Survival traits of Cronobacter sakazakii and Cronobacter maloniticus exposed to lethal Far-UVC 222nm and blue light 405nm

Cronobacter spp., recognised as a foodborne pathogen, presents a significant risk to neonates and vulnerable groups emphasizing the necessity for effective management. This study investigates the efficacy of a dual light system, combining far-UVC (222nm) and blue light (405nm), in combating this bacterial threat. We explored the survival phenotypes of Cronobacter sakazakii and Cronobacter maloniticus under lethal dual light exposure, after 30 minutes of light treatment, strain-dependent sensitivity was observed, with 21 out of 27 strains being fully inactivated within 290mJ/cm2 of far-UVC and 23800mJ/cm2 of blue light. Survivors, identified as Cronobacter spp., were detected, indicating bacterial resistance. Pigment production was also examined before and after the light treatment. All tested Cronobacter sakazakii exhibited yellow colonies on tryptic soy agar, whereas creamy white colonies were seen on milk agar. Colorimetric analysis verified the difference in pigment production on the two agar types. Light treatments had minimal impact on survivor pigment production, except for one isolate that lost pigment after light treatment on tryptic soy agar, resulting in failure to grow during subsequent enrichment. This research provides new evidence to understand the impact of dual light treatment on bacterial survival, contributing to the development of safer and more effective strategies in pathogenic bacteria control.


INTRODUCTION
Cronobacter sakazakii and Cronobacter maloniticus are persistent pathogens found in dairy food processing environments 1 , and often exhibits relatively small amounts of pigment production.Their ability to survive in dry conditions poses a significant risk, particularly as contaminants in infant formula products.Their pigment production is assumed to influence the fitness and persistence of Cronobater spp.Infections in infants resulting from the consumption of contaminated milk products can lead to serious conditions such as neonatal meningitis, septicemia, and necrotizing enterocolitis, with a high lethality rate ranging from 40 to 80 % 2 .Powdered infant formulas (PIF) are identified as the primary vehicles for transmission, prompting the need for new sanitization strategies to effectively control these pathogens in both food manufacturing and household food processing 2 .
Antimicrobial light sanitizations have proven effective in combating bacterial pathogens, with far-UVC and visible blue light emerging as promising alternatives for use in food processing environments.These wavelengths are considered safer than conventional germicidal UVC (254 nm), which poses the risk of carcinogenic harm to operators.Visible blue light is already applied clinically to treat human skin infections 3 .However, caution is needed due to potential photochemical damage to the retina and retinal pigment epithelium at high doses or prolonged exposure, necessitating the use of eyewear with filtration to mitigate these risks 3 .In contrast, far-UVC, even at high doses (up to 10 J/cm 2 ), does not cause biological harm to humans 4 .Moreover, it eliminates the need for human absence during operation, a requirement for conventional UVC.In the SARS-CoV-2 post-pandemic era, there is a growing exploration of alternative light sources beyond conventional UVC, indicating the potential for new methodologies in light sanitization 5 .
Our previous research successfully developed a dual light strategy 6 involving far-UVC and blue light, showcasing synergistic effects against extended spectrum β-lactamase producing Escherichia coli strains 7 .Notably, the study highlighted the predominant role played by far UVC in these outcomes.Given that far-UVC is invisible light, the integration with blue light serves as an additional indicator, facilitating practical applications.In addition to its antimicrobial effect, the blue light acts as a visual cue, indicating whether the light set is switched on or off in real-world scenarios.This study further advanced our comprehension of the dual light bactericidal effect by evaluating its efficacy against two dairy borne Cronobacter spp.(specifically Cronobacter sakazakii and malonaticus).The research explored the potential bactericidal effects of dual light exposure, combining far-UVC (222 nm) and blue light (405 nm).The correlation between temperature-dependent yellow pigment and the cell survival of Cronobacter spp. is explored under the applied light stress.This study reported specific bacterial phenotypes in response to the applied light treatment, prompting discussion on the necessity of clarifying bacterial responses and resilience under lethal light stress.

METHODOLOGY 2.1 Bacterial strains
A total of twenty-three Cronobacter sakazakii and four Cronobacter malonaticus isolates were used (as listed in Table 1

Dual light system
The light system utilised in this study was obtained from our previous research 6 , consisting of three blue LED (405 nm) bars with a total radiant flux of 36 W, and four USHIO far-UVC (222 nm) mercury-free excimer lamps rated at 12 W each.The light intensities of two light source were determined by the irradiance measurements using a SpectriLight ILT950 Spectroradiometer (International Light Technologies, Peabody, MA, USA).

Screening the antimicrobial efficacy of dual light using drop plating test
Drop plating method was employed for the investigation.An overnight cell culture with a concentration of approximately 9 log10 CFU/mL for each isolate was serially diluted in PBS.Approximately 2.8 to 3.0 log10 CFU cells (equivalent to 10 µl of a ~5 log10 CFU/ml cell diluent) were then individually applied to Tryptic Soy Agar (TSA) and Milk Agar (MA) plates (Fort Richard Laboratories Ltd).These plates were subsequently subjected to the dual light treatment for durations of 5, 10, 15, 20, 25, and 30 min.The distance between the plates and the light source was maintained at 25 cm.
The cell-seeded plates were prepared in duplicate, each containing 4-5 strains on a single plate.One of these duplicate plates was exposed to the light, while the other was covered with black paper and positioned under the same light source, serving as the control group.To assess resuscitation after light application, all plates were subsequently incubated at 37 °C for up to 48 hours.The presence of colonies within each isolate's designated drop zone were recorded.The absence of visible colonies was interpreted as undetectable and indicative of complete inactivation, as illustrated in Figure 1.The test was conducted in duplicate for all 27 strains on TSA and MA, respectively.The incidence of survivors was enumerated for assessing the bacterial tolerance to the applied light.Sampled survivors were revived in 10 ml of TSB overnight for subsequent antimicrobial testing or storage at -80°C for future use.The recovered colonies were also analysed using the Bruker Daltonik MALDI Biotyper 3.0 Real Time Classification (RTC) program to identify the survivor at genus level.

Ozone detection
During the light treatment, ozone levels in the vicinity of the agar surface (at a lighting distance of 25 cm) were monitored using a handheld Aeroqual Gas Sensor model AER-500L equipped with an interchangeable ozone sensor head (positioned 20 cm below the light source).Ozone concentrations were measured in parts per million (ppm) and recorded every minute throughout the illumination period.These measurements were conducted in duplicate.

Pigment production of C. sakazakii isolates
The ability to produce a yellow pigment 8 on TSA and MA at 25 °C, which occurs between 24 to 72 hours, was assessed for all tested isolates.To determine the pigment production of these isolates, control drop plates from the antimicrobial screening experiment were employed for colourimetric analysis.The colour of pigmented colonies was evaluated through visual observation and quantified in the Lab* (CIELAB) colour space using a CR20 colorimeter (Minolta, Osaka, Japan), positioned at the back of agar plates.Calibration was conducted using a white tile before each new sample measurement.This measurement process was carried out in triplicate, and the average values were employed to assess the presence of pigment.A 3D Scatter plot was generated using Minitab18.
After light treatment, survivor colonies' pigmentation was determined through visual observation, comparing them with colonies from control plates as a reference.Colourimetric analysis was impractical for survivors with limited colonization on agar (diameter 1-2 mm) as the colour meter's camera (diameter ~ 1 cm) captured them with poor resolution, hindering accurate color differentiation.

Antimicrobial efficacy of the dual light
Out of the 27 tested strains, 21 strains were completely inactivated within 30 minutes of light treatment, receiving 290 mJ/cm 2 of far-UVC and 23.80 J/cm 2 of blue light.Additionally, 11 strains were efficiently inactivated within 5 min of light treatment, receiving 48 mJ/cm 2 of far-UVC and 3.96 J/cm 2 of blue light.Illumination occurred at six different time points, ranging from 5 minutes to 30 minutes (refer to Table 3).Across all tested strains and time points, most seeded cells in each drop were eliminated, with sporadic survivor cells observed (see Figure 1).The revived colonies were recorded after 30 min of light treatment for each strain.Table 3 summarizes the survivor counts for the TSA and MA groups respectively, while Figure 2 presents the total survivor counts from both groups for each strain, revealing a strain-dependent light sensitivity pattern.Notably, 11 strains, including isolates 607, 54390, 3465-4A, 4.10C, 50C, AQ7, BAA-894, ERL073901, ERL1068, NZRM2029, and ES235, showed no survivors in the testing, classifying them as comparatively light-sensitive isolates.Among these, 5 strains (3465-4A, 4.10C, 50C, AQ7, and ES235) are dairy-borne isolates, while the remaining 6 strains are clinical strains.The second group consists of 16 isolates that required a 30-minute treatment with a higher dose, indicating light tolerance.Most of these isolates are sourced from dairy environments, except for two clinical isolates, ERL104073 and NZRM202a.The relationship between strain origin and light tolerance remains unclear.However, it is assumed that environmental isolates, accustomed to harsh conditions, might possess more sophisticated survival mechanisms that could be adapted to withstand antimicrobial light stress.
Table 3.The number of colonies that survived after the 30-min dual light treatment.

Strain
No.

Dual
Figure 2. The occurrence of cell survival for the tested 27 Cronobacter spp.after dual light treatment.
All surviving cells were identified as Cronobacter spp.using MOLDI-TOF.Throughout the treatment period, environmental ozone (O3) levels in the lighting area were monitored and remained within the range of 0.04-0.08ppm (Figure 3).The detected ozone levels in the study did not adversely affect cell viability.
Figure 3.The level of ozone (O3) during dual light treatment period.

Dual light treatment against randomly selected survivors
Survivors were randomly selected and enriched overnight in 10 ml of TSB broth at 37 °C.The overnight cultures were used to prepare drop plates, which were then tested for their susceptibility to light using the same dual light treatment.Each tested survivor isolate was labelled with a format like "mother isolate number, S+number (representing different generations of survivors), survived from which treatment time."For example, "25-S1-30" represents the first-generation survivor of isolate No. 25 from the 30 min light treatment.
All the survivors were drop-plated on TSA and MA and exposed to light treatments ranging from 5 to 30 min.As shown in Figure 4, the occurrence of cell survival was counted and compared between the mother strain and the first-generation survivor.Among the 13 first-generation survivors, 11 showed equal or fewer instances of cell survival than their mother isolates, while isolates 7, 8, 9, 10, 15, 22, and 25 typically exhibited fewer instances of survival, indicating decreased light tolerance.Survivors of isolates 24 and 13, on the other hand, displayed an increased occurrence of survival, suggesting the development of light tolerance.The strain-specific nature of the surviving phenotype cannot be conclusively proven at this time.The continuation of survivor passaging in future work could provide insights into how bacteria develop light resistance over generations by examining associated changes at both the genomic and functional levels.

Pigment production measured among the isolates.
The pigment-producing phenotype of all Cronobacter sakazakii strains were initially confirmed by observing the colouration of the grown colonies in the drop zone on TSA agar plates.These plates were incubated at 25 °C for up to 72 hours.All tested strains exhibited yellow colonies, indicating the presence of pigment production.However, there was variability in pigment production between the two tested agar types, as shown in Figure 5 (control groups).While all strains displayed yellow-pigmented colonies on TSA, they exhibited white creamy colonies on MA agar.The pigment production of each isolate on TSA and MA was quantified using colourimetric analysis in the Lab* colour space.In this colour space, L* represents lightness on a scale from black to white (ranging from zero to 100), while a* and b* represent chromaticity with no specific numeric limits.A 3D scatter plot clearly illustrated the differentiation between the TSA group and the MA group, indicating distinct pigment production on the two agar plates.Additionally, it was observed that the applied light treatments did not significantly affect the pigment production of the survivors in all the antimicrobial screenings.The only exception was one survivor from isolate No. 6, which formed a white colony on TSA.Unfortunately, this colony could not be enriched in subsequent broth cultivation, suggesting that the loss of pigment may have affected the cell's functionality and overall fitness.

CONCLUSION
This study assessed the effectiveness of a dual light system, combining far-UVC and blue light, against Cronobacter sakazakii and Cronobacter maloniticus.Notably, 21 out of 27 strains were fully inactivated within 30 min of dual light exposure, indicating strain resistance in 6 of the strains that continued to grow after treatment.Pigment production analysis on tryptic soy agar and milk agar revealed distinctive colours, minimally impacted by light treatments, except for one isolate experiencing pigment loss.The research highlighted specific bacterial phenotypes in response to treatment, generating new candidates used to understand bacterial responses and resistance under lethal light stress in the future.The efforts on clarifying the bacterial survival mechanism will give further scientific output to facilitate the development of safer and more effective strategies for controlling pathogenic bacteria.

Figure 4 .
Figure 4.The cell survival occurrence from light treatment on 1 st generation survivors of the selected isolates.Solid colour bars represent tests using survivor cells, while greyed colour bars represent tests of the individual mother isolate.

Figure 5 .
Figure 5. Colourimetric analysis of the pigmented cell groups for all isolates.Blue dot: Milk Agar group; Red dot: TSA group.Left: the plot analysis is presented; Right, the visual observation of colony colour is displayed (upper row, TSA Group; bottom row, Milk Agar).

Table 1 .
). Clinical Cronobacter spp.isolates were sourced from the Institute of Environmental Science and Research, Ltd. (ESR) in Porirua, New Zealand.Isolates 6-15 were acquired from AsureQuality Limited, New Zealand, in 2011.Single colonies of each isolate were statically incubated under aerobic conditions in tryptic soy broth (TSB) at 37°C for 20 h to obtain overnight bacterial culture (~9 log CFU/ml).Bacterial isolates used in this study.

Table 2 .
Applied light doses of two light sources during the dual light treatment.