Antimicrobial Efficacy and Spectrum of Phosphorous-Fluorine Co-Doped TiO2 Nanoparticles on the Foodborne Pathogenic Bacteria Campylobacter jejuni, Salmonella Typhimurium, Enterohaemorrhagic E. coli, Yersinia enterocolitica, Shewanella putrefaciens, Listeria monocytogenes and Staphylococcus aureus

Contamination of meats and meat products with foodborne pathogenic bacteria raises serious safety issues in the food industry. The antibacterial activities of phosphorous-fluorine co-doped TiO2 nanoparticles (PF-TiO2) were investigated against seven foodborne pathogenic bacteria: Campylobacter jejuni, Salmonella Typhimurium, Enterohaemorrhagic E. coli, Yersinia enterocolitica, Shewanella putrefaciens, Listeria monocytogenes and Staphylococcus aureus. PF-TiO2 NPs were synthesized hydrothermally at 250 °C for 1, 3, 6 or 12 h, and then tested at three different concentrations (500 μg/mL, 100 μg/mL, 20 μg/mL) for the inactivation of foodborne bacteria under UVA irradiation, daylight exposure or dark conditions. The antibacterial efficacies were compared after 30 min of exposure to light. Distinct differences in the antibacterial activities of the PF-TiO2 NPs, and the susceptibilities of tested foodborne pathogenic bacterium species were found. PF-TiO2/3 h and PF-TiO2/6 h showed the highest antibacterial activity by decreasing the living bacterial cell number from ~106 by ~5 log (L. monocytogenes), ~4 log (EHEC), ~3 log (Y. enterolcolitca, S. putrefaciens) and ~2.5 log (S. aureus), along with complete eradication of C. jejuni and S. Typhimurium. Efficacy of PF-TiO2/1 h and PF-TiO2/12 h NPs was lower, typically causing a ~2–4 log decrease in colony forming units depending on the tested bacterium while the effect of PF-TiO2/0 h was comparable to P25 TiO2, a commercial TiO2 with high photocatalytic activity. Our results show that PF-co-doping of TiO2 NPs enhanced the antibacterial action against foodborne pathogenic bacteria and are potential candidates for use in the food industry as active surface components, potentially contributing to the production of meats that are safe for consumption.


Introduction
Foodborne pathogenic bacteria can seriously influence the safety and quality of meats. They not only can cause diseases and death, but also represent an economic burden [1]. Some species are normal microbiota members present on livestock or the skin of humans and animals, while others are ubiquitous in the surrounding environment of the processed animal.
One such study investigated the antimicrobial efficacies of undoped, doped or codoped TiO 2 NPs on different meats such as lamb and fish [40,41]. Their antibacterial efficacies were revealed by targeting only a few important foodborne pathogen bacteria: Listeria monocytogenes, Salmonella Typhimurium, Campylobacter jejuni [42,43]. Until now, no systematic study has been carried out examining the survival of the most important foodborne pathogenic bacteria in the presence of co-doped TiO 2 NPs.
In a recent study we produced and thoroughly characterized the physical and chemical properties of PF-co-doped TiO 2 NPs, where both P and F were used as dopants, and revealed their enhanced antibacterial activities against carbapenem resistant Klebsiella penumoniae isolates [44]. In order to investigate the possible applicability of PF-co-doped TiO 2 in the food industry, here we have compared the antibacterial efficacy of PF-co-doped TiO 2 NPs on seven foodborne pathogenic bacterium isolates: C. jejuni, S. Typhimurium, Enterohaemorrhagic Escherichia coli, Y. enterocolitica, S. putrefaciens, L. monocytogenes and S. aureus. With these tests we examined both the pure photoinduced antimicrobial efficacies and how the duration of the hydrothermal treatments influenced the antibacterial features of the produced NPs on multiple foodborne pathogenic bacteria.

Synthesis and Characterization of PF-Co-Doped TiO 2 NPs
Synthesis of PF-co-doped TiO 2 NPs (PF-TiO 2 ) was performed as described previously [44]. Briefly, 46 g of TiCl 4 was suspended in 100 mL of 2-propanol and sonicated for 5 min. The light yellow solution was diluted with 200 mL of highly purified deionized water. These Ti precursors were hydrolysed by adding 500 mL of 1.5 M NaOH solution dropwise under vigorous stirring. The resulting white precipitate was centrifuged and washed three times with 100 mL of deionized water. After that, 0.38 mol/L HPF 6 solution (7.3 mL) was added to the dispersion while stirring. Aliquots (50 mL) of the dispersion were treated hydrothermally at 250 • C for 1, 3, 6 or 12 h. Samples were denoted PF-TiO 2 /0 h, PF-TiO 2 /1 h, PF-TiO 2 /3 h, PF-TiO 2 /6 h and PF-TiO 2 /12 h, respectively. The dispersions were then centrifuged and washed with deionized water followed by 2-propanol. The obtained sediments were dried at 50 • C in an oven for 12 h.

Antibacterial Tests
In order to compare the susceptibility of seven foodborne pathogens against different PF-co-doped TiO 2 NPs, antibacterial tests were performed. A starter culture was made on the day of the test from an overnight culture of the relevant bacterium grown in 20 mL of MH medium as described in Section 2.2. At mid logarithmic phase (OD 600 = 0.4-0.6), the culture of the relevant bacterium was centrifuged 10,000× g for 2 min, and then washed twice with 0.9 w/v% NaCl solution. At the last washing step, the suspension was centrifuged, and the optical density was set to OD 600 = 1 (1 × 10 8 Colony Forming Units (CFU)/mL) in 0.9 w/v% NaCl solution. Tests were carried out in a flat bottom non-adhesive 24-well tissue culture plates containing 990 µL of dispersion of PF-co-doped TiO 2 NPs in 0.9 w/v% NaCl and 10 µL of bacterium suspension. The final concentration of the bacterium suspension was~10 6 CFU/mL. Three different concentrations (500 µg/mL, 100 µg/mL, 20 µg/mL) of the PF-co-doped TiO 2 NPs were compared for their antibacterial efficacies.
After thorough mixing by pipetting with a 1 mL tip, PF-co-doped TiO 2 and bacterial suspensions were incubated in a closed dark box for 15 min. Then, the photocatalytic reactions were carried out using a 15-W UV-A lamp (F15W/T8/BL368 fluorescent lamp, Sylvania, Wilmington, MA, USA). For bacterial enumeration, 10 µL of sample aliquots were taken after 15 min dark incubation and 30 min after UV exposure. Experiments were also performed under daylight conditions by using a 15 W lamp (Viva-Lite T8/W15/5500K), or under dark conditions for 30 min.
In every test, concentration-matched bacterium suspensions in 0.9 w/v% NaCl solution without any PF-TiO 2 NPs served as bacterium controls, while AEROXIDE TiO 2 P 25 (Evonik, Essen, Germany) acted as a control.
After incubation, the numbers of viable cells were determined by dropping and running off 10 µL of suitably diluted aliquots onto MH agar plates and then counting the colonies after 24 h incubation at the appropriate temperature (30, 37 or 42 • C) and growth condition (aerobic or microaerophilic), depending on the bacterium species under investigation. Each test was performed in triplicate and repeated on a separate day.

Statistical Analysis
Statistical analysis was performed using Microsoft Excel 2016 MSO software (Microsoft Corp., Redmond, WA, USA). Students t-test was applied to all pairwise comparisons. The level of significance was p < 0.05. Data were expressed as mean ± standard deviation.

Results and Discussion
The antibacterial activity of PF-co-doped TiO 2 NPs on Campylobacter jejuni, Salmonella Typhimurium, Enterohaemorrhagic E. coli, Yersinia enterocolitica, Shewanella putrefaciens, Listeria monocytogenes and Staphylococcus aureus, which are among the most important bacterial foodborne pathogens, were studied [2,3,5,7,10,12,14]. The applied PF-TiO 2 NPs were characterized recently [44]. Therefore, this study has strictly focused on the comparative antibacterial analysis of the NPs on the afore-mentioned foodborne pathogenic bacteria. The main structural and morphological features of PF-TiO 2 NPs are summarized in Figure 1. X-ray powder diffraction measurements revealed that the crystalline phase of all PF-TiO 2 samples was solely the anatase itself (JCPDS card No. 78-2486). The prolonged hydrothermal treatment resulted in higher intensities of d 101 diffraction peaks while the peak broadening decreased gradually over time ( Figure 1a). These changes in the peak shape indicate that hydrothermal treatment enhanced the crystallinity and increased the crystallite size of PF-TiO 2 samples. The average crystallite size was calculated from the full width at half maximum (FWHM) of d101 peak by using Scherrer equation. Without treatment,~6 nm anatase crystallites were formed while treatments for 1-12 h resulted in 10-24 nm crystallites ( Figure 1b). In addition to producing larger crystallites, treatment also increased the anatase content, thus the proportion of amorphous phase reduced ( Figure 1b). It should be noted that even without hydrothermal treatment, high crystallinity was observed (76 wt%). The high crystallinity of the samples is crucial for the effective photocatalytic generation of ROS [45]. Crystal defects determine the photoelectric properties of semiconductors, and they can significantly affect the charge carrier recombination [46]. Figure 1c,d shows representative TEM images of PF-TiO 2 /0 h and PF-TiO 2 /12 h samples. For the untreated sample, tiny particles are barely distinguishable (Figure 1c). After 12 h hydrothermal treatment, mainly spherical but also anisotropically shaped nanocrystals are visible ( Figure 1d). In accordance with the XRD measurements, TEM images also showed that the particle size increased sharply during the hydrothermal treatment, however, the mixed morphology was retained. Consequently, PF-TiO 2 nanoparticles with both spherical and polyhedral (faceted) shapes have been observed for all the treated samples [44]. TEM images suggest that polyhedral nanoparticles possess truncated tetragonal bipyramidal geometry, which is typical for the anatase nanocrystallites.
semiconductors, and they can significantly affect the charge carrier recombination [46]. Figure 1c,d shows representative TEM images of PF-TiO2/0 h and PF-TiO2/12 h samples. For the untreated sample, tiny particles are barely distinguishable (Figure 1c). After 12 h hydrothermal treatment, mainly spherical but also anisotropically shaped nanocrystals are visible (Figure 1d). In accordance with the XRD measurements, TEM images also showed that the particle size increased sharply during the hydrothermal treatment, however, the mixed morphology was retained. Consequently, PF-TiO2 nanoparticles with both spherical and polyhedral (faceted) shapes have been observed for all the treated samples [44]. TEM images suggest that polyhedral nanoparticles possess truncated tetragonal bipyramidal geometry, which is typical for the anatase nanocrystallites. Results of antibacterial tests show that none of the bacterium species were sensitive to the PF-TiO2 NPs at any concentrations under dark conditions, indicating that their antibacterial effects stem from photocatalytic reactions ( Figure 2). This is in agreement with previous studies [44] where TiO2 NPs did not exhibit antibacterial effects under dark conditions [38,39,42,43]. Without light excitation, TiO2 NPs were not toxic for bacteria as CFU values indicated ( Figure 2). Under dark conditions, TiO2 NPs can adsorb on the cell wall of bacteria. In order to ensure adsorption-desorption equilibrium, PF-co-doped TiO2 NPs had to be thoroughly mixed with bacterial suspensions under dark conditions and incubated for 15 min. The duration of this phase depends on the applied NP and typically ranges from 10-40 min [38]. However, light with appropriate energy can induce charge separation in the TiO2 NPs and subsequently redox reactions can take place on their surface. Recently, we studied the formation of the main ROS in the PF-TiO2 dispersions by means of electron paramagnetic resonance (EPR) spectroscopy. During the photocatalytic reactions, the presence of hydroxyl (OH • ) and superoxide radicals (O2 •− ), as well as singlet Results of antibacterial tests show that none of the bacterium species were sensitive to the PF-TiO 2 NPs at any concentrations under dark conditions, indicating that their antibacterial effects stem from photocatalytic reactions ( Figure 2). This is in agreement with previous studies [44] where TiO 2 NPs did not exhibit antibacterial effects under dark conditions [38,39,42,43]. Without light excitation, TiO 2 NPs were not toxic for bacteria as CFU values indicated ( Figure 2). Under dark conditions, TiO 2 NPs can adsorb on the cell wall of bacteria. In order to ensure adsorption-desorption equilibrium, PF-co-doped TiO 2 NPs had to be thoroughly mixed with bacterial suspensions under dark conditions and incubated for 15 min. The duration of this phase depends on the applied NP and typically ranges from 10-40 min [38]. However, light with appropriate energy can induce charge separation in the TiO 2 NPs and subsequently redox reactions can take place on their surface. Recently, we studied the formation of the main ROS in the PF-TiO 2 dispersions by means of electron paramagnetic resonance (EPR) spectroscopy. During the photocatalytic reactions, the presence of hydroxyl (OH • ) and superoxide radicals (O 2 •− ), as well as singlet oxygen ( 1 O 2 ) was confirmed [44]. Among them, the most reactive and damaging OH • radical was the predominant form. Similar to our recent study [44], high bacterial cell number (10 6 CFU/mL) was applied in order to reveal antibacterial efficacy of TiO 2 NPs, although such a high bacterial CFU is usually not present on the surface of meats.   Even though TiO 2 are considered safe in food applications by the FDA, no comparative study has been reported on the antibacterial potential of TiO 2 NPs on various foodborne pathogen bacteria. Even though some data are available for L. monocytogenes, S. Typhimurium, C. jejuni, S. aureus [42,43,[47][48][49], there is no available data for EHEC, Y. enterocolytica and S. putrefaciens. These studies focused on the photocatalytic-based antibacterial activity of standard nano-TiO 2 (P25) [42,47,48], or its combination with Ag, an antibacterial metal [43]. Even though the enhanced antibacterial efficacies of TiO 2 NPs doped with other metals and metal oxides, including Cr, Cu, CoO and ZnO, were demonstrated under laboratory conditions [19,[50][51][52][53], their application raises some toxicity issues. Several metals are not only toxic for the bacterial cell, but also for humans and eukaryotic organisms [53][54][55][56][57][58].
A plausible explanation for the extreme susceptibility of C. jejuni is its lack of the classical oxidative stress response regulatory elements SoxRS and OxyR [60]. Our current results indicate that the defence mechanism of C. jejuni is unable to cope with the ROS generated by PF-co-doped TiO 2 NPs. To best of our knowledge no previous study has investigated the survival of this bacterium species under direct UVA exposure. However, our data are partially comparable with a previous study [61] where a 5 log CFU/mL reduction was observed after the application of 405 nm light for 30 min in a suspensionbased model. Even though we did not see a 5 log CFU/mL reduction in our experiments, but we demonstrated that PF-co-doped TiO 2 NPs drastically enhance the antibacterial effects of UVA treatments.
S. Typhimurium was the second most sensitive bacterium species, showing significant bacterial cell number loss when incubated with PF-TiO 2 NPs (Figure 2b, Supplementary Figure S1b/1-3). The most effective photocatalytic NPs were PF-TiO 2 /3 h and TiO 2 /6 h if applied together with UVA, as they completely eradicated S. Typhimurium (Figure 2b). Coincubation and UVA treatment of this bacterium species with PF-TiO 2 /1 h or PF-TiO 2 /12 h resulted in a drastic 5 log CFU reduction from 2 × 10 6 , to 2 × 10 1 and 1 × 10 1 , respectively. PF-TiO 2 /0 h was more effective than the TiO 2 P25 (Figure 2b). The antibacterial effect of the NPs decreased with decreasing NP concentration. Concerning the comparison of the sensitivities of C. jejuni with S. Typhimurium to NPs, our results are in agreement with the results of the available published studies which showed that the sensitivity of C. jejuni was 8-to 16-fold lower than that of S. Typhimurium [59,62].
Until now, very few results are available based on the antibacterial effect of UVA (315-400 nm) as research mostly focuses on the antibacterial efficacy of UVC [49,63,64] with a wavelength in the germicide range (200-280 nm). A recent study, however, has investigated the UVA-induced antibacterial effect of TiO 2 [42]. The experimental set-up was almost identical to that used by us allowing direct comparison of results. Long et al. [42] found that 500 µg/mL TiO 2 P25 decreased the living S. Typhimurium cell count by nearly 1.5 log in 30 min, in line with our results. However, our results went beyond this as we showed that antibacterial activity of a TiO 2 NP can be further enhanced through PF-codoping, from among PF-TiO 2 /3 h and PF-TiO 2 /6 h formulations were the most effectives. Antibacterial activity of these co-doped, NPs were almost comparable to the bactericide activity of CuO and was better than that of ZnO demonstrated by a recent study [59]. It should be however noted that in this study the applied bacterial number was less (10 4 CFU/mL) and the exposure times were longer (16 h) in contrast to our experimental setup where the bacterial concentration was 10 6 CFU/mL while the exposition times were 30 min. Silver NPs, one of the most widely used antimicrobial nanomaterials [65], had the highest anti-microbial efficacy according to this study.
C. jejuni was the only tested species where the survival was also affected by the applied UV light alone. A 3.21 and 3.63 log CFU/g decrease in S. Typhimurium and EHEC, respectively, was achieved on the surface of cheese if UVA was applied in combination with citric acid [66,67]. This evidence generally supports our results, although the experiments were performed on solid surfaces, while ours employed a mixed liquid system.
In contrast, being one of the most serious foodborne pathogens under EU surveillance [6], several Listeria monocytogenes eradication practices have been investigated based on germicidal UVC irradiation [66,69] or on UVA-combined photocatalysis [42,66]. Our results clearly demonstrated the drastic antibacterial activity of the PF-TiO 2 NPs on L. monocytogenes (Figure 2f, Supplementary Figure S1f/1-3) and this species was the most sensitive bacterium species followed C. jejuni and S. Typhimurium. Concerning the photocatalytic activity of TiO 2 P25, our results match with the previous results of Long et al. [42] as after 30 min incubation a 2 log magnitude decrease in the bacterial count of L. monocytogenes was detected, however this decrease in the living bacterial cell numbers could be further enhanced if the PF-TiO 2 NPs, especially PF-TiO 2 /3 h and PF-TiO 2 /6 h (Figure 2f), were applied in the photocatalytic reaction. In a previous study, doping of TiO 2 with CoO resulted in a <100 log decrease of the L. monocytogenes cells in 24 h when a 4 mg/mL NP suspension was applied at a 10 6 CFU/mL final bacterium concentration [50]. Even though this antimicrobial effect of CoO was revealed both under dark and light conditions, it was undetectable after 6 h exposure.
Sensitivity of the other tested Gram-positive bacterium species S. aureus (Figure 2g, Supplementary Figure S1g/1-3) to the PF-TiO 2 NPs was most comparable to the patterns of Y. enterocolitica and S. putrefaciens, with almost all NPs able to induce a 1-3 log CFU decrease. Due to its great importance in human health, possible elimination of S. aureus has previously been investigated by other authors. By using UVA irradiated TiO 2 -based photocatalysis in submerged coated systems, a~1 log reduction in CFU was observed after 4 h [48], a result that was in line with our control (TiO 2 P25) results. Another study showed that silver doped TiO 2 nanotubes (Ag/TNTs) had superior antibacterial features with agar diffusion, compared to TiO 2 (P25) and TNT alone [70]. Unfortunately, we could not find any data in the study if silver alone, as control, was tested or not. It would have been necessary as the antibacterial feature of silver is well known. A more relevant study was presented by Jalvo et al. [47], where a 3 log reduction in the living cell number in a S. aureus biofilm was obtained by using a TiO 2 -activated glass surface and 2 h irradiation treatments with 290-400 nm light. This study demonstrated that photocatalytically active surfaces have great potential in food industrial applications to hinder the contamination of meats with foodborne pathogenic bacteria. Moreover, doping with non-metal elements such as P and F, is a promising method to increase the antimicrobial efficacy of TiO 2 NPs, as we have demonstrated here. Highly crystalline PF-TiO 2 NPs with an anatase crystal phase proved to be good candidates for prevention of foodborne illness as they have superior antimicrobial effects.

Conclusions
All seven tested bacterium species are typical foodborne pathogenic bacteria that can enter the food chain during meat processing and proliferate during transportation and storage. One option to eliminate these pathogens, or at least decrease their number, is the use of antibacterial surfaces both on the tools used for processing and the food packaging itself. In this study, we have demonstrated that PF-co-doped TiO 2 NPs have a wide antibacterial spectrum and display enhanced light inducible antibacterial activity. Therefore, they are potential candidates for application in the food industry as active surface components.