C. jejuni intake shortened life-span of C. elegans
The harm of foodborne pathogens to C. elegans was usually reflected in the life-span of the nematodes. A C. elegans life-span experimental model was used to measure the responses of the worm to C. jejuni infection (Fig. 2A). E. coli OP50 was used as the food that normally sustains nematodes on reaching the L4 stage. As shown in Fig. 2B, C. jejuni killed about 50% of the worms within 5 days of their transferral to the lawn of the pathogen (C/day0), whereas 80% of the worms fed OP50 were still alive after 8 days (E/day0). All the nematodes in the E/day0 group died within 21 days; the equivalent period in the C/day0 group was 13 days. When the L4 stage nematodes were fed OP50 for 3 days before C. jejuni treatment (E/day0 + C/day3), they proved more resistant to C. jejuni: nearly 50% of these worms were still alive on day 10. However, their life-span was shorter than that of the worms fed OP50 only (E/day0). In addition, the initial number of C. jejuni cells recovered from the worms in the E/day0 + C/day3 group was smaller than that of the C. jejuni cells recovered from the worms in the C/day0 group (Fig. S1). This indicated that C. elegans at day 0 of the L4 stage was most sensitive to C. jejuni infection. Therefore, worms at day 3 of the L4 stage were chosen for the life-span assay due to the moderate life-span of the worm, pathogenicity of C. jejuni at this time point and available time space for LAB intervention.
The body size of C. elegans directly reflects the growth and development of nematodes which is closely related to their energy intake. In addition to pathogenicity, pathogens may also affect the life-span of nematodes due to their inability to be metobolized by C. elegans. To determine whether worms’ life-span shorten by C. jejuni was due to caloric intake or pathogenicity of C. jejuni, the body size of the nematodes fed C. jejuni was compared with that of the worms in the E/day0 group (Fig. 2C). The (E + C)/day0 group nematodes were fed equal amounts of E. coli OP50 and C. jejuni concurrently from day 0 of L4 stage. For 8 days, the nematodes in the (E + C)/day0 group and the E/day0 + C/day3 group were nearly the same size as those in the E/day0 group, which indicated that the three groups had almost the same caloric intake during this period. Furthermore, although the intestinal load of C. jejuni in the nematodes in the E/day0 + C/day3 group was much larger than the load in the (E + C)/day0 group, the load of C. jejuni in each of the two groups remained stable (Fig. 2D), which indicated that the nematodes showed no preference for ingesting C. jejuni versus E. coli. Therefore, substituting C. jejuni for E. coli did not lead to fasting, which may have resulted in worm death.
Some LAB strains prolonged the life-span of C. elegans treated with C. jejuni
A prolonged nematodes’ life-span after infection is a common indicator of antibacterial ability in C. elegans. To enable rapid evaluation of the defensive effects of LAB, forty-four LAB were assessed on their ability to protect C. elegans from C. jejuni infection mediated death. On day 13, C. elegans fed only E. coli OP50 displayed 50% survival (LD50) whereas the survival of nematodes fed only C. jejuni was only 20%. As shown in Fig. 3, S2 and Table 1, S2, the LAB isolates varied in their ability to protect the live worms, with survival rates ranging from 15–47%. Among tested isolates, Z5, 13M2, N9, L103, G20, 1132 and 13 − 7 provided high levels of protection (each producing an approximately 40% worm survival rate), whilst 422, B and Z6 did not show significant protection.
Some LAB strains decreased the C. jejuni load in the intestine of C. elegans
Diminishing pathogenic bacterial colonisation in the intestinal tract is probably one of underlying mechanisms of LAB in prolonging nematodes’ life-span. Also, the live LAB cells colonized in the C. elegans intestine played a role in decreasing the number of pathogenic bacteria. To investigate whether association exist between the life-span of C. elegans and the load of different bacteria in nematodes’ intestine, the number of LAB, C. jejuni and E. coli OP50 in intestine were counted. Twenty-six LAB strains which showed various levels of protection were evaluated on their ability to persist in the worm intestine from day 2 to day 6 (Fig. 4 and S3). The loads of LAB strains such as Z5, 427, N34 and X13, which showed a strong ability to protect C. elegans against death (37.52%-40.36% survival rate) exceeded log105 CFU/worm during the assay. In contrast, the loads of B, G14, ZX7 and Z7 were found to be low (log104 CFU/worm), also showed low levels of nematode protection in the life-span assay. The C. jejuni loads in these worms from day 2 to day 6 were also checked. Similarly, different LAB strains were associated with different levels of C. jejuni loads (log103.5–4.5 CFU/worm) in the nematode intestine during the assay (Fig. 5 and S4). The C. jejuni load in C. elegans treated with Z5, X13, 13 − 7, 427, G20 and N9 (offering high levels of protection in terms of worm life-span, 38.02%-43.28% survival rate) was almost 1.5 orders of magnitude smaller than that in the control group (E/day0 + C/day3, log104.5), whereas B, Z6, LGG and 422 (offering low levels of protection in terms of worm life-span) did not significantly decrease the C. jejuni load. The results of correlation analysis showed that the LAB load was highly positively correlated and conversely the C. jejuni load was highly negatively correlated with the survival of C. elegans. In addition, the load of LAB was moderately correlated with the load of C. jejuni in C. elegans (Table 2). It is worth noting that, there were no obvious differences in OP50 load among the E/day0 + C/day3 group and other LAB intervention groups (log101.2–1.5 CFU/worm) on day 6 (Fig. S5).
Longevity effects of LAB on C. elegans treated with C.jejuni were not due to caloric reduction
The body size of C. elegans directly reflects the growth and development of nematodes, which is closely related to their energy intake. Except the body size, pharyngeal pumping represents the feeding capacity of C. elegans and is a key index for the physiological activities. Also, it is an important parameter to evaluate the toxicity of drugs in C. elegans.To determine whether the longevity effects of LAB were the result of caloric reduction, the body size (Fig. 6A, B and Fig. S6) and pharynx pumping (Fig. 6C and Fig. S7) of nematodes fed 26 LAB strains and C. jejuni (L/day0 + C/day3 groups) was compared with that of nematodes in the E/day0 + C/day3 group. The worms in the E/day0 + C/day3 group and those in the LAB intervention groups showed little differences in body size, with the exceptions of the worms treated with N34, 675, 427 and 676, which were smaller. However, the N34, 675, 427 and 676 strains had varying protective effects (37.52%, 22.67%, 38.02% and 27.05% survival rate on day 13, Table S2). These were not the top survival rates in the life-span assay. The same situation also appeared in the determination of nematodes’ pharynx pumping. The pharynx pumping of worms was at the range of 50 to 58 per 30 s in the E/day0 + C/day3 group and most LAB intervention groups except for N34, 675, 427 and 676 treated groups. There were no significant differences in body size and number of pharynx pumping of nematodes treated by these LAB which showed varied longevity effects on C. elegans treated with C. jejuni.
Influence on C. jejuni growth by co-culturing E. coli OP50
To investigate whether the substance produced by E. coli OP50 such as certain bacteriocins would kill C. jejuni, the viability of C. jejuni cultured with or without live E. coli OP50 was measured. As showed in Fig. S8, the number of C. jejuni elevated in both tests after a 24-h incubation did not show significant difference. Furthermore, the pathogen was uninfluenced after the live E. coli OP50 were added to the growing C. jejuni after 48 h of incubation.
Some LAB strains upregulated immune gene transcription to protect C. elegans against C. jejuni infection
Regulation on the host’s immune system through pivotal signalling pathways is also an underlying mechanism by LAB on prolonging nematodes’ life-span against pathogen infection. To determine the mechanism by which LAB protected C. elegans against C. jejuni infection, the transcription levels of the 14 immune genes (tir-1, nsy-1, sek-1, pmk-1, spp-1, clec-85, abf-2, clec-60, lys-7, daf-16, age-1, dbl-1, skn-1 and bar-1) of C. elegans on day 6 were compared between the E/day0, E/day0 + C/day3 and L/day0 + C/day3 groups. As shown in Fig. 7 and S9, the transcription of tir-1, pmk-1 and bar-1 (respectively MAPK signalling pathway genes and an antioxidant gene) was to some extent increased when nematodes were infected with C. jejuni. In addition, slight increases were observed in some of the defence immune genes of C. elegans infected with C. jejuni, such as daf-16 and age-1 (Daf-16 signalling pathway genes) and dbl-1 (a TGF-β signalling pathway gene).
The 14 immune genes of C. elegans were also examined after treatment with 11 LAB strains, which showed variation in the protection against C. jejuni-induced worm death (Fig. 7, S9 and Table S3). It was found that the immune genes of C. elegans did not change significantly after LAB intervention without C. jejuni infection, which indicated that LAB strains were safe to healthy host (Table S3). As shown in Fig. 7 and S9, when nematodes were treated with LAB offering low levels of protection for survival, the transcription levels of their defence genes were almost identical to those of nematodes infected by C. jejuni alone. For these LAB strains, such as PC-T7 and B, only some genes (such as tir-1 and skn-1) were transcribed at a slightly higher rate than their counterparts in the group infected with C. jejuni alone, and the expression of spp-1 and bar-1 was even inferior to that in the control group. The transcription levels of some genes (such as tir-1, pmk-1 and bar-1) were much lower in some of the groups treated with poorly protective LAB (422, G14) than in the group infected with C. jejuni only. On the contrary, for those LAB strains, which protected the nematodes against C. jejuni-induced death in the life-span assay, the transcription levels of genes assayed were increased considerably on day 6. For example, treatment with 13 − 7, N9 and Z5 significantly enhanced the transcription of the MAPK signalling pathway genes nsy-1, sek-1 and pmk-1, the antioxidant gene skn-1 and the Daf-16 signal pathway genes age-1 and daf-16. The levels of transcription of the antibacterial peptide genes spp-1, clec-85 and lys-7 in these LAB intervention groups were 3–4 times higher than those in the group infected with C. jejuni alone. These data indicated that the ability of LAB to protect nematodes against C. jejuni-induced death was correlated with their influence on the levels of transcription of immune genes.
LAB strains which prolonged the life-span of C. elegans decreased the C. jejuni load in mice with T. gondii induced acute ileitis
LAB that showed protection of nematodes against bacterial infection might not applied to mammals due to obvious differences between the two organisms. To investigate whether the LAB screened from C. elegans model had the same effects in mammal, 11 LAB strains with different effects on C. elegans immune gene transcription were further investigated, to determine their capabilities to decrease the load of C. jejuni in mice. Seven days after T. gondii infection, the mice developed acute ileitis and likely to die, so the C. jejuni loads were checked on day 5. The load of C. jejuni in the faeces of mice in the C. jejuni infected group reached 108 CFU/g (Fig. 8A). LAB strains 13 − 7, Z5 and G20, which had already shown an outstanding ability to protect the nematodes, also exerted superior suppressive effect on C. jejuni load (less than 106 CFU/g faeces) in the mouse intestinal tract. Strains 422 and G14, which showed poor protective effects in terms of worm life-span, correspondingly played an inconspicuous role in suppressing C. jejuni load (around 108 CFU/g faeces). In addition, PC-T7 and Z6, which showed slight depressive effects on C. jejuni load (107-108 CFU/g faeces) had previously offered moderate and low protection, respectively, to the worms. Meanwhile, N9 and 430, which had moderate depressive effects on C. jejuni load (106-107 CFU/g faeces), had previously offered the worms high and moderate levels of protection, respectively. Therefore, the effects of all the 11 LAB strains apart from B and 427 were consistent across the C. elegans and mice samples.
Correlation analysis was conducted to examine the relationship between the ability of the 11 LAB strains to clear C. jejuni in mice and the survival rate of C. jejuni-infected nematodes. The relative index R2, which reached 0.79093, indicated that the C. jejuni-antagonistic activity of LAB in C. elegans was significantly co-related to their C. jejuni-antagonistic activity in mice (Fig. 8B).
LAB strains which prolonged the life-span of C. elegans decreased the C. jejuni load in chicken
Outbreaks of campylobacteriosis can occur if humans ingest undercooked poultry contaminated by the C. jejuni. LAB applied in fodder could reduce Campylobacter colonization in poultry and stop the disease outbreak at its source. To investigate whether the LAB screened from C. elegans had the same effects in poultry, 11 LAB strains with different effects on C. elegans immune gene transcription were investigated to determine their abilities to clear C. jejuni in chicken. The inhibitory effect of LABs on C. jejuni colonization in chicks’ cecum was examined. Approximately 24 h after hatching, chicks were inoculated orally with C. jejuni, and then LAB was administered daily for two weeks. CFU of C. jejuni in chicken cecum in all groups were evaluated on day 23. The average value of C. jejuni increased to 108 CFU/g cecal content in the C. jejuni infected group (Fig. 9A). Z5 and 427, exerted most significant suppressive effects on C. jejuni colonisation in the chicken cecum, which resulted in C. jejuni loads fell to below 104 CFU/g cecal content in these two groups. Correspondingly, Z5 had demonstrated an outstanding ability to protect the nematodes and decrease C. jejuni load in the mouse intestinal tract, while the antagonizing ability by 427 in mice was reversed. Meanwhile, 430, B and G14 showed poor abilities to clear C. jejuni in chicken cecum. The C. jejuni loads in the last three groups were higher than 107 CFU/g cecal content. The effects of these 3 strains on protecting the nematodes were similar to that of elimination of C. jejuni in chicken. However, 430 and B exhibited moderate strength of C. jejuni antagonization in mice, which was different from their performance in the chicken. In addition, 13 − 7, N9, G20, PC-T7, 422 and Z6 showed moderate scavenging activity on C. jejuni in the chicken cecal contents with the load of C. jejuni at the range from 104.8 to 105.5 CFU/g faeces. It is worth noting that a few strains (422 and Z6) showed a certain degree of inconsistency in the C. jejuni antagonism in different models..
Correlation analysis was conducted to examine the relationship between the ability of the 11 LAB strains to clear C. jejuni in chicken and the survival rate of C. jejuni-infected nematodes. The relative index R2, which reached 0.50071, indicated that the C. jejuni-antagonistic activity of LAB in C. elegans was related to their C. jejuni-antagonistic activity in chicken (Fig. 9B).