Salmonella enterica Typhimurium (ATCC 13311) was used in this study. Salmonella Typhimurium was grown from a single colony in Lennox Broth (LB broth) overnight in a shaking incubator at 37˚C, at a speed of 200 rpm. Overnight bacterial cultures were centrifuged at 5500 rpm for 10 min at 4˚C and re-suspended in PBS twice. The bacterial populations were then adjusted to an OD of 600_nm, which equates to 1.0 in PBS. Salmonella suspensions were then further diluted to 10³ cfu/mL.

Packages of triple washed spinach leaves were purchased from local supermarkets in Auburn, AL. The leaves, with a total weight of 125 grams, were collected and rinsed with filtered water in a sterilized beaker. Under a biosafety cabinet, the leaves were sprayed with 70% EtOH and air dried under the cabinet. Each batch of spinach leaves (now 25 grams/ batch) was sterilely picked up, placed inside a sterile plastic tote bag, sprayed with a fresh Salmonella Typhimurium suspension (total 100 cfu) with a sterile adjustable sprayer (Spray Anywhere, Fisher Scientific, Pittsburgh, PA.) The bag of leafy greens (25 grams) with the final Salmonella suspension of 4 cfu/g was then mixed manually for 10 min to be homogenized in the biosafety cabinet.

Three different pre-enrichment broths were used in spinach study. They are lactose broth, LB, and MPW. Each broth (50 ml), was added into the bag and mixed well. The sterile bag was folded loosely to ensure oxygen aeration for Salmonella growth. The pre-enrichment solution was held in room temperature for 1 hour to stabilize the pH and then incubated at 37˚C for 5, 7, and 22 hours. At each incubation time, the pre-enrichment solution was centrifuged at 10,000 rpm for 10 minutes twice. The centrifuged pellet was re-suspended to a 1.0 mL final volume using filter sterilized water.

Boxes of Campari tomatoes were also purchased from local supermarkets in Auburn, AL. Two Campari tomatoes were collected as a group. Each group of tomatoes was then weighted, rinsed, and cleaned with 70% EtOH as previously described. Tomatoes were placed in a sterile plastic bag and sprayed with Salmonella Typhimurium suspension at (4 cfu/g) using a sterile adjustable Sprayer. After the spiking of Salmonella on tomatoes, the three pre-enrichment broths were used. They are UP broth, LB broth, and MPW broth. Each broth, at a ratio of 1:2 (tomatoes: broth, w/v), was added into the bag with contaminated tomatoes and mixed well. The sterile bag was folded loosely to ensure oxygen aeration for Salmonella growth, and the rest of pre-enrichment steps were performed the same as in the previous paragraph.

Magnetoelastic (ME) biosensors were fabricated by a commercially available Metaglas 2826MB ribbon (Metaglas, Inc). Further detailed descriptions of sensor fabrication, dicing, and final treatment processes are included in Horikawa et al., 2015 [16] . The ME sensors used in this study had a final layer coated with Au (gold), and were cut into a strip shape at the size of 1 mm × 0.2 mm × 0.028 mm. Phages are bound to the gold-coated sensor layer due to physical adsorption. Phage E2 was an fd-tet filamentous phage and was selected through Phage Display method. This phage was used as bio-recognition elements in this study for specific binding to Salmonella Typhimurium in produce samples. The detail bio-panning procedures, sensitivity tests, and specificity of this phage were described in Sorokulova at al., 2005 [23] . Phage sensors were prepared by coating the ME sensor with 1 × 10¹¹ virons of phage solution in TBS for one hour at room temperature on a rotator. The phage coated sensor was then washed three times with TBS. Bovine Serum Albumin (BSA) at 0.1% was used as a blocking reagent for preventing non-specific binding. The phage sensor was put in 0.1% BSA solution for 1 hour at room temperature on a rotator. Sensors coated with 0.1% BSA (without phages) served as negative controls in the frequency measurement experiments.

The final 1.0 mL solution from pre-enriched spinach leaves or tomato samples (Section 2.2) was further concentrated down to 330 µL in filtered water by centrifugation. After the phage ME biosensor was incubated with Salmonella suspensions for one hour at room temperatures, each sensor was washed with 1× TBS three times and taken out for frequency measurements and data analysis. The procedures for frequency measurements were the same as described in Wang et al., 2017 [22] . Each batch of inoculated produce was tested by 8 sensors and the experiment was repeated twice. Three non-phage sensors with BSA only served as controls in each group.

Overall, data collected from frequency measurements was analyzed by an on-tailed unpaired student’s t-test with p < 0.05 and α = 0.05 (as described in Wang et al., 2017 [22] ). The degree of dissimilarity between control and measurement sensors was calculated and the shortest incubation time was decided for sensor detection. Confidence level of difference equated to (1 − p value) × 100%.

XLD agar were used in this section of experiments. Frist, the 10 µL of the final 1.0 mL bacterial suspension concentrated from each test group (various media types and incubation times) was diluted and plated on XLD agar. The resulted plates were incubated at 37˚C for 16 - 18 h for total Salmonella counts in pre-enrichment (as Total Salmonella Counts). The rest of final 1mL bacterial suspension was then incubated with a phage coated sensor in a 1.5 mL micro-tube for 1 hour at room temperature with gentle rocking. The sensor was washed three times by 1 × TBS. Salmonella cells on the sensor were eluted by 0.1 M Glycine buffer (pH 2.2) for 10 min and neutralized by adding 1M Tris-HCl (pH 9.1). The neutralized bacterial suspension was diluted with PBS and plated on XLD agar plates for recorded as Salmonella number on sensor. The Salmonella capture rate was calculated by Salmonella capture rate in broth = (Salmonella number on one sensor/Total Salmonella Counts in broth) × 100%.

Each test group had duplicate plates. Each experimental parameter (various broth types and incubation times) was tested by four sensors and the whole experiment was repeated twice.

Table 1 shows the data of the frequency shift of biosensors for Salmonella detection on spiked spinach leaves in MPW, Lac broth, and LB broth for 7 and 22 hours pre-enrichment times. In this study, a one-tailed student t test with p value smaller than 0.05 (α = 0.05) was used. The confidence level of differences (CLD) of 95% or more was needed to achieve the significant Salmonella positive signal from the differences of frequency shifts between measurements and control sensors. According to Table 1, after 7 hours of incubation, the value of CLD

Note: ^a-frequency shifts (kHz) of measurement sensors; ^b-frequency shifts (kHz) of control sensors; ^c-confident level of difference (%).

of both MPW and Lac broths did not reach or exceed 95% and was lower than the CLD of 96.76% in LB broth. This data showed that LB broth is the only broth among the three to effectively yield Salmonella positive signals in artificially spiked spinach at a 7 hours incubation period. After 22 hours of incubation, all three broths produced significant Salmonella positive signals in frequency shifts with high CLD values exceed 95% (Table 1).

As for tomatoes samples, the detail study of resonant frequency changes in three broths (MPW, UP, and LB) after 5, 7, and 22 hours of pre-enrichment are presented in Table 2. By analyzing the data with a CLD above 95%, tomato samples in LB broth demonstrated significant differences in frequency shift of measurement sensors in compared to controls as early as 5 hours of incubation time. However, tomato samples in MPW and UP after 5 hours and 7 hours pre-enrichment did not show any significant frequency differences between measurements and control sensors. After an incubation time of 22 hours, significant frequency differences were observed in all three broths.

In order for easy understanding, CLD values, measured by frequency shift (Table 1 and Table 2) and passed 95% significant differences between the measurement and control sensors in various broths, are expressed as Salmonella positive signals. Otherwise, the CLD values lower than 95% were categorized as Salmonella negative signals. Table 3 and Table 4 are the summary tables of Salmonella detection signals of artificially spiked spinach leaves tomatoes in four different broths for 7 and 22 hours of incubation using phage ME biosensor platform. This summary data clearly demonstrated that LB is the best pre-enrichment medium to be use for early detection of 5-7 hours incubation for detection low Salmonella contaminations in both tomato and spinach samples.

Note: ^a-frequency shifts (kHz) of measurement sensors; ^b-frequency shifts (kHz) of control sensors; ^c-confident level of difference (%).

Note: Negative Salmonella detection signal (-) represented CLD is under 95%; positive signal (+) represented CLD equals or larger than 95%. Eight sensors were used for each test condition and the experiment was repeated twice.

¹Note: Negative Salmonella detection signal (−) represented CLD is under 95%; positive signal (+) represented CLD equals or larger than 95%. Eight sensors were used for each test condition and the experiment was repeated twice.

In order to understand the detail performances of phage biosensors in each broth, the number of Salmonella on the sensor in all three broths for 7 and 22 hour incubation times were evaluated. The data are shown in Figure 2 for spinach samples and Figure 3 for tomato samples. According to the report of Li et al., 2010 [14] , the Salmonella detection limit of phage ME biosensor was 500 cfu/mm² as determined by the direct detection of Salmonella on the tomatoes surfaces. Their study of phage biosensor direct detection of Salmonella was conducted by air-dried Salmonella cells on the surface of tomatoes. Therefore, the detection unit was expressed as cfu/mm². In this study, the same Salmonella amount was used for set up the detection threshold of Salmonella. Since this study was performed in the liquid detection format, the unit was expressed as cfu/sensor. After a 7 hour incubation period, the phage biosensor was able to capture 937 cfu/sensor of Salmonella with spinach samples in LB, which passed the detection threshold of Salmonella counts (present as a red line in Figure 2). In MPW and Lac broth enrichment, Salmonella numbers did not exceed 500 cfu/sensor. Therefore, these two broths failed to pass the detection threshold and also didn’t show Salmonella positive signals in frequency tests. At 22 hours incubation, all three broths were able to demonstrate a high Salmonella number of 10⁴ - 10⁵ cfu/sensor (Figure 2), which triggered the Salmonella positive signal in biosensor frequency detections with high CLD values. With the early detection of 7 hours incubation time in the 1st pre-enrichment step of FDA procedures, LB appeared to be the best among the three media for spinach samples to use in Salmonella detection by ME biosensors.

The microbiological analysis of contaminated tomato samples in MPW, UP, and LB broths after 5, 7, and 22 hours of pre-enrichment time are shown in Figure 3. According to Figure 3, LB broth was the only broth in the three, which is able to capture enough Salmonella cells (719 cfu/sensor) and passed the biosensor detection threshold in the fifth hour of the early incubation stage. MPW and UP did not show enough Salmonella numbers on the sensor until 22 hours of incubation time. This data may indicate that MPW and UP didn’t promote Salmonella growth in tomato samples after short hours of incubation. As the same concept mentioned previously in the study of Wang et al, in 2015 [24] , MPW and UP may not be the suitable broth for quick incubation of phage ME biosensors for detecting Salmonella in tomatoes.

When cross referencing the microbiological data of Salmonella numbers on the sensors and the data of frequency shifts, it yields interesting findings. In the LB broth with both 7 and 22 hours of incubation time, the CLD values in tomatoes (Table 3) were higher than the values in spinach samples (Table 1). However, the Salmonella number on the sensors was lower in tomatoes (Figure 3) than the number in spinach (Figure 2). The frequency measurements and microbiological data of LB in spinach and tomatoes samples seemed to contradictory to each other. Therefore, it is curious to know whether the affinity actions of the phages to Salmonella was still performed normally in the same broths, but different produce types. In the report of Qiang et al., in 2017 [25] , proteins and components in different blocking buffers would inhibit the affinity of phage probes to its target and promote the non-specific binding in ELISA assays. To answer this question, Salmonella capture rates of the phage biosensors were studies. The Salmonella capture rate was calculated by the percentages of Salmonella numbers captured by phages on the sensor and divided by the total Salmonella growth in each test broth with 7 hours of incubation period. The data truly revealed the fruit types played as a factor to affect the phage affinity to capture target pathogens in LB. Figure 4 shows that in 7 hours of pre-enrichment of LB, 9.606% of Salmonella was captured by phages on the biosensors in tomatoes samples, while the capture rate was only 8.57% in spinach. Apparently, substances in spinach samples (such as chlorophyll or flavonoids) may slightly inhibit phage affinity to Salmonella. More studies are needed to seek a conclusive statement about this finding. Besides, in this set of data, LB again demonstrated the highest capture rates among all broths, while MPW showed the lowest Salmonella capture ability for phage biosensors to detect Salmonella in two tested produce samples.

Overall, the phage ME biosensors were able to detect Salmonella in the LB broth within 5 hours of incubation in tomatoes samples and within 7 hours in low Salmonella spiked spinach samples. MPW may not be a suitable short time pre-enrichment medium for detecting Salmonella in both produce samples using phage ME biosensor. The comparison of the phage ME biosensors to FDA BAM culture method and qRT-PCR method are summarized in Table 5. The detection limit of Salmonella by phage ME sensor method may not be as low as two other methods, but the pre-enrichment time is tremendously decreased. This may be compensated by extending the pre-enrichment time less than one hour to reach the same detection limit. The cost of the ME sensor method is also much lower than the two other methods. Our data also demonstrated that it is possible to implant phage ME biosensors into FDA BAM methods for screening and shortening the Salmonella pre-enrichment times and then perform the BAM culture or PCR methods.