Assessing the cytotoxicity and apoptosis-inducing ability of solar irradiated Salmonella Typhimurium in the RAW264.7 cell line in vitro

Abstract

Solar water disinfection (SODIS) reduces diarrheal disease in resource-limited settings. However, the host cell interactions of solar-inactivated pathogens remain ambiguous. This study assessed the effects of natural solar radiation on the viability, intracellular survival, cytotoxicity, and apoptotic responses of Salmonella enterica serovar Typhimurium in RAW 264.7 macrophages. Bacteria were exposed to (i) natural solar ultraviolet radiation (SUVR), (ii) identical conditions but shielded from SUVR, and (iii) heat-chemically inactivated with 0.5% phenol and 65 °C. Solar-irradiated S. Typhimurium for 4 h and 8 h was completely inactivated and showed no intracellular recovery in macrophages, whereas the non-irradiated controls showed a high intracellular replication rate. Solar-treated S. Typhimurium induced significantly lower cytotoxicity and fewer necrotic macrophages, while the non-irradiated bacteria triggered high LDH release and extensive cell death. Flow cytometric analysis showed that solar-inactivated S. Typhimurium induced attenuated necrotic responses compared to the substantial necrosis caused by viable bacteria. The findings demonstrated that solar irradiation neutralises S. Typhimurium infectivity and reduces its capacity to damage or kill macrophages, supporting the safety of SODIS-treated water.

Introduction

Salmonella enterica serovar Typhimurium (S. Typhimurium) is an enteric zoonotic pathogen that infects humans and animals. Ingestion of S. Typhimurium through contaminated food or water causes gastroenteritis¹. Globally, typhoidal and non-typhoidal Salmonella infections are a leading cause of morbidity, with high rates of mortality, in resource-limited settings, particularly in sub-Saharan Africa and parts of the Asian sub-continent². Solar disinfection of water is an effective method of reducing waterborne infections, such as gastrointestinal diseases, in developing countries³. During solar disinfection, UV-A rays and visible light induce oxidative stress by generating reactive oxygen species (ROS) that damage DNA, proteins, and lipids. The success of SODIS has been attributed to solar ultraviolet inactivation of disease-causing microbes in water⁴. Despite the advantages of SODIS, the influence of solar-inactivated microorganisms on the host’s immune cells remains unclear. Salmonella Typhimurium is an intracellular pathogen that can induce cytotoxic effects in both phagocytic and non-phagocytic cells⁵. Once S. Typhimurium invades a phagocytic cell such as a macrophage, it replicates and produces cytotoxins within the cell and induces an apoptotic response⁶. It is firmly established that the proteins encoded by the Salmonella pathogenicity islands (SPIs) on the S. Typhimurium chromosome are important for both invasion and intracellular survival⁷. SPIs encode several proteins that facilitate the invasion of epithelial cells by mediating actin cytoskeletal rearrangements and the internalization of bacteria⁸. The live form of S. Typhimurium can induce caspase-1-dependent pyroptosis⁹. Pyroptosis is dependent on the inflammasome, which regulates caspase-1 activation. This reaction leads to proteolytic processing of cytokines IL-1β and IL-8, which play crucial roles in acute and chronic inflammation and stimulate the recruitment of immune cells, driving the inflammatory response¹⁰.

Many studies have demonstrated the ability of solar irradiation to eliminate the cultivability of pathogenic microorganisms, including Salmonella species, in water. However, current research has almost exclusively focused on microbial inactivation in water rather than on the biological consequences of solar-inactivated pathogens in host immune cells¹². The fate of solar-inactivated pathogens after ingestion is still unknown. It remains unclear whether these bacteria retain the ability to invade host cells, induce cytotoxicity, or trigger inflammatory cell death pathways. This knowledge gap is crucial because there is still a possibility that solar-attenuated bacteria can potentially interact with immune cells, thereby influencing inflammation, immune activation, and host susceptibility to secondary infections. To date, no studies have comprehensively evaluated how solar-inactivated S. Typhimurium affects macrophage viability, intracellular survival, cytotoxicity, or apoptotic pathways.

This study addresses this important gap by evaluating, for the first time, the effects of solar irradiated S. Typhimurium on macrophage function and cell death pathways. Specifically, the study examined whether SODIS-treated S. Typhimurium retains the ability to invade and replicate within RAW264.7 macrophages, to induce cytotoxicity, apoptotic, pyroptotic, or necrotic responses. The study also compared solar-irradiated, heat-chemically inactivated, and non-irradiated bacteria, and it offers novel insights into the post-irradiation virulence properties of S. Typhimurium and the immunological consequences of consuming SODIS-treated water. Thus, the objectives were (i) to evaluate the effectiveness of the SODIS method for the inactivation of S. Typhimurium, (ii) to assess whether solar-inactivated S. Typhimurium can resuscitate within host macrophages, and (iii) to determine the cytotoxic and cell death responses of macrophages exposed to solar-irradiated S. Typhimurium.

Methodology

Culture of S. Typhimurium

Salmonella enterica serovar Typhimurium (ATCC^® 29629) was grown on Luria-Bertani (LB) agar. A single colony of S. Typhimurium was then inoculated in 33% LB broth and incubated at 37 °C with shaking at 150 rpm for 24 h until an exponential growth phase was reached. The culture was then diluted to an OD₅₄₈ of 0.002 in pre-warmed LB broth and incubated at 37 °C with shaking at 150 rpm overnight (18 h) until the stationary phase was reached¹¹. All bacterial culturing steps were performed in triplicate.

Preparation of solar and non-solar irradiated S. Typhimurium

Salmonella Typhimurium that reached the stationary phase was harvested by centrifugation at 4000 × g for 15 min and washed three times with autoclaved, still-bottled mineral water (Oasis, Vanderbijlpark, South Africa). The bacteria were diluted with the still bottled mineral water to an OD₅₄₆ of 0.1 (approximately 1 × 10⁷ CFU/mL) in a total volume of 15 mL in 25 cm³ transparent tissue culture flasks. Flasks were placed on aluminum foil and exposed to the sun for 0, 2, 4, 6, and 8 h on the rooftop of a building at the Vaal University of Technology (26°42’39.1"S 27°51’46.2"E -26.710858, 27.862820), on sunny cloudless days from 08:00–16:00. Solar ultraviolet irradiance (UVA + UVB radiation) was measured at 2 h intervals using a Lutron 340 A UV Light Meter (Lutron Electronics Company, Coopersburg, PA). For non-solar-irradiated controls, the samples were exposed to identical atmospheric conditions, except for SUV, by enclosing them in an opaque, ventilated box¹². Immediately upon completion of each 2 h irradiation interval, the flasks were retrieved and transported to the cell culture facility; suspensions were kept on ice and processed for infection within 60 min. No storage beyond this window or freeze-thaw cycles occurred before infection assays. Viability after treatments was evaluated using the Miles and Misra drop method and expressed as Log₁₀ CFU/mL. All solar irradiation and enumeration procedures were done in triplicate.

Preparation of heat-chemical inactivated S. Typhimurium

Stationary-phase Salmonella Typhimurium was harvested as above and washed 3 times with autoclaved 1× PBS to remove LB broth. The bacterial suspension was adjusted to an OD₅₄₆ of 0.1 (approximately 1 × 10⁷ CFU/mL) in 1× PBS and inactivated using a combination of heat and chemical treatment (0.5% phenol at 65 °C for 1 h)¹³. The heat-inactivated culture was incubated at room temperature with shaking at 150 rpm for 48 h. The viability was assessed on LB agar plates using the Miles and Misra drop counting technique¹⁴. Following the 48-h incubation, the cultures were washed, resuspended in infection medium (See "Invasion and intracellular growth analysis of S. Typhimurium inRAW264.7 cells" section), and used for infection within 60 min of final processing. All the chemical inactivation steps and enumeration were performed in triplicate.

Testing of bacterial viability

For heat/chemical-attenuated, solar-irradiated, and non-irradiated controls, 100 µL of each sample was serially diluted 1:10 in sterile PBS to a final dilution factor of 10^− 6. Then 20 µL was spotted on LB agar plates in numbered sectors, air-dried for 30 min, and incubated at 37 °C for 24 h. Colonies were counted in drop areas containing the largest number of discrete colonies without confluence or gross diminution due to overcrowding. The CFUs were expressed as Log₁₀ CFU/mL. In addition, at the time of infection (See "Invasion and intracellular growth analysis of S. Typhimurium inRAW264.7 cells" section), an aliquot of each inoculum was plated to verify viability. Viability assays for all conditions were performed in triplicate.

Establishment of the macrophage cell line

The murine macrophage cell line RAW 264.7 (Cellonex, Separations Scientific, Randburg, South Africa) was maintained in Dulbecco’s Modified Eagle’s Medium (DMEM), supplemented with 10% foetal bovine serum (FBS) and 1% Penicillin /Streptomycin at 37 °C in a humidified 5% CO₂ incubator.

Invasion and intracellular growth analysis of S. Typhimurium in RAW264.7 cells

RAW264.7 macrophages were seeded in 6-well plates at 1 × 10⁵ cells/ml and incubated for 24 h at 37 °C in a humidified 5% CO₂ incubator. On the day of infection, the monolayers were washed once with 1× DPBS and 1000 µL of infection media (DMEM-I) containing S. Typhimurium (MOI 10:1). A negative control (DMEM + 10% FBS) was added to the 6-well plates and incubated in a humidified incubator at 37 °C with a 5% CO₂ atmosphere for 4 h.

For MOI standardization across treatments, the inoculum volume for each condition (NS, SI4, SI8, HA) was calculated based on the pre-treatment CFU/mL estimate (OD-to-CFU conversion verified by plating representative aliquots from the batch culture before treatment). Thus, the inactivated groups (SI4, SI8, HA) received the same inoculum volume, corresponding to a MOI of 10:1 based on pre-treatment CFU, ensuring equivalent bacterial particle input across groups. At the time of infection, the viability of each inoculum was verified by plating (NS inoculum yielded colonies consistent with the target MOI, whereas SI4, SI8, and HA inocula yielded 0 CFU, confirming inactivation).

Infection assays commenced within 1 h of completing the irradiation and attenuation procedures; the 3, 24, and 48 h post-infection time points were counted from the time of inoculum addition. After the 4 h infection period, unbound bacteria were washed off with DPBS. Then, DMEM containing 10% FBS and 20 µg/ml gentamicin was added, and cultures were incubated for 3, 24, or 48 h at 37 °C in a humidified CelCulture^® CO₂ Incubator (ESCO, Singapore) at 37 °C with 5% CO₂¹⁵. All invasion and intracellular growth assays were performed in triplicate.

Harvest of infected macrophage lysate for CFU plating

At 3, 24, and 48 h post-infection, the culture media were aspirated, and the cells were lysed by adding 1000 µL sterile lysis buffer (0.5% Triton X-100) per well, followed by incubation at room temperature for 5 min¹⁶. The lysates were mixed and transferred to sterile 96-well plates for serial 1:10 dilutions in LB broth (25 µL into 225 µL). Then 20 µL of each dilution was spotted in triplicate onto LB agar plates and incubated at 37 °C for 24 h before enumeration.

Cytopathic and cytotoxic effects of S. Typhimurium in RAW264.7 cells

The potential for the (i) heat and chemically inactivated, (ii) non-solar irradiated, and (iii) solar irradiated for 4 and 8 h S. Typhimurium cells to induce cytopathic effects in the macrophages after 3, 24, and 48 h post-infection was investigated. The macrophages for each treatment were monitored for cell detachment, rounding, and floating using an inverted bright-field light microscope at 20× magnification¹⁷. Cytotoxicity was assessed using the lactate dehydrogenase (LDH) assay obtained from Thermo Fisher Scientific (Waltham, MA) at 3 and 24 h post-infection according to the manufacturer’s instructions. LDH release was expressed as percent cytotoxicity using the formula provided (Eq. 1). Inoculum volumes were matched across treatments to maintain a pre-treatment MOI of 10:1, enabling comparison of cellular responses to equivalent bacterial inputs despite differences in post-treatment viability. All cytopathic and cytotoxicity assays were performed in triplicate.

Equation (1)

.

Experimental value (Exp. Value) stands for LDH released by macrophages infected with S. Typhimurium; effector cells spontaneous control (Eff cells control) is the LDH control for S. Typhimurium cells; Target cells Spontaneous control (Target cells control) represents the LDH released by non-infected macrophages, and the Target Cell Maximum Control (Target cell max. control) represents the LDH maximum release by lysis of macrophages using 10% (vol/vol) of 10× lysis buffer.

Apoptosis assay of treated RAW264.7 cells

Apoptosis and necrosis of the treated RAW264.7 macrophages were assessed at 3 and 24 h post-infection using flow cytometry on a Guava EasyCyte 8HT instrument (Merck/Millipore, Molsheim, France) with a MultiCaspase SR kit (Merck/Millipore) according to the manufacturer’s instructions. Cells were incubated for 1 h at 37 °C in MultiCaspase SR solution containing an SR-Peptide fluorophore, washed twice with apoptosis wash buffer, stained with 7-AAD, incubated at room temperature for 10 min, and analyzed. Melphalan (50 µg/mL) served as a positive control for apoptosis. All cell death assays were performed in triplicate.

Statistical analysis

All experiments were conducted with three independent biological replicates unless otherwise specified. Data were presented as mean ± SEM. and two-way ANOVA (for analysis involving treatment and time factors), followed by appropriate post hoc tests to control for multiple comparisons (Šidák’s and Tukey’s multiple comparisons). Analyses were performed using GraphPad Prism 10.

Results

Viability assessment of S. Typhimurium

The sensitivity of S. Typhimurium to natural sunlight is shown in Fig. 1. The lowest solar irradiance was observed at the beginning of the experiment at 8.00 (15.7 ± 0.3 W/m²), and the highest of 47.4 ± 0.8 W/m² was reached at midday. Salmonella Typhimurium was completely inactivated within four hours of exposure to sunlight. However, the non-solar-irradiated samples remained viable at 0, 4, and 8 h. There was a statistically significant (p < 0.001) difference in mean viability between the solar and non-solar treatments. The heat- or chemical-attenuated S. Typhimurium was not viable (results not shown in the graph).

Fig. 1.

Viability (Log₁₀ CFU/ml) of S. Typhimurium following 0, 4, and 8 h exposure to solar irradiation (SI) or non-solar irradiated controls (NS). Measurements were taken immediately after each exposure interval. Error bars indicate ± SEM (n = 3 independent biological replicates). Two-way ANOVA (treatment × time) with Šidák’s multiple comparisons; SI vs. NS at 2–8 h, p < 0.001; 0 h not significant.

Based on the inactivation profile observed in Fig. 1, both the 4-hour and 8-hour solar irradiation treatments, representing the minimal timepoint at which S. Typhimurium became fully inactivated (4 h) and prolonged exposure control (8 h), were selected for subsequent infection, intracellular survival, cytotoxicity, and apoptosis assays.

Invasion and intracellular growth assessment of S. Typhimurium

This study used the Gentamicin Protection Assay to assess the potential for the (i) heat/chemical attenuated (HA), (ii) non-solar irradiated (NS), and (iii) solar irradiated (SI4 and SI8) bacteria to recover and propagate in the macrophages. Co-cultures of macrophages and S. Typhimurium that were solar irradiated for 4 and 8 h showed no bacterial growth after 3, 24, and 48 h of culture (Fig. 2). Similarly, S. Typhimurium that were heated and chemically inactivated did not propagate in the macrophages. However, non-solar-irradiated S. Typhimurium were able to multiply within macrophages at 3, 24, and 48 h. The highest level of intracellular growth of S. Typhimurium occurred after 3 h of stimulation with the non-solar irradiated samples (Fig. 2). However, at 24 h, growth reduced slightly. Intracellular bacterial growth increased after 48 h of macrophage infection with non-solar irradiated S. Typhimurium (NS) (Fig. 2).

Fig. 2.

Intracellular growth assay of macrophages that were (i) untreated, (ii) treated with heat and chemically attenuated S. Typhimurium (HA), (iii) treated with non-solar irradiated S. Typhimurium (NS), (iv) treated with 4 h- and 8 h-solar irradiated S. Typhimurium, (SI4 and SI8, respectively). The intracellular growth analysis was conducted at 3-, 24-, and 48-hours post-infection. Bars represent mean Log₁₀CFU/mL ± SEM (n = 3).

Macrophage morphology was assessed at 3, 24, and 48 h after exposure to heat attenuated (HA), non-solar irradiated (NS), and solar irradiated S. Typhimurium (SI4 and SI8). At 3 h, the macrophages in all treatment groups showed normal morphology with no observable cytopathic effects (Fig. 3).

Fig. 3.

Cytopathic effects of RAW264.7 macrophages at 3 h that were (A) untreated, (B) treated with heat and chemically attenuated S. Typhimurium (HA), (C) treated with non-solar irradiated S. Typhimurium (NS), (D) treated with 4 h-solar irradiated S. Typhimurium (SI4), (E) treated with 8 h-solar irradiated S. Typhimurium (SI8). (Scale bar = 50 μm).

However, at 24 h post-infection, morphological differences were observed. Macrophages exposed to NS bacteria showed pronounced cytopathic damage, including rounding, shrinkage, membrane disintegration, and detachment from the culture surface (Fig. 4C). In contrast, macrophages treated with solar irradiated (SI4, SI4) and HA bacteria showed milder changes. The non-treated (M) (Fig. 4A) and HA-treated cells (Fig. 4B) maintained an intact morphology, with occasional enlargement and elongation observed in HA-treated cells (Fig. 4B). Solar irradiated (SI4 and SI8) S. Typhimurium (Fig. 4D and E) resulted in modest swelling and rounding of some cells, but without extensive fragmentation observed in the NS-treated group (Fig. 4C).

Fig. 4.

Cytopathic effects of RAW264.7 macrophages at 24 h i. that were (A) untreated (B) treated with heat and chemically attenuated S. Typhimurium (HA), (C) treated with non-solar irradiated S. Typhimurium (NS), (D) treated with 4 h-solar irradiated S. Typhimurium (SI4), (E) treated with 8 h-solar irradiated S. Typhimurium (SI8). Note: Macrophages infected with solar irradiated S. Typhimurium exhibit reduced cytopathic effects. KEY: Red arrows show macrophages with pseudopods; Yellow arrows show shrunken macrophages (Scale bar = 50 μm).

After 48 h, the distinctions between treatments became more pronounced. The non-treated cells (M) (Fig. 5A) proliferated and maintained a healthy cellular morphology. Whereas the NS-treated macrophages showed severe cytopathic effects marked by extensive debris (Fig. 5C). In contrast, macrophages exposed to SI4, SI8, or HA bacteria maintained considerable structural integrity. HA-treated cells continued to show minor elongation or activation-like morphology (Fig. 5B), while SI4 and SI8 treatments produced mild rounding but no primary detachment or lysis (Fig. 5D-E). Unlike the NS-treated macrophages, no widespread cell destruction was observed in any of the inactivated treatments.

Fig. 5.

Cytopathic effects of RAW264.7 macrophages at 48 h i. that were (A) untreated (B) treated with heat and chemical attenuated S. Typhimurium (HA), (C) treated with non-solar irradiated S. Typhimurium with (NS), (D) treated with 4 h-solar irradiated S. Typhimurium (SI4), (E) treated with 8 h-solar irradiated S. Typhimurium (SI8). Note: Macrophages infected with irradiated S. Typhimurium exhibit reduced cytopathic effects. KEY: Green arrows show elongated macrophages; Red arrows show macrophages with pseudopods; Yellow arrows show shrunken macrophages. (Scale bar = 50 μm).

In the cytotoxicity analysis, the macrophages co-cultured with both the heat and chemically attenuated and solar irradiated S. Typhimurium did not release any LDH in the first three hours (Fig. 6). Non-solar irradiated S. Typhimurium induced the highest level of cytotoxicity, with LDH release of 55% ± 6% at 3 h and 75% ± 1% at 24 h post-infection (Fig. 6). In contrast, macrophages exposed to solar-irradiated bacteria (SI4 and SI8) exhibited significantly lower LDH release.

Fig. 6.

LDH Cytotoxicity in RAW264.7 macrophages at 3 and 24 h post-infection with S. Typhimurium under four treatment conditions (i) heat and chemically attenuated (HA), (ii) non-solar irradiated S. Typhimurium (NS), and (iii) solar irradiated samples after 4 (SI4) and 8 (SI8) hours. Bars represent mean % cytotoxicity ± SEM (n = 3). Letters denote homogenous subgroups identified by Tukey’s HSD test (α = 0.05) following significant two-way ANOVA.

Apoptotic assays for macrophages infected with S. Typhimurium

Flow cytometric analysis of cellular states across the six treatment conditions showed significant alterations in cell viability and death pathways. The untreated macrophages (M) showed high viability at 3 h p.i. (99 ± 0.6%) with minimal cell death. Moreover, Melphalan treatments also showed high viability (85% ± 0.4%) and induced moderate necrotic cell death (12% ± 1%) and minimal late apoptosis (2% ± 1%). The NS, SI4, and SI8 exhibited substantially reduced viability (49% ± 4%, 59% ± 0.1%, and 67% ± 9%, respectively), with prominent necrotic and late-apoptotic populations. The HA treatments showed moderate viability (71% ± 1%) and high mid-apoptotic populations compared to other treatments. Melphalan, included as a positive apoptosis control, showed a shift towards mid-apoptotic and late-apoptotic populations.

Two-way ANOVA revealed significant effects of treatment and cell-death state (F _{(15, 48)} = 53.56, p < 0.0001), indicating that the effects of each treatment differed across the cellular states examined. Within-state post hoc testing showed distinct statistical groupings: NS clustered uniquely in the necrotic state, SI4 and SI8 formed intermediate groups, and Melphalan was the only group with a high proportion of mid-apoptotic cells. Therefore, this showed that solar irradiation attenuates the cytotoxic effects of S. Typhimurium on macrophages, producing cell death profiles significantly different from NS but not entirely equivalent to HA or untreated controls (Fig. 7).

Fig. 7.

Flow cytometry analysis (A) Representative fluorescence plot RAW264.7 of macrophages following 3 h of stimulation with S. Typhimurium that were (i) untreated (M) (ii) treated with heat and chemical attenuated S. Typhimurium (HA), (iii) treated with non-solar irradiated S. Typhimurium (NS), (iv) treated with 4 h- and 8 h-solar irradiated S. Typhimurium (SI4 and SI8, respectively) and (v) treated with 50 ug/mL of Melphalan (positive control). (B) Data = mean ± SEM (n = 3). Letters denote statistical groupings (Tukey’s HSD, α = 0.05) following significant two-way ANOVA.

After 24 h post-infection, flow analysis using the Multi-caspase SR kit revealed distinct cell death profiles across the treatments (Fig. 8). The results show that heat- and chemical-treated S. Typhimurium, as well as non-solar-irradiated and solar-irradiated S. Typhimurium, showed an increase in necrotic cells compared to those treated for 3 h. (Fig. 8) The heat/chemical-treated S. Typhimurium exhibited the lowest proportion of necrosis (9%). There was a significant (p < 0.001) increase in the number of necrotic macrophages that were infected with the non-irradiated S. Typhimurium (49%±1%). The macrophages treated with S. Typhimurium that was solar irradiated for 8 h (SI8) had fewer necrotic cells than the macrophages treated with non-irradiated S. Typhimurium. The differences in the percentage of necrotic cells were statistically significant (p < 0.001). However, there were non-significant differences in necrotic cells between the macrophages infected with S. Typhimurium irradiated for 4 and 8 h (SI4 and S8) (43%±2% and 35%±0.3%, respectively). No significant differences were found in necrotic cells between the NS and SI4 samples (49%±1% and 43%±2%, respectively) (Fig. 8). Melphalan treated macrophages demonstrated a distinct pattern characterized by a high percentage of mid apoptotic and late apoptotic cells (71% ± 1% and 21% |± 0.3%, respectively) with minimal viability (7% ± 0.2%). This apoptotic pattern was unique to Melphalan and absent in all the bacterial treatments. At 48 h post-infection, no cells were detected by flow cytometry in the non-solar-irradiated control; thus, the results at 48 h were not considered.

Fig. 8.

Flow cytometry analysis (A) Representative fluorescence plot RAW264.7 of macrophages following 24 h of stimulation with S. Typhimurium that were (i) untreated (M) (ii) treated with heat and chemical attenuated S. Typhimurium (HA), (iii) treated with non-solar irradiated S. Typhimurium (NS), (iv) treated with four h- and 8 h-solar irradiated S. Typhimurium (SI4 and SI8, respectively) and (v) treated with 50 ug/mL of Melphalan (positive control). (B) Data = mean ± SEM (n = 3). Letters denote statistical groupings (Tukey’s HSD, α = 0.05) following significant two-way ANOVA.

Two-way ANOVA showed highly significant effects of treatment, cell death state, and their interaction (F _(15,48) = 1810.36, p < 0.0001), confirming that treatment-dependent differences varied strongly across states. Post-hoc comparisons showed that NS, SI4, and SI8 formed distinct statistical groups in the necrotic state. In contrast, all treatments except Melphalan clustered together in mid-apoptosis and late apoptosis due to uniformly low apoptotic activity (Fig. 8). This showed that solar irradiation substantially reduces but does not eliminate cytotoxicity of S. Typhimurium, and the attenuation pattern is more pronounced at 24 h than at 3 h (Figs. 7 and 8).

Discussion

Viability of S. Typhimurium

One of the main findings of this study is that the viability of S. Typhimurium was significantly (p < 0.001) reduced when exposed to chemical and heat treatment (positive control) and solar radiation for either 4–8 h (Fig. 1). The rapid loss of cultivability reinforces the effectiveness of SODIS as a low-cost water treatment method particularly for communities relying on untreated surface water^18,19. Some of the reasons proposed for the complete loss of viability of micro-organisms in SODIS-treated water are the effects of reactive oxygen species (ROS) generated by the UV-A radiation^20,21, loss of membrane potential²², and protein damage²³. Moreover, the complete elimination of viable bacteria after 4 h occurred under natural light rather than in controlled laboratory UV exposure, highlighting the effectiveness of SODIS in real-world conditions. The results confirm that the disinfection times commonly recommended for SODIS users are sufficient for neutralizing pathogenic Salmonella under typical African climatic conditions.

Intracellular growth assessment of S. Typhimurium

A significant contribution of this study is the evidence that solar-irradiated S. Typhimurium not only loses cultivability in water but also fails to resuscitate or replicate within macrophages (Fig. 2). Both the 4-h- and 8-h -irradiated bacteria displayed complete attenuation, with no intracellular recovery after 3, 24, and 48 h of infection. This indicates that solar irradiation reduces virulence functions such as invasion and resistance to macrophage death^51,53.

In contrast, the non-irradiated controls showed high intracellular replication, consistent with various studies showing that Salmonella species are invasive pathogens capable of replicating within host cells and enhancing host cell survival to prolong infection^{25,] 26}). This persistence and growth of the Salmonella species within host cells is the leading cause of the severity of gastroenteritis²⁷. A slight decrease in CFUs of the bacteria was observed from 3 to 24 h in non-solar-irradiated samples, and thereafter the numbers increased from 24 to 48 h. The initial decrease in CFUs in the macrophages is probably due to their phagocytic and bactericidal activity, which enables the ingestion and destruction of invading pathogens²⁴. The subsequent increase at 48 h (Fig. 2) indicates bacterial adaptation, replication, and macrophage lysis, which are the main characteristics of virulent Salmonella infection^50,55,58.

These results provide original evidence that S. Typhimurium cannot regain infectivity once internalized. Notably, this excludes the possibility that solar-inactivated pathogens might resuscitate within host cells, addressing a longstanding safety concern about SODIS water.

Cytopathic and cytotoxic effects of S. Typhimurium in RAW264.7 cells

Cytopathic analysis of the macrophages during early infection (3 h) for all the treatments generally showed that the cells exhibited less pronounced cytopathic effects (Fig. 3). However, at 24 and 48 h post-infections (Figs. 4 and 5), solar irradiated S. Typhimurium significantly reduced cytopathic effects compared with non-irradiated bacteria. In contrast, macrophages exposed to viable S. Typhimurium displayed morphological disruption, detachment, and fragmentation, whereas cells treated with solar-irradiated and heat-attenuated bacteria remained intact (Figs. 4B-E and 5B-E). Instead, they exhibited morphological features consistent with controlled inflammatory cell death rather than catastrophic lysis.

Moreover, macrophages stimulated with solar-irradiated cells demonstrated moderate swelling, rounding, and occasionally pseudopod formation (Figs. 4D-E and 5D-E). These features are commonly associated with pyroptotic activation rather than uncontrolled necrosis⁴⁹. This suggests that while solar-injured bacteria cannot invade or replicate, they still retain molecular patterns capable of activating macrophage immune pathways.

The LDH cytotoxicity assays strengthened these observations (Fig. 6). After 3 h, only the viable Salmonella caused significant cytotoxicity. In comparison, solar-irradiated and heat-attenuated samples showed no cytotoxicity (Fig. 6). By 24 h, moderate LDH release was observed in solar-irradiated conditions (Fig. 6), likely reflecting GSDMD-pore mediated leakage typical of pyroptosis, or secondary necrosis of activated macrophages (Devant et al.³⁶; Yeisley et al.³⁷. Nonetheless, cytotoxicity remained significantly lower than in macrophages infected with viable S. Typhimurium.

Moreover, although solar-irradiated S. Typhimurium was non-viable and non-culturable, the observed cytotoxicity cannot be attributed solely to active infection, intracellular replication, or classical virulence mechanisms. Instead, the responses also likely reflect macrophage recognition of residual pathogen-associated molecular patterns (PAMPs), such as LPS, flagellin, and outer membrane proteins, that remain intact even after inactivation^54,57,59.

Cell death assays for macrophages infected with S. Typhimurium

Upon bacterial infection, macrophage cell death can occur through a variety of pathways, including apoptosis, necrosis, autophagic cell death, necroptosis, oncosis, pyronecrosis, and pyroptosis⁴¹. Flow cytometric analysis was done to ascertain the type of cell death induced by the various treatments. The results demonstrated that solar-irradiated bacteria induced significantly less necrosis than non-solar-irradiated S. Typhimurium at both 3 and 24 h. Viable Salmonella rapidly induced high levels of cell death, consistent with the activation of inflammasome-driven pyroptosis and later-stage necrosis resulting from intracellular bacterial replication and macrophage lysis^9,52. The solar-irradiated S. Typhimurium samples showed signs of pyroptotic cell death, with characteristics like necrotic cell death, including increased membrane permeabilization that allows the 7-AAD dye to permeate and intercalate into the DNA (Ryder et al. 2025). Pyroptosis arises from the caspase-1-dependent inflammasome activation and or recognition by the macrophage NLRP3 and NLRC4 receptors. Several studies have proposed that pyroptosis may benefit the host during infection, as it is an inflammatory form of cell death that recruits neutrophils in vivo⁴³. Caspase-1 activation also influences the development of adaptive immune responses by inducing the production of IL-18 which plays a significant role in stimulating the differentiation of T helper cells⁴⁴.

In contrast, solar-irradiated and heat/chemical-attenuated S. Typhimurium induced a more controlled pattern of cell death, consistent with limited pyroptotic activation . However, without progression to extensive necrosis (Fig. 7). The reduced necrotic response after 8 h of solar irradiation suggests that prolonged irradiation causes deeper protein denaturation or antigenic degradation, potentially lowering the bacteria’s inflammatory capacity^48,56.

Heat/chemical inactivation and solar irradiation inactivated S. Typhimurium, and the heat/chemical-inactivated, solar-irradiated S. Typhimurium also showed signs of necrotic cell death, but to a lesser extent than viable S. Typhimurium. The increase in necrotic cells in the flow cytometry samples could also be due to efferocytosis. When macrophages die, they are engulfed and digested by other macrophages through efferocytosis⁴⁵. Efferocytosis can, therefore, aid in eliminating inflammatory conditions. However, in the current study, efferocytosis was likely not occurring as rapidly as it should, and the macrophages released danger signals, which probably led to secondary necrosis⁴⁶.

Importantly, this study provides the first evidence that solar-irradiated S. Typhimurium elicits a measurable, weaker necrotic response in macrophages. This finding carries clear implications for SODIS users. Although immune recognition may occur, the magnitude of host cell damage is significantly lower than in viable infections. This can potentially reduce the likelihood of excessive inflammation or tissue injury following ingestion of SODIS-treated water.

Limitations

This study focused solely on cell death outcomes using morphological analysis, LDH release, and flow cytometry. While these approaches reliably distinguish broad forms of membrane damage and cell viability loss, they do not identify the precise molecular pathways leading to macrophage death. For instance, upstream inflammatory mediators, chemokines, or inflammasome-specific markers, such as the IL-1 family of cytokines and gasdermin-D cleavage. As a result, pyroptosis, necrosis, or secondary necrosis cannot be fully differentiated, especially in treatments that showed limited but detectable membrane permeabilisation. The mechanistic insights were beyond the scope of this study, which primarily aimed to compare the cytotoxicity profiles of viable versus solar-inactivated S. Typhimurium. Therefore, future investigations incorporating inflammasome signaling assays and chemokine profiling would provide more detailed insight into the pathways involved in macrophage responses to solar-inactivated bacteria.

Conclusion

This study shows that solar irradiation effectively eliminates the viability of S. Typhimurium and deprives it of its capacity to invade and replicate within macrophages. Importantly, this study shows, for the first time, that solar-inactivated Salmonella significantly reduces cytotoxicity and markedly attenuates a necrotic response in RAW264.7 cells compared with viable bacteria. While solar-irradiated organisms retain some ability to stimulate macrophage activation, the resulting cell death resembles controlled pyroptotic signaling rather than destructive lysis, suggesting limited inflammatory risk. These findings extend the current understanding of SODIS beyond pathogen activation alone by clarifying how solar-damaged microorganisms interact with host immune cells after ingestion. The absence of intracellular resuscitation and lower cytotoxicity provides strong evidence supporting the safety of SODIS-treated water contaminated with S. Typhimurium. Therefore, future work may involve investigating cell death mechanisms in more detail by profiling cytokine responses, assessing VBNC states using molecular methods, and using pyroptosis inhibitors to confirm pathways.

Author contributions

P.C. conception of work, data analysis, drafting the manuscriptC.S. Data analysis, critical review of manuscriptE.U-J. critical reviewM.P. Supervision of research, critical review and final approval.

Data availability

The data from this research will be made available upon request from the first author Patience Chihomvu or it is available on request.

Declarations

Competing interests

The authors declare no competing interests.