The C. elegans strains used in this study are listed in Supplementary Table S1. Worms were cultured at 20°C on a nematode growth medium (NGM) agar (Brenner, 1974). Plates were seeded with pre-cultured bacterial strains according to the probiotic supplementation method. C. elegans were age-synchronized using the egg laying technique and incubated at 20°C until the larvae reached the desired stage of development for subsequent experimentation.

The bacterial strains used in this study are listed in Supplementary Table S2. A solution of the probiotic supplement Lactococcus lactis and Leuconostoc mesenteroides was prepared in liquid NGM buffer. The culture was grown overnight at 35°C, concentrated, and resuspended at 15.24 mg/mL. The probiotic solution was added to OP50 at 10% (w/v) and seeded on NGM agar plates using a final concentration of 8 mg/mL. CeMbio cultures were prepared according to the previously designed methods (Dirksen et al., 2020) and seeded on NGM agar plates according to the protocol mentioned previously.

All survival analysis were performed at 20°C. The L4 stage worms were transferred to fresh plates and used on day 3 for the survival assay (Amrit et al., 2014). The worms were transferred every day until they ceased producing progeny, after approximately 3–5 days and then every 2 days until all worms died, unless indicated otherwise (the plates were spotted for use every 2 days from fresh cultures). For each experiment, at least three plates (25 worms per plate) per bacterial strain were analyzed for the CeMbio survival analysis, and for OP50 supplementation experimentation, five plates (at least 25 worms per plate) per bacterial strain were analyzed. A death event was determined via ceased pharyngeal pumping and no response to gentle prodding with a platinum worm pick. The worms were examined daily. If the worms were unintentionally lost, AVID (age-associated vulval integrity defects frequently described as ruptured) (Leiser et al., 2016), or had undergone matricide, these were censored and excluded from the survival analysis. Statistical analyses were performed using GraphPad Prism 9.5.1 for statistical log-rank (Mantel–Cox) and Gehan–Breslow–Wilcoxon analysis, in all cases p <0.05 was considered significant.

Fecundity was measured with five individual L4 synchronized hermaphrodites (five repeats/25 worms in total/bacterial composition). Each individual adult was transferred to fresh plates daily (one worm per plate) until reproduction ceased. The total number of viable offspring was counted per day per worm.

Body characteristics and locomotion were measured from three plates of (at least 20) age-synchronized worms per bacterial strain, at day 1 of adulthood. Videos were taken using a stereo microscope (Nikon S74747) with a D1000 camera and then analyzed using WormLab software (MBF Bioscience). The software analyzed the free roaming locomotion patterns of the worms with the speed metric being reported for this study. These assays were established according to previous recorded methods (Amrit et al., 2014; Keith et al., 2014; Mack et al., 2018; Dirksen et al., 2020).

The animals were raised as described previously for lifespan assays. On day 8, the animals were removed from the NGM plates and suspended for 3 h in liquid cultures with blue food dye (FD&C Blue #1, B0790, TCI, 5.0% wt/vol in liquid NGM). The animals were then washed with M9 to remove the unabsorbed dye. Then, the images were captured using a stereo microscope (Nikon S74747) with a D1000 camera for the presence or absence of blue food dye in the body cavity and analyzed using LAS X software (Leica). The following calculation was used to determine the percent of intestinal leakage “permeability”: S m u r f   a s s a y : % = i n t e s t i n e + l e a k a g e + l e n g h t   o f   l e a k a g e   μ m b o d y   c a v i t y + l e n g h t   μ m .

Three or more independent experiments were carried out, equaling 8–10 animals per condition. This is as was adapted from the previous methods (Gelino et al., 2016; Kim and Moon, 2019). Data were analyzed using GraphPad Prism version 9.5.1 (GraphPad Software, San Diego, California, United States).

The expression of the stress reporters was measured according to Manjarrez and Mailler, 2020, with supporting evidence for heat shock induction of these stress reporters from An and Blackwell (2003), Bar-Ziv et al. (2020), Bischof et al. (2008), Chen et al. (2023), Labbadia et al. (2017), Taylor et al. (2021), Yoneda et al. (2004). The hsp-4::GFP positive control was treated with tunicamycin for 6 h at 20°C, with a 24-h recovery at 20°C prior to imaging (Yoneda et al., 2004; Bischof et al., 2008). As a hsp-6p::GFP positive control, 1-day-old worms were heat-shocked for 6 h at 30°C, with a 2-h recovery period at 20°C. All experimental measurements were taken under basal conditions: tunicamycin with 50 ng/mL, or heat-shocked at 35°C, for 30 min followed by a 1-h recovery period at 20°C prior to imaging. The images were acquired using a Leica DMi8 fitted with a SpectraX illuminator (Lumencor), an ORCA Flash4.0 v2 sCMOS camera (Hamamatsu), and LAS X software (Leica). Relative fluorescence units (RFUs) were calculated using a LAS X relative fluorescence calculator using a 200 × 200-µm square as a background measurement for the fluorescence intensity of the worm. F(t) = fluorescence channel/region of interest (ROI); F(0) = fluorescence channel/background (Bkg), and K is set to 1 as normalized EGFP (Stepanenko et al., 2008): F = d F F 0 = K * F t F 0   F 0 F b .

Upregulation of the positive control for each stress reporter was used to obtain the F_max (maximum reporter intensity) (Manjarrez et al., 2020; Manjarrez and Mailler, 2020). The normalized values were plotted, and p-values were generated by the nested t-test using GraphPad Prism version 9.5.1 (GraphPad Software, San Diego, California, United States).

Prism 9.5.1 software was used for the survival analysis, using the log-rank (Mantel–Cox) method which analyzed the significance of difference in the overall curve. The Gehan–Breslow–Wilcoxon method was used to assess the significance of survival earlier versus later in the survival timeline. The statistical analysis resulting from the Mantel–Cox, Gehan–Breslow–Wilcoxon, and nested and Student’s t-test, in all cases, showed that p <0.05 was considered significant. An asterisk, in the figures, indicates statistical significance of the aforementioned statistical analysis as compared to its indicated reference. At least three biological replicates comprise all the referenced datasets.

The effect of CeMbio, the laboratory-derived microbiome based on natural isolates, and CeMbio supplemented with L. lactis or L. mesenteroides on the survival of C. elegans was compared to that of the commonly used E. coli, OP50. The results showed that all three CeMbio treatments exhibited significant differences in survival compared to OP50 (Figure 1). While CeMbio showed the longest median survival and overall lifespan when supplemented with L. lactis or L. mesenteroides, it demonstrated a reduction in the median survival and overall lifespan, contrary to our initial expectation (Figure 1). This survival analysis suggests that the supplementation of L. lactis or L. mesenteroides to CeMbio had a negative effect on the balance of the differentiated CeMbio microbial community.

This led to the possibility that supplementation of either L. lactis or L. mesenteroides to the undifferentiated OP50 laboratory strain would improve the lifespan and median survival of the nematodes compared to the OP50 alone, in which both lactic acid bacteria strains and combined OP50 conditions are shown to colonize the C. elegans gut (S1). After investigating the effect of supplementing OP50 with L. lactis or L. mesenteroides, a positive correlation was discovered with the extension of the median and overall lifespan, without showing any signs of developmental arrest associated with either potential probiotic strain (Figures 2A–C; Supplementary Figure S2). These results suggest that the nutrients/metabolites derived by supplementing L. lactis or L. mesenteroides with OP50 must have advantageous effects by differentiating the C. elegans axenic OP50 strain. Most of the lactic acid bacterial strains or supplementations exhibited extensions in the median and overall lifespan within 13.33%–33.33% and 25%–29%, respectively. The L. lactis-supplemented OP50 or L. lactis monoculture only shows significant differences when analyzed for early death events by the Gehan–Breslow–Wilcoxon test. The log-rank test proved insignificant between the supplementation and the monoculture for L. lactis. However, additional support for the beneficial contribution of nutrients/metabolites of L. mesenteroides intensified with the growth on the monoculture, which exhibits a lifespan extension that exceeds of all biomes tested (Figure 2C), with an 87% increase in the median survival and a 67% increase in the overall lifespan beyond the standard OP50. While L. mesenteroides is not known to produce antimicrobials such as nisin, L lactis has been reported to produce nisin (Khelissa et al., 2021). A significant reduction in the survival rate of C. elegans has been observed with exposure to nisin concentrations higher than 0.2 mg mL⁻¹ (Boelter et al., 2023). However, since the addition of lactic acid bacteria to OP50 has led to a significant improvement in the median and overall survival in simulated biomes, the deleterious effect of nisin produced (if any) by L. lactis was not observed.

In the OP50 + L.m. group, the nematodes were found to be morphologically distinct, being shorter, thinner, and possessing a smaller area than their counterparts in the OP50 and OP50 + L.l. groups (Figures 3A–C; Supplementary Figure S3). Additionally, these nematodes displayed slower locomotor behavior compared to those in the OP50, OP50 + L.l., and L.m. groups (Figure 3D; Supplementary Figure S3). Nematodes in the OP50 + L.l. group were shorter and wider than those in the OP50 group, yet longer and wider than those in the OP50 + L.m. group (Figures 3A, B; Supplementary Figure S3). They were significantly larger in area and displayed faster locomotion than those in the OP50 + L.m. group, but did not significantly differ from the OP50 group in these aspects (Figures 3C, D; Supplementary Figure S3). The L.m. group nematodes were shorter, thinner, and smaller than their counterparts in the OP50, OP50 + L.l., and L.l. groups. However, they displayed faster locomotive behavior than the OP50 + L.m., OP50 + L.l., and L.l groups (Figures 3A–D; Supplementary Figure S3). In the L.l. group, nematodes were longer, wider, and larger in area than their L.m. counterparts (Figures 3A–C; Supplementary Figure S3). Interestingly, two distinct widths were observed in this group, with measurements varying around the mean (Figure 3B). These nematodes exhibited slower locomotive behaviors than those in the L.m. group (Figure 3D).

In terms of progeny production, the OP50 + L.m. group, despite their reduced speed, produced a higher number of progeny than the L.m. monoculture (Figure 4A). The OP50 + L.l. group produced progeny equivalent to those of the OP50 group and at a higher level than those of the L.l. monoculture (Figure 4A). The L.l. group produced fewer progeny than both the OP50 and OP50 + L.l. groups (Figures 4A, B). The L.m. group showed a decrease in progeny production on the third day of the reproductive cycle compared to the OP50 group and produced fewer total progeny than the OP50 and OP50 + L.m. groups (Figures 4A, B). Despite this, the L.m. group continued to produce progeny for a longer duration at a higher level than those of the other groups (Figure 4B).

Assessing intestinal permeability using the Smurf assay revealed an increase in 8-day-old L.m. worms compared to the OP50 group (Figure 5). Similarly, an increased intestinal permeability was observed in the 8-day-old L.l. group, indicating that these longer-lived worms also had increased intestinal permeability akin to the L.m. monoculture group (Figure 5). However, there was no significant increase in the intestinal permeability that was observed in the OP50 + L.m. or OP50 + L.m. group (Figure 5).

In the context of reactive oxygen species (ROS) stress responses, the data showed that the basal and heat shock (HS) levels in the OP50 + L.m. group were elevated compared to those in the L.m. group (Figure 6; Supplementary Figure S4). Despite this increase, the basal and HS levels remained relatively unchanged upon extrinsic heat shock insults. In the OP50 + L.l. HS group, the ROS stress response was found to be elevated compared to that in the L.l. HS group. However, similar to the OP50 + L.m. group, the basal and HS levels remained relatively unchanged upon insults (Figure 6; Supplementary Figure S4). In the L.m. group, the basal ROS levels were found to be below those in the OP50 and OP50 + L.m. group, as well as the HS groups for these culture groups (Figure 6; Supplementary Figure S4). The basal and HS ROS response levels in the L.m. group remained relatively unchanged upon stress insults as measured by the gcs-1 reporter strain, indicating the lowest measured stress levels (Figure 6; Supplementary Figure S4). The ROS stress response in the L.l. HS group was found to be below that of the OP50 HS and OP50 + L.l. HS group (Figure 6; Supplementary Figure S4). However, the basal group showed a slight increase over the HS group but was otherwise unchanged upon insult (Figure 6; Supplementary Figure S4).

Concerning the UPR_cyt stress responses, interesting patterns were observed across different groups. In the OP50 + L.m. group, the basal stress response levels were significantly higher than those in the OP50 and L.m. group. However, these levels were decreased in comparison to the OP50 + L.m. HS and L.m. HS response (Figure 7; Supplementary Figure S5). For the OP50 + L.l. group, the basal stress response levels were notably decreased compared to both the OP50 + L.l. HS and L.l. groups (Figure 7; Supplementary Figure S5). Thus, the OP50 + L.l. group displayed a reduced UPR_cyt basal stress response. In the L.m. group, the UPR_cyt basal stress response was significantly decreased compared to the OP50, OP50 + L.m., and L.l. group (Figure 7; Supplementary Figure S5). However, the L.m. HS stress response in L.m. showed a robust increase over basal L.m. levels, with OP50 + L.m. HS, and L.l. HS, suggesting an elevated UPR_cyt stress response upon heat shock in the L.m. group. Lastly, in the L.l. group, the UPR_cyt basal level was decreased relative to both the L.l. HS, OP50, and OP50 + L.l. groups (Figure 7; Supplementary Figure S5). The L.l. HS stress response decreased compared to the OP50 HS and L.m. HS stress responses, indicating a lowered UPR_cyt response upon heat shock in the L.l. group.

In terms of UPR_ER stress responses, short-term treatment with tunicamycin showed only marginal increases although not significant for OP50, OP50 + L.m., and L.l. While, showing decreases with short-term exposure for OP50 + L.l and L.m. compared to controls (Figure 8A; Supplementary Figure S6A). OP50 + L.l did show an increase amount of basal stress over OP50 + L.m under DMSO treatment but did not show significant differences upon tunicamycin treatment (Figure 8A). However, the OP50 tunicamycin treated group showed a significant increase in stress over the L.m. treated group upon short-term exposure (Figure 8A; Supplementary Figure S6A). The other possible induction of the hsp-4 transgene according to the CGC, heat shock, shows a diverse trend across the different groups studied. For the OP50 + L.m. group, the UPR_ER basal stress response levels were found to be elevated above those in the OP50 + L.l. group. A slight elevation in the HS to basal level was observed, although the changes in these levels upon extrinsic heat shock insults remained relatively unchanged (Figure 8B; Supplementary Figure S6B). In the OP50 + L.l. group, the levels in the UPR_ER HS group were significantly elevated compared to the basal response levels in the same group. These levels were also found to be decreased relative to the OP50 and OP50 HS groups. Furthermore, the OP50 + L.l. response level decreased compared to the OP50 + L.m. response level (Figure 8B; Supplementary Figure S6B). In the L.m. group, the UPR_ER basal and L.m. HS stress responses remained relatively unchanged upon insult, suggesting that this group had robust resistance to UPR_ER stress (Figure 8B; Supplementary Figure S6B). No significant differences were observed between these responses and those of the other groups. In the L.l. group, the UPR_ER HS group was found to decrease relative to the OP50 HS group, suggesting a reduced response upon heat shock in the L.l. group. Interestingly, the basal groups showed a slight increase over the HS groups or slight increase or decreases in most groups with tunicamycin treatment, however; these changes in most treatments were not significant, and the responses were otherwise relatively unchanged (Figures 8A, B; Supplementary Figures S6A, B).

The UPR_mt stress responses across various groups demonstrated intriguing patterns. In the OP50 + L.m. group, the HS response showed a marked decrease compared to the OP50 HS and OP50 + L.l. HS responses (Figure 9; Supplementary Figure S7). However, under these conditions, the basal level of response was slightly elevated over the OP50 + L.m. HS levels, but this elevation was not significant (Figure 9; Supplementary Figure S7). In the context of the OP50 + L.l. group, the level of the UPR_mt HS group was elevated compared to the basal response level (Figure 9; Supplementary Figure S7). While the level of the OP50 + L.l. HS group was increased above the level of the OP50 + L.m. HS group, there were no significant differences between OP50 HS or L.l. HS groups (Figure 9; Supplementary Figure S7). Investigating the L.m. group, the UPR_mt showed that the L.m. basal and L.m. HS stress responses were relatively unchanged upon insults, indicating the lowest measured stress levels. Basal and HS stress responses in L.m. were found to be decreased below those in the OP50 and OP50 HS groups (Figure 9; Supplementary Figure S7). Moreover, L.m. HS stress response levels decreased relative to those in the L.l. HS group (Figure 9; Supplementary Figure S7). In the L.l. group, the UPR_mt showed a slight increase in the HS response over the basal condition upon insult, but this was not significant (Figure 9; Supplementary Figure S7). However, the L.l. HS group exhibited an increased response compared to the L.m. HS group, while maintaining an overall higher response in the basal and HS level (Figure 9; Supplementary Figure S7).

This study highlights the importance of significantly lower basal and stress levels as indicators of an early health response in the C. elegans model system. The results emphasize the potential probiotic applicability of these biomarkers for predicting early health responses, whereas a balanced and tightly modulated stress response was found to be associated with the longest survival probability, which demonstrated significantly longer survival rates than those with altered stress responses. The efficient adaptation mechanisms that maintain homeostasis ultimately lead to an increased survival probability. The relatively unchanged stress responses exhibited better overall health span parameters, contributing to the ability to maintain physiologically balanced condition in the face of stressors, which is a key feature in determining the longevity and overall health of an organism provided with L. lactis and L. mesenteroides as a therapeutic probiotic supplement.