Why not cut cucumbers with a steel knife?

Cutting knives are the major harborage site for bacteria. Once the cucumbers are cut with a contaminated knife, the microorganisms can attach to cut surfaces 4 , 5 , 6.

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Evaluation of kitchen knife inoculation

Concentration of E. coli inoculated on a kitchen knife was shown in Table 1. When the inoculation concentrations were 1.09 ± 0.08, 3.34 ± 0.21, 5.20 ± 0.13 and 7.06 ± 0.25 log CFU/mL, the amount of E. coli on knife were 0.36 ± 0.10, 2.85 ± 0.20, 4.70 ± 0.28 and 6.49 ± 0.01 log CFU/knife. The number of E. coli inoculated on the knife increased with the increase of the inoculation level, the number was about 0.5-log less than the inoculation solution. Kusumaningrum et al.20 reported that the amount of Salmonella Enteritidis and Staphylococcus aureus on stainless steel surfaces declined with the decrease of the inoculation concentration, the change pattern was similar to this study. Furthermore, previous reports pointed out that the decay model describing the transfer behavior of pathogenic bacteria during slicing of vegetable was less accurate when using the low inoculum15. Thus, the transfer number of E. coli on the knife was small under low inoculation concentration, which was not conducive to researching and monitoring the transfer of E. coli. In order to reveal E. coli transfer, higher inoculation concentrations (3.34 ± 0.21, 5.20 ± 0.13 and 7.06 ± 0.25 log CFU/mL) were selected for subsequent experiments. Table 1 Concentration of E. coli inoculated on a kitchen knife. Full size table

E. coli transferred from kitchen knife to cucumber

Experimental data showed that the transfer amount and rate of E. coli exhibited a fluctuating decay pattern during cutting process (Table 2). It was the highest at the first cut in all treatments, after that data showed a sharp decrease, then slightly increased and leveled off, finally decreased again as the number of slices increased. Moreover, the number of E. coli remaining on cucumber slices after 9 cuts was below 100. At low inoculum level (3.34 ± 0.21 log CFU/mL), the recovery amount (slice 1: 0.95 ± 0.10; slice 2: 0.68 ± 0.14; slice 3: 0.31 ± 0.14 log CFU/cucumber slice) and transfer rate (slice 1: 1.259; slice 2: 0.676; slice 3: 0.288%) of E. coli on the first 3 slices decreased significantly as the cutting number increased. Then they increased significantly at the fourth slice (recovery amount: 0.65 ± 0.11 log CFU/cucumber slice; transfer rate: 0.631%) and the changes on the following data points became stable. When the ninth slice was reached, the recovery amount (0.26 ± 0.16 log CFU/cucumber slice) and transfer rate (0.257%) of E. coli were decreased rapidly. This was similar to the previous study, the bacterial transfer that occurs after cutting food with a blade inoculated with 104 CFU/mL bacterial solution showed a fluctuating decrease trend23. At moderate and high inoculation levels (5.20 ± 0.13 log CFU/mL and 7.06 ± 0.25 log CFU/mL), higher amount and transfer rate of E. coli appeared (3.36 ± 0.03 and 3.75 ± 0.08 log CFU/cucumber slice; 4.571% and 0.182%). The number of E. coli recovered from the fourth slice under moderate-concentration and fifth slice of cucumber under high-concentration were reduced by approximately 2.8 and 4.05 log CFU/cucumber slices, respectively, were about 3.3 and 4.62 log CFU lower than the inoculum level on the knife. The amount of E. coli transferred on the next slice tended to be flat, then slowly decreased. The transfer of bacteria depends on the nature of the contaminated surface, cutting speed, force and cutting action24,25. Stainless steel is a hydrophilic surface, which can be used as a medium for bacteria to attach, may promote the release of pathogens during the food preparation process and relocate them to the surface of high-moisture food26. The cutting speed and action were kept as consistent as possible in this study, all cutting action were the longitudinal cutting that perpendicular to the cutting board, therefore cutting speed and action might have little effect on the transfer of E. coli. However, the force was variable during the cutting process, the fluctuating transfer of E. coli might be affected by force7. The similar trends were observed by other researchers with regard to other pathogenic bacteria that transfer through surface of stainless steel to cucumbers, lettuce and celery20,27,28. Furthermore, in previous researches, the transfer behavior of pathogenic bacteria during the slicing process of fish, meat and vegetables were similar which could use the decay model to describe29,30,31. The transfer of E. coli was probably because the shearing force on the knife surface, which was not determined in our study, might have affected the removal of loosely attached cells, resulting in the transfer of bacterial cells on the knife to the cucumber slices32. As the slice continued, the number of cells transferred back to the knife gradually decreased and cells transferred to cucumer slice with the next cut gradually decreased, this might be because the knife was simultaneously affected by adhesion and hydrophobicity. When the attachment between E. coli and knife was not as strong as E. coli and cucumber, the cells might detachment from the knife and movement to cucumber slice so that transfer becomes more feasible. The remain of exudate released from the cucumber slices altered the hydrophobicity of the knife surface and affected the transfer of E. coli. This caused some transferred cells to move back onto the knife33. Furthermore, the dual- or multiple- species of endophytes in cucumber exudate might affect the adhesion of E. coli and stainless steel34. Changes in the transfer rate during the continuous cutting process, which might be because consecutive cutting was not a static, neither an easy-to-control process21. Table 2 The transfer of E. coli from the kitchen knife to the cucumber surface. Full size table

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BL images of E. coli transfer and distribution on cucumber slice after cutting The high inoculation (7.06 ± 0.25 log CFU/mL) was used to reveal the transfer of E. coli during the cross-contamination process according to previous research33. BL images of E. coli transferred and distributed on the cross-section of cucumber were shown in Fig. 1a. Our results suggested that the adhesion of the initial cleavage site was strongest due to mechanical action, most E. coli transferred to the upper section. The cells transferred back to the knife due to the hydration of the exudate, E. coli would be randomly distributed on the tissues in the middle and lower parts of the cucumber slices. The E. coli luminescence signal detected on the vascular bundles, xylem vessel and placental tissues was the strongest at the site of inoculation, representing the most transfer of strains. This might be because these tissues have xylem tissues that could transport nutrients and water to cucumbers12, which provided nutrients for growth of E. coli. These tissues would accumulate and adhesion of E. coli more easily than other tissues and the detected luminescent signal will be stronger than other tissues. The 3D surface chart further showed that more luminescence signals were detected at the initial cleavage site and stronger luminescence signals were detected in the xylem tissues (Fig. 1b). Figure 1 Immediate BL images of E. coli transfered and distributed on the cross-section of cucumber. (a) The left side was the control of uncontaminated cucumber slices, the right side was the cucumber slice after cutting horizontally by a knife inoculated with E. coli (7.06 ± 0.25 log CFU/mL). (b) 3D surface chart of the same processed sample. The red color represented a strong signal and the blue color represented a weak signal. IndiGo software (https://www.nchsoftware.com/accounting/index.html?kw=sage%20software&gclid=AIaIQobChMI3MvFj-zr7wIVj2gqCh14fAmLEAEYASAAEgJPqPD_BwE, https://softwaretopic.informer.com/). Full size image

Survival of E. coli on cucumber slices during storage

BL images showed that the contaminated area of E. coli on all cucumber slice samples gradually decreased with the decrease of storage temperature and the extension of storage time. More specifically, the luminescent signal of the E. coli under 37 °C storage condition was strongest (Fig. 2). This might be because 37 °C was the suitable temperature for the growth of E. coli. E. coli was irregularly distributed on vascular bundles, xylem vessels, placenta and mesophyll tissues where the signal color were the reddest and the signal were the strongest. The survival of E. coli on cucumber slice after cutting 5 times was significantly reduced, the luminescent signal of E. coli was below the minimum detection limit. Compared with other storage temperatures, the distribution area of E. coli under 37 °C at the edge decreased sharply as the storage time. The reduction of contaminated area and the weakening of the bioluminescent signals might be caused by E. coli death and internalization. The death of E. coli was verified by the fluorescence microscope images in Fig. 3. It was found that more number of living E. coli were observed on the cucumber slices in the early stage of inoculation 30 min after cross-contamination (Fig. 3a), while more E. coli deaths were observed after 2 h of inoculation (Fig. 3b). The death of E. coli might be due to the gradual consumption of water and nutrients on the surface of cucumber slices with the extension of storage time. This might also be caused by the release of hydrogen peroxide from damaged plant tissue that causes oxidative stress in the bacterial cells and the presence of competing microorganisms20, thereby harming or inactivating E. coli35. However, the antibacterial hydrogen peroxide produced by the wound will temporarily affect the attachment of E. coli and tends to decrease after injury36, so some bacteria will survive37. Under other temperature conditions (25 °C, 10 °C, 4 °C), E. coli mainly colonized and distributed on the margins, placenta and vascular system tissues. However, the attenuation degree of E. coli in the same contaminated area was much lower than 37 °C with the storage time. These temperatures (25 °C, 10 °C, 4 °C) are not suitable for the growth of E. coli, leading to differences in the growth and attenuation metabolism of E. coli on cucumbers. However, previous studies have shown that pathogenic bacteria could survive at 4 °C and grow at 10 °C, indicating that unintentional abuse of temperature could cause the growth of pathogenic bacteria, enough to reach potentially hazardous levels38. The survival ability of E. coli was stable due to the decreased ability of self-protection and regulation of plant cells during low temperature storage of cucumber39. The increased activity of POD and SOD could kill the superoxide free radical produced in adversity40, reduce the damage to E. coli. Meanwhile, the exudate released from the cucumber slices provided sufficient nutrition for the survival of the bacteria and maintained their activity. These results showed that there were still potential safety hazards in cutting cucumbers with contaminated knives even under low temperature conditions, the distribution of E. coli tended to be on the initial cutting site and nutrient-rich tissues. In addition, it was observed that the transfer area and change trend of E. coli after cutting cucumber using a kitchen knife inoculated with 105 and 103 CFU/mL of E. coli during storage was similar to the concentration of 107 CFU/mL (data not shown). Figure 2 BL images of the transfer and distributed of E. coli on the cucumber slices under different storage temperatures and times. (a) The first row of cucumber slices from left to right represented cut numbers of 1, 2, 3; the second row represented 4, 5, 6; the third row represented 7, 8, 9. The scales are the same under the same temperature. All scales represent 3 cm. (b) The bar graph showed the contaminated area of E. coli on cucumber slices after cutting under each storage condition. Different contaminated areas were indicated by different colors. IndiGo software (https://www.nchsoftware.com/accounting/index.html?kw=sage%20software&gclid=AIaIQobChMI3MvFj-zr7wIVj2gqCh14fAmLEAEYASAAEgJPqPD_BwE, https://softwaretopic.informer.com/). OriginPro 8 software (https://en.freedownloadmanager.org/users-choice/Origin_8_Free_Download_Full_Version.html, https://softwaretopic.informer.com/). Full size image

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Figure 3 Fluorescence microscopy images of E. coli (× 100). Fluorescence microscopy image of live/dead bacteria on cucumber slices inoculated with E. coli after 30 min (a) and 2 h (b) from inoculation. Image-Pro plus 6.0.0 (https://www.xrayscan.com/software-image-pro-plus/, https://www.xrayscan.com/). Full size image

BLI of E. coli internalization during storage

E. coli was internalized from the attachment point of cucumber, then, spread to the surrounding tissues (Fig. 4a). The maximum internalization distance was 2–3 mm after 30 min from inoculation. The 3D surface chart further clarified that the bioluminescent signals were the strongest on the inoculated surface, and became weaker with an increase in internal distance (Fig. 4b). Regarding the internalization distance, the result obtained in the present study was in agreement with previous research findings41 indicating that microorganisms could penetrate into the medium, the penetration distance was in the first 2–3 mm of the medium surface, rendering the bacteria more difficult to detect. The internalization of E. coli at different storage times and temperatures was shown in Fig. 5. The internalized area of all samples decreased rapidly with storage time, being constant stable after 60 min from inoculation. This might be due to the temporarily affect the growth of E. coli by cucumber exudate, which led to a rapid decrease in internalized E. coli. After a period of storage, the bacterial cells had adapted to the growth environment and could survive then stabilized. E. coli reached the maximum internalization distance after 30 min from inoculation at 37 °C without change with the extension of storage time. The internalization distance of E. coli declined with temperature decreased, and remained unchanged after 30 min. Less E. coli internalization distance and area was observed at refrigeration temperatures (4 and 10 °C), but the viability of E. coli was weaker at 4 °C. The internalization is probably due to the fact that vascular bundles, xylem vessels function in transporting nutrients and water, thus providing access to E. coli to the internal tissues42,43. But the refrigeration temperatures were not suitable for bacterial growth, they could reduce the activity of E. coli. Results further proved that the decreased of the contaminated area on the cucumber slice and the luminescence signal were caused by the internalization of E. coli as well as the death of E. coli. Figure 4 BL images of longitudinal section of E. coli internalized from the cross-section of cucumber. (a) Bacterial internalization image of cutting cucumber horizontally (cut surface of cucumber is on the right, 7.06 ± 0.25 log CFU/mL) with a bacterial knife and then cutting longitudinally. (b) The 3D surface chart of the same processed sample. The red color represented a strong signal and the blue color represented a weak signal. IndiGo software (https://www.nchsoftware.com/accounting/index.html?kw=sage%20software&gclid=AIaIQobChMI3MvFj-zr7wIVj2gqCh14fAmLEAEYASAAEgJPqPD_BwE, https://softwaretopic.informer.com/). Full size image

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