.0) was used to dissolve the LPS pellet. LPS solution of the seven Cronobacter spp. was prepared by mixing an equal volume of each LPS solution for use as an immunogen in New Zealand white rabbits.

Formalin-killed cell (FKC) mixture was also prepared as an immunogen. Cultures of the seven Cronobacter spp. were applied as treatments with 0.5% formalin for 24 h, followed by centrifugation (3,000 × g, 4°C, for 30 min) according to the method of Song and Kim (2013). First injection was administered with a mixture of immunogen and Freund’s complete adjuvant (1:1, v/v). Second and third injections were administered using the same mixture of immunogen and Freund’s incomplete adjuvant (1:1, v/v) at 4 weeks after the first injection. Immunogen was injected into the back of rabbits at four sites at a concentration of 0.25 mL/site. Blood samples were taken every week until 18 weeks after the first injection.

Blood samples were centrifuged (10,000 × g) for 30 min at 4°C to separate the anti-sera. Anti-Cronobacter IgG was purified from the anti-sera by caprylic acid and ammonium sulfate precipitation as described by McKinney and Parkinson (1987) and according to further modified methods of Shukla et al. (2012) and Song and Kim (2013).

The purity of various anti-Cronobacter IgGs preparations was checked using SDS-PAGE (Atto, Tokyo, Japan) under reduced conditions as described by Park et al. (2012) with some modifications.

Rabbit anti-Cronobacter IgG was used to develop an INC-ELISA method for detection of Cronobacter spp. Standard response curve of the seven Cronobacter spp. was constructed using the following procedure. First, fresh cultures of all tested Cronobacter spp. were diluted decimally with 0.05 M carbonate buffer (pH 9.5) in order to coat 96-well plates (SPL Life Sciences, Gyeonggi-do, Korea), whereas only carbonate buffer was used as a negative control. Simultaneously, coated cultures of Cronobacter spp. were counted on NA and VRBG agar plates for determination of concentration. After coating at 4°C overnight, 96-well plates were washed three times with 0.01 M PBS (pH 7.0) and blocked with 200 μL of 5% skim milk at 37°C for 2 h. Ninety six-well plates were then washed with 0.01 mol/L PBS-0.05% Tween 20 (PBST), added with rabbit anti-Cronobacter IgG, and incubated at 37°C for 1 h. Then, plates were washed again with 0.01 M PBST, after which 100 μL of phosphatase-labeled goat anti-rabbit IgG was added and the plates incubated at 37°C for 1 h. Plates were washed again with 0.01 M PBST, after which 50 μL of pNPP liquid substrate was added to each well for a 30 min enzyme-substrate reaction. Finally, 50 μL of 0.01 M NaOH solution was added to stop the reaction, after which the final reaction mixture was analyzed using an Infinite M200 (Tecan; Seestrasse, Switzerland). A detection limit of the developed INC-ELISA was determined by using a data linearity function and standard deviation of responses for the calibration standard and samples, respectively (Hubaux and Vos, 1970).

Seven common Cronobacter foodborne pathogens (Table 1) were used for the specificity test of the developed INC-ELISA. Cultures of tested foodborne pathogens were serially diluted to final concentrations of 10⁰ to 10⁸ CFU/mL and then used to check sensitivity of the developed INC-ELISA. All experiments were repeated three times.

Firstly, the seven Cronobacter strains were separately cultured in NB at 37°C for 20 h, after which the cultures were serially diluted with BPW to obtain the lowest final concentration (<10 CFU/mL) of Cronobacter live cells for use in further experiments. To examine the applicability of the developed anti-Cronobacter IgG against each Cronobacter strain, 25 g of PIF sample was aseptically placed into a flask, mixed with 225 mL of BPW as a pre-enriched broth, and separately spiked with 1 mL (less than 10 cells/25 g of PIF) of each Cronobacter species, followed by pre-enrichment incubation at 37°C for 8 h. Following this, 1 mL of the pre-enrichment culture was inoculated into EE broth and incubated for 8 h of enrichment (Korea Food and Drug Administration [KFDA], 2014). Further, enriched aliquots of pre-treated food samples were tested using the developed INC-ELISA and standard USFDA methods. For control, non-spiked PIF samples were analyzed using the developed INC-ELISA and standard microbiological USFDA methods (United States Food and Drug Administration [USFDA], 2002). All experiments were performed in three replicates in order to maintain reliability of the work.

The detection limit was calculated as the average value of absorbance at zero concentration with three standard deviations (Park et al., 2012). All experiments and results were analyzed in at least three trials for proving reliable and reproducible data.

As shown in Figure 1, anti-serum containing anti-Cronobacter IgG developed by the LPS mixture showed no titer for any Cronobacter species. Figure 1 provides the fact that LPS antigen of Cronobacter is not appropriate for the production of anti-Cronobacter antibody. For antibodies developed using FKC and SCP mixtures of Cronobacter spp., titers of the developed rabbit anti-Cronobacter serum containing IgG were determined by INC-ELISA after 18 weeks of immunization, as shown in Figures 2 and 3. Titers of the developed anti-Cronobacter seru containing IgG increased after the first injection as well as gradually increased after the second and third injections. Titers of anti-Cronobacter serum containing IgG developed from the FKC mixture of Cronobacter spp. (Figure 2) were in the following order: C. muytjensii, C. turicensis, C. malonaticus, C. condimenti, C. sakazakii, C. dublinensis, and C. universalis. Comparatively, anti-Cronobacter serum containing IgG developed from an SCP mixture of the seven Cronobacter spp. showed higher titers against the seven Cronobacter spp. (Figure 3), which were in the following order: C. muytjensii, C. condimenti, C. turicensis, C. sakazakii, C. malonaticus, C. dublinensis, and C. universalis. It is well known that an increased titer of developed antibody results in effective performance during immunoassay. The developed and purified anti-Cronobacter IgG showed very high purity as compared to the commercial rabbit IgG (Figure 4). Developed antibody as well as commercial rabbit IgG (lanes 2, 3, 4, and 5) showed a strong band around 51 kDa and a light band around 25 kDa, respectively. These results confirm that our developed rabbit anti-Cronobacter IgG with high purity is comparable to commercial rabbit IgG.

An INC-ELISA method was developed to detect the seven Cronobacter spp. using rabbit anti-Cronobacter IgG developed from the aforementioned SCP mixture. The developed INC-ELISA method showed excellent sensitivity toward Cronobacter spp., including C. condimenti, C. dublinensis, C. malonaticus, C. muytjensii, C. sakazakii, C. turicensis, and C. universalis, confirming its ability to detect all seven Cronobacter spp. in a single test (Table 1). Furthermore, the developed INC-ELISA was highly sensitive to all seven different Cronobacter spp. with a detection limit ranging from (5.6 ± 0.30) × 10³ to (2.1 ± 0.01) × 10⁵ CFU/mL (Table 1).

According to Table 2, the developed INC-ELISA showed no cross-reactivity with any of the other tested bacterial genera. The recognition difference between targeted bacteria (positive value; P value) and non-targeted bacteria (negative value; N value) is an important parameter affecting the results of the immunoassay. In the present study, non-targeted bacteria (non-Cronobacter) showed a P/N value of <2 (Table 2), which is considered as a negative result, whereas targeted strains showed a P/N value of >2, which is considered as a positive result (Table 1). In support of this, the P/N value for F. pulveris was around 1.942 (Table 2) where a P/N value >2 is considered as a positive result. Compared to several other methods, our newly developed INC-ELISA based on rabbit anti-Cronobacter IgG is less expensive and easier to perform since it does not require expensive reagents or probes and has an assay time of only 36 h comprising 8 h of pre-enrichment, 8 h of enrichment, and 20 h of INC-ELISA. During assay development, accuracy of the rapid detection method was confirmed by standard plate colony counting techniques. Moreover, the developed rapid assay is accurate enough to avoid repeated colony counting steps to save detection time.

To detect small amounts of Cronobacter spp. cells using the developed method, PIF samples were separately spiked with each of the seven Cronobacter spp. at low concentrations (<10 cells/25 g). All PIF samples spiked separately with C. condimenti, C. dublinesis, C. malonaticus, C. muytjensii, C. sakazakii, C. turicensis, and C. universalis showed the presence of Cronobacter spp. in both the INC-ELISA and USFDA/KFDA methods, whereas unspiked samples showed the absence of Cronobacter spp. Specific enrichment was carried out in EE broth as per KFDA standard methods (Korea Food and Drug Administration [KFDA], 2014). In the present study, the detection limit of the developed INC-ELISA against Cronobacter spp. was found to be <10 cells/25 g of PIF after 16 h of enrichment (Table 3).

Further, to confirm the results obtained using the developed antibody and INC-ELISA for Cronobacter detection, conventional standard plate count technique using the spreading method on NA and VRBG agar plates was carried out. The PIF samples artificially contaminated with each Cronobacter spp. separately (<10 cells/25 g) after 8 h of pre-enrichment and 8 h of enrichment in EE broth showed similar bacterial counts for each Cronobacter spp. in the range of 1.1 × 10⁵ – 2.9 × 10⁵ CFU/mL on specific VRBG agar plates (Table 3). These results can give an idea to reconfirm the detection limit obtained in pure culture of Cronobacter spp. using the developed antibody and INC-ELISA assay (Table 1).