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Lactic acid bacteria that activate immune gene expression in Caenorhabditis elegans can antagonise Campylobacter jejuni infection in nematodes, chickens and mice

Abstract

Background

Campylobacter jejuni is the major micro-bacillary pathogen responsible for human coloenteritis. Lactic acid bacteria (LAB) have been shown to protect against Campylobacter infection. However, LAB with a good ability to inhibit the growth of C. jejuni in vitro are less effective in animals and animal models, and have the disadvantages of high cost, a long cycle, cumbersome operation and insignificant immune response indicators. Caenorhabditis elegans is increasingly used to screen probiotics for their anti-pathogenic properties. However, no research on the use of C. elegans to screen for probiotic candidates antagonistic to C. jejuni has been conducted to date.

Results

This study established a lifespan model of C. elegans, enabling the preselection of LAB to counter C. jejuni infection. A potential protective mechanism of LAB was identified. Some distinct LAB species offered a high level of protection to C. elegans against C. jejuni. The LAB strains with a high protection rate reduced the load of C. jejuni in C. elegans. The transcription of antibacterial peptide genes, MAPK and Daf-16 signalling pathway-related genes was elevated using the LAB isolates with a high protection rate. The reliability of the lifespan model of C. elegans was verified using mice and chickens infected with C. jejuni.

Conclusions

The results showed that different LAB had different abilities to protect C. elegans against C. jejuni. C. elegans provides a reliable model for researchers to screen for LAB that are antagonistic to C. jejuni on a large scale.

Background

Infection by pathogenic Campylobacter may result in symptoms such as bloody diarrhoea, abdominal pain and fever. In many countries, Campylobacter species is the major microbacillary pathogen responsible for human coloenteritis [1]. In under developed countries, diarrhoeic disease is 10 times more likely to result from Campylobacter infection than from infection with Escherichia coli O157: H, Shigella species or Salmonella species [2, 3]. Some peripheral neuropathies, such as Guillain-Barré syndrome and Miller Fieher syndrome, are long-term consequences of Campylobacter infection [2]. Campylobacter jejuni (C. jejuni) is also one of the primary reasons for microbacillary food-borne disease in some developed countries [3]. To date, all therapies for Campylobacter infection involved antibiotics, especially in poultry industry. There is an urgency to develop alternative approaches due to a gradual increase in antibiotic-resistant Campylobacter [4]. It is also necessary to illustrate the mechanisms underlying the functions of such alternatives to support their development and implementation.

Research has increasingly shown that lactic acid bacteria (LAB) colonise the human gastrointestinal tract and play a vital role in maintaining intestinal function and well being of the host. Among the beneficial effects of LAB are bacteriostatic activities targeting pathogens such as Escherichia coli (E. coli), Salmonella and Listeria monocytogenes [5, 6]. LAB have been shown to protect against Campylobacter infection. Nishiyama reported that LAB inhibited Campylobacter and colonisation of this pathogen was reduced by Lactobacillus gasseri SBT2055 isolated from healthy chicken [7]. Another study reported a decrease in C. jejuni invasion in the gut of turkey poults after treatment with Lactobacillus salivarius NRRL B-30514, suggesting that competitive exclusion can play a role [8]. Wagner et al. conducted a simulation experiment with immunodeficient and immunocompetent mice, they found that bifidobacteria and lactobacilli can increase colonisation resistance. Specifically, the results showed that bifidobacteria and lactobacilli can resist C. jejuni enteric persistence in the human gut [9]. To date, studies of antagonism against C. jejuni by LAB have mainly been conducted in vitro or in vivo. Unfortunately, some of the strains of LAB that showed a good ability to inhibit the growth of C. jejuni in vitro are less effective in antagonising C. jejuni in animals. Whether poultry or mice, all animal models have the disadvantages of high cost, a long cycle, cumbersome operation and insignificant immune response indicators, and do not permit rapid large-scale screening for LAB capable of effective antagonising C. jejuni.

The small, free-living (non-parasitic) soil worm Caenorhabditis elegans (C. elegans) has been widely used in biological studies as in vivo model, due to its short generation time, diminutive form and clear genetic background. To date, C. elegans has been used to study the effect of pathogenic microorganism, such as Salmonella enterica, Pseudomonas aeruginosa, Enterococcus faecalis and Staphylococcus aureus [10,11,12,13,14]. C. elegans is also increasingly used to screen for antimicrobials and probiotics [15]. In addition, institutional studies of the effects of LAB can be processed using C. elegans. However, no research on the use of C. elegans to screen for probiotic candidates antagonistic to C. jejuni has been conducted to date. Moreover, the molecular mechanisms underlying the protective mechanism of LAB have yet to be established. In this study, a C. elegans life-span experimental model was developed to investigate the response of nematodes to C. jejuni infection, enabling rapid evaluation of the protective effects of LAB and understanding of microbe-host interactions. At the same time, in this article, mice (with acute ileitis induced by Toxoplasma gondii (T. gondii) infection which abrogating the colonisation resistance) and chicken were also used to validate the C. elegans model individually [52].

Results

C. jejuni intake shortened the lifespan of C. elegans

The harm caused by foodborne pathogens to the Caenorhabditis elegans is usually reflected in its lifespan. An experimental lifespan model of C. elegans was used to measure its response to C. jejuni infection (Fig. S1). The OP50 strain of Escherichia coli was used as the food source as this normally sustains nematodes on reaching the L4 stage. As shown in Fig. 1A, approximately 50% of the C. elegans transferred to the C. jejuni group died within 5 days, whereas 80% of the C. elegans fed with OP50 (OP50 group) were still alive after 8 days. All of the nematodes in the OP50 group died within 21 days, whereas this was 13 days for the C. jejuni group. When the nematodes were fed OP50 for 3 days before being treated with C. jejuni (OP50/C. jejuni group), they were more resistant to C. jejuni and nearly 50% were still alive on day 10. However, the lifespan of C. elegans was shorter in the OP50/C. jejuni group than in the OP50 group. In addition, the initial number of C. jejuni cells that were recovered from the nematodes in the OP50/C. jejuni group was lower than that recovered from the nematodes in the C. jejuni group (Fig. 1C). This may be due to the protective effect of OP50 and/or shorter time of C. jejuni intake. However, to allow available time for lactic acid bacteria (LAB) intervention, C. elegans at day 3 after the L4 stage was chosen for the lifespan assay. Although the OP50/C. jejuni group was less effective in infecting C. elegans with C. jejuni than the C. jejuni group, the lifespan of C. elegans was still considerably shortened compared with the OP50 group.

Fig. 1
figure1

Establishment of a life-span, body size and C. jejuni load in C. elegans assay. A Life-span of C. elegans treated with E. coli and C. jejuni in different ways. Day 0 marked the arrival of the nematodes at the L4 stage before being fed thallus. B Body size of C. elegans treated with E. coli and C. jejuni in different ways. Day 0 marked the arrival of the nematodes at the L4 stage before being fed thallus. C C. jejuni load in C. elegans treated with E. coli and C. jejuni in different ways. Day 0 marked the point at which the nematodes were first fed C. jejuni

The body size of C. elegans directly reflects its growth and development, which are closely related to its energy intake. In addition to directly causing disease, pathogens may also affect the lifespan of C. elegans due to their inability to be metabolised by this nematode. To determine whether the lifespan of C. elegans was shortened due to the pathogenicity of C. jejuni or reduced caloric intake, the body size of C. elegans fed with C. jejuni was compared with that of C. elegans fed with OP50 (Fig. 1B). The nematodes in the OP50 + C. jejuni group were fed equal amounts of OP50 and C. jejuni concurrently at the L4 stage. For 8 days, the nematodes in the OP50 + C. jejuni and OP50/C. jejuni groups were nearly the same size as those in the OP50 group, which indicated that the three groups had almost the same caloric intake during this period. Although the intestinal load of C. jejuni in the nematodes in the OP50/C. jejuni group was much larger than that in the OP50 + C. jejuni group, the load of C. jejuni in both groups remained stable (Fig. 1C), indicating that the nematodes showed no preference for ingesting C. jejuni over E. coli. Therefore, substituting C. jejuni for E. coli did not lead to fasting in C. elegans, which would have affected its lifespan.

LAB strains prolonged the lifespan of C. elegans infected by C. jejuni

The lifespan of C. elegans after bacterial infection is a common indicator of this nematode’s antibacterial ability. To enable the rapid evaluation of the defensive effects of LAB, 44 LAB were assessed for their ability to protect C. elegans from C. jejuni infection-mediated death. On day 13, the C. elegans that were only fed OP50 displayed a 50% survival rate (DT50), whereas the DT50 of the C. elegans that were only fed C. jejuni was only 20%. As shown in Table 1, the LAB isolates varied in their ability to protect the live nematodes, with survival rates ranging from 15 to 60%. Among the tested isolates, some LAB provided high levels of protection, especially 13–7, Z5 and N9 (each leading to a survival rate of > 40%), and the lifespans of the nematodes in these three groups were significantly longer than the lifespan of the nematodes the OP50/C. jejuni group (Fig. 2).

Table 1 Statistical analysis of the protection effects of LAB stains on C. elegans infected by C. jejuni NCTC 11168a
Fig. 2
figure2

Differential effects of LAB on the survival of C. elegans infected with C. jejuni. OP50/C. jejuni: treated with E. coli OP50 for the first 3 days and then with C. jejuni after the L4 stage. LAB/C. jejuni: treated with LAB for the first 3 days and then with C. jejuni after the L4 stage. Day 0 marked the arrival of the nematodes at the L4 stage before being fed thallus

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 the underlying mechanisms of LAB in prolonging the lifespan of nematodes. In addition, LAB cells colonising the intestine of C. elegans play a role in decreasing the number of pathogenic bacteria. To investigate whether an association exists between the lifespan of C. elegans and the loadings of different bacteria in their intestines, the numbers of LAB, C. jejuni and E. coli OP50 in the intestines of C. elegans were counted. The 44 LAB strains that showed various levels of protection were evaluated for their ability to persist in the intestines of C. elegans from day 2 to day 6. The loads of LAB on 13–7, Z5 and N9, which showed a strong ability to protect C. elegans against death, exceeded log 104.5 colony-forming unit (CFU)/nematode during the assay (Fig. 3A and B). In contrast, the groups showed low levels of protection for the nematodes in the lifespan assay, and had the lowest colonisation levels of LAB (Table S2). The C. jejuni loads in C. elegans from day 2 to day 6 were also determined. As shown in Fig. 3C and D, the C. jejuni loads in C. elegans treated with 13–7, Z5 and N9 were almost 1.5 orders of magnitude lower than that in the OP50/C. jejuni group (log 104.5 CFU/nematode). In contrast, the groups offering low levels of protection in terms of lifespan did not significantly decrease the C. jejuni load (Table S3). There were no obvious differences in the OP50 load between the OP50/C. jejuni group and the other LAB intervention groups (log 101.2–101.5 CFU/nematode) on day 6 (Fig. 3E and Table S4). The results of a correlation analysis showed that the LAB and C. jejuni loads were highly positively correlated and highly negatively correlated with the survival of C. elegans, respectively. In addition, the OP50 load was not correlated with the survival of C. elegans (Table 2).

Fig. 3
figure3

LAB, C. jejuni and OP50 load in the intestine of C. elegans. A and B LAB load. Day 0 marked the arrival of the nematodes at the L4 stage before being fed thallus. C and D C. jejuni load. Day 0 marked the point at which the nematodes were first fed C. jejuni. E OP50 load. Day 0 marked the arrival of the nematodes at the L4 stage before being fed thallus. The graphs show means ± SDs. Column labelled with different superscript letters (a, b) showed significant differences (p < 0.05). Any two columns with same superscript letter

Table 2 Correlation analysis of LAB colonization and C.jejuni load in the intestine and survival of C. elegans

No change on body size and pharyngeal pumping of C. elegans infected by C. jejuni under LAB intervention

As mentioned, the body size of C. elegans directly reflects its growth and development, which are closely related to its energy intake. In addition to body size, pharyngeal pumping represents the feeding capacity of C. elegans and is another key index for physiological activities. To determine whether the longevity effects of LAB were the result of caloric reduction, the body size (Table S5) and pharyngeal pumping rate (Table S6) of C. elegans fed with 44 LAB strains and C. jejuni (LAB/C. jejuni groups) were compared with those of the nematodes in the OP50/C. jejuni group. The nematodes in the OP50/C. jejuni group and those in the 13–7, Z5 and N9 intervention groups showed little difference in body size (Fig. 4A and B). Similarly, little difference was observed in pharyngeal pumping between the groups. Figure 4C showed that the pharyngeal pumping rate ranged from 50 to 58 per 30s in the OP50/C. jejuni group and 13–7, Z5 and N9 intervention groups. There were no significant differences in body size or pharyngeal pumping of nematodes treated by these LAB, thus demonstrating the varied longevity of C. elegans treated with C. jejuni.

Fig. 4
figure4

Effects of LAB on body size and pharynx pumping of C. elegans. A and B Body size (area) of C. elegans treated with LAB and C. jejuni on day 8. Day 0 marked the arrival of the nematodes at the L4 stage before being fed thallus. C Effects of LAB on the pharynx pumping of C. elegans infected by C. jejuni. Pharynx pumping (per 30s) of C. elegans treated with LAB and C. jejuni on day 8. All day 0 marked the arrival of the nematodes at the L4 stage before being fed thallus. The graphs show means ± SDs. Column labelled with different superscript letters (a, b) showed significant differences (p < 0.05). Any two columns with same superscript letter

Influence on C. jejuni growth by co-culturing with E. coli OP50

To investigate whether the substances produced by E. coli OP50, such as certain bacteriocins, could kill C. jejuni, the viability of C. jejuni cultured with and without live E. coli OP50 was measured. As shown in Fig. 5, the number of C. jejuni increased in both tests after 24 h of incubation and did not exhibit a significant difference between the tests. Furthermore, the number of C. jejuni was uninfluenced after the live E. coli OP50 were added to the growing C. jejuni after 48 h of incubation.

Fig. 5
figure5

Live cell counts of C. jejuni in the co-culture with E.coli OP50 strain

LAB strains upregulated the immune gene transcription

Regulation of the host’s immune system through pivotal signalling pathways is an underlying mechanism by which LAB can prolong the lifespan of a nematode and protect it against pathogen infection. To determine the mechanism by which LAB protected C. elegans against C. jejuni infection, the transcription levels of 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 OP50, OP50/C. jejuni, and LAB/C. jejuni groups. As shown in Fig. 6A, B and C, the transcription of tir-1 and pmk-1 (MAPK signalling pathway genes) and bar-1 (an antioxidant gene) increased to some extent when C. elegans was 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).

Fig. 6
figure6

Differential effects of LAB on transcription of immune genes of C. elegans on day 6. Day 0 marked the arrival of the nematodes at the L4 stage before being fed thallus. * Indicates statistically significant differences at p < 0.05. ** Indicates statistically significant differences at p < 0.01

The 14 immune genes of C. elegans were also examined after treatment with 44 LAB strains, which revealed a variation in the protection against C. jejuni-induced nematode mortality (Table S7). It was found that the expression level of the immune genes of C. elegans did not change significantly after LAB intervention without C. jejuni infection, indicating that the LAB strains were safe for a healthy host (Table S8). As shown in Fig. 6A, B and C, the treatments of nematodes 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 signalling pathway genes age-1 and daf-16. The transcription levels 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 suggest that the ability of certain LAB to protect nematodes against C. jejuni-induced mortality was correlated with their influence on the transcription levels of immune genes. In contrast, when C. elegans was treated with LAB offering low levels of protection for survival, the transcription levels of their defence genes were almost identical to those of C. elegans infected with C. jejuni alone (Table S7).

LAB strains that prolonged the lifespan of C. elegans decreased the C. jejuni load in mice with T. gondii-induced acute ileitis

The LAB that protect nematodes against bacterial infection might be inapplicable to mammals due to the obvious differences between the organisms. To investigate whether the LAB screened from C. elegans had the same effects in mammals, 11 LAB strains (including 13–7, Z5 and N9) with different effects on the immune gene transcription of C. elegans were further investigated to determine their abilities to decrease the C. jejuni load in mice. Toxoplasma gondii was used to induce acute ileitis to avoid colonisation resistance in normal mice. Seven days after T. gondii infection, the mice developed acute ileitis and most died; thus, the C. jejuni loads were determined on day 9. The C. jejuni load in the faeces of mice in the C. jejuni-infected group reached 108 CFU/g (Fig. 7A). The LAB strains that showed the strongest ability to protect C. elegans (i.e. 13–7, Z5 and G20) also exerted a superior suppressive effect on the C. jejuni load (< 106 CFU/g of faeces) in the intestinal tract of mice. Strains 422 and G14, which showed poor protective effects in terms of the lifespan of C. elegans, correspondingly played an inconspicuous role in suppressing the C. jejuni load (~ 108 CFU/g of faeces). Consistently with this, PC-T7 and Z6, which had previously offered moderate and low levels of protection, respectively, to C. elegans, showed slight suppressive effects on the C. jejuni load (107–108 CFU/g of faeces). Meanwhile, N9 and 430, which had previously offered high and moderate levels of protection to C. elegans, respectively, had moderate suppressive effects on the C. jejuni load (106–107 CFU/g of faeces).

Fig. 7
figure7

Differential effects of LAB on C. jejuni load in mice infected by C. jejuni and correlation analysis between C. elegans and mice models. A Culturable C. jejuni in mice faeces on day 9. The graphs show means ± SDs. Column labelled with different superscript letters (a, b, c) showed significant differences (p < 0.05). Any two columns with same superscript letter. B Correlation analysis of ability of LAB strains to clear C. jejuni in infected mice on day 5 and the survival rate of C. jejuni-infected nematodes under LAB treatment. C Correlation analysis of ability of LAB strains to clear C. jejuni in infected mice on day 9 and upregulate immune gene transcription to protect C. elegans against C. jejuni infection

A correlation analysis was conducted to examine the relationship between the ability of the 11 LAB strains to remove C. jejuni in mice and the survival rate of C. jejuni-infected nematodes or the uprefulation ability of immune gene (spp-1) in C. elegans by LAB against C. jejuni infection. The correlation coefficient R2 reached 0.79093 and 0.71604, indicating that the C. jejuni-antagonistic activity of LAB in C. elegans by upregulating worms immune gene transcription to prolong life was significantly correlated with the C. jejuni-antagonistic activity of LAB in mice (Fig. 7B and C).

LAB strains that prolonged the lifespan of C. elegans decreased the C. jejuni load in chickens

Outbreaks of campylobacteriosis can occur if humans ingest undercooked poultry contaminated with C. jejuni. Studies have shown that LAB applied to fodder could reduce Campylobacter colonisation in poultry and stop the outbreak of this disease 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 immune gene transcription in C. elegans were investigated to determine their abilities to remove C. jejuni in chickens. The inhibitory effect of LAB on C. jejuni colonisation in the cecum of chickens was examined. Approximately 24 h after hatching, chickens were inoculated orally with C. jejuni, and then LAB were administered daily for 2 weeks. The CFU count of C. jejuni in the cecum of chickens in all groups was evaluated on day 23. The average value of C. jejuni increased to 108 CFU/g of cecal content in the C. jejuni-infected group (Fig. 8A). Strains Z5 exerted significant suppressive effect on C. jejuni colonisation in the cecum of chickens, which resulted in the reduction of the C. jejuni load to below 104 CFU/g of cecal content in this group. Meanwhile, strains 430, B and G14 showed poor abilities to remove C. jejuni from the cecum of chickens. The C. jejuni loads in these groups were > 107 CFU/g of the cecal content. The effects of these three strains in eliminating C. jejuni in C. elegans were similar to their effects in eliminating C. jejuni in chickens. However, strains 430 and B exhibited moderate C. jejuni antagonism in C. elegans, which were stronger than their performance in chickens. In addition, 13–7, N9, G20, PC-T7, 422 and Z6 showed moderate ability to eliminate C. jejuni in the cecal content of chickens, with a C. jejuni load of 104.8–105.5 CFU/g of faeces. Two of those strains (422 and Z6) presented a certain degree of inconsistency in the C. jejuni antagonism in chickens and nematodes.

Fig. 8
figure8

Differential effects of LAB on C. jejuni load in chicken infected by C. jejuni. A Culturable C. jejuni in chicks’ cecal contents on day 23. The graphs show means ± SDs. Column labelled with different superscript letters (a, b, c) showed significant differences (p < 0.05). Any two columns with same superscript letter. B Correlation analysis of ability of LAB strains to clear C. jejuni in infected chicks’ cecal contents and the survival rate of C. jejuni-infected nematodes under LAB treatment. C Correlation analysis of ability of LAB strains to clear C. jejuni in infected chicks’ cecal contents and upregulate immune gene transcription to protect C. elegans against C. jejuni infection

A correlation analysis was conducted to examine the relationship between the ability of the 11 LAB strains to remove C. jejuni in chickens and the survival rate of C. jejuni-infected nematodes or the uprefulation ability of immune gene (spp-1) in C. elegans by LAB against C. jejuni infection. The R2 value reached 0.50071 and 0.63811, indicating that the C. jejuni-antagonistic activity of LAB in C. elegans by upregulating worms immune gene transcription to prolong life was correlated with their C. jejuni-antagonistic activity in chickens (Fig. 8B and C).

Discussion

The model organism C. elegans has been treated with numerous pathogenic microorganisms from animals and humans to examine the relationships between pathogenic bacteria and their hosts [10,11,12,13,14]. In this study, a lifespan experimental model of C. elegans was established to examine the response of this nematode to C. jejuni infection. Our experiments revealed that LAB strains varied in their ability to defend C. elegans against infection from C. jejuni (NCTC 11168). In addition, the antagonistic effects of LAB isolates on C. jejuni in C. elegans were verified in both mice and chickens. The lifespan experimental model of C. elegans not only provided a useful method of screening for LAB candidates with the potential to mitigate C. jejuni infection, but also helped to explain the defence mechanism imparted by the LAB isolates. The findings also indicated that LAB defended C. elegans not only by inhibiting C. jejuni colonisation in the intestines, but also by activating the defence immune genes of C. elegans.

The natural physiological activities of nematodes are usually halted by the colonisation of invading pathogens [17,18,19]. Some LAB, such as Lacticaseibacillus rhamnosus [20], Lactobacillus acidophilus [21], Lactobacillus fermentum [13], Lactobacillus gasseri and Lactobacillus plantarum [22], have been reported to show high shielding efficiencies to nematodes from pathogens, to diminish the risk of bacterial colonisation in the intestinal tract, and to prolong the lifespan of nematodes. The intestinal colonisation of C. jejuni may occur in humans and animals, and is often observed in poultry. The binding of fimbrial adhesins to the host’s intestinal tract is a precursor to C. jejuni nosogenesis, which leads to diseases such as diarrhoea [23, 24]. Many studies have shown that C. jejuni colonisation and growth in the host’s intestinal tract can be reduced by some LAB, such as Bifidobacterium and Lactobacillus salivarius [25, 26]. The results of the present study confirmed that the level of C. jejuni in the intestinal tract of C. elegans was affected differentially by LAB strains, which offered strong to weak levels of protection. The C. jejuni load in C. elegans was strongly correlated with its lifespan, which indicated that the inhibition of C. jejuni colonisation in the intestinal tract was one of the mechanisms through which LAB offered protection to C. elegans. As reported, some foodborne pathogens, such as Salmonella enterica serovar Typhimurium [21] and entero-invasive E. coli [27], can colonise the intestinal tract of nematodes to form a persistent lethal infection. The colonised LAB in the intestine continuously produce antibacterial products, and can also occupy the adhesion sites of pathogens, which jointly exert antibacterial effects [54]. However, in this study, the LAB load and C. jejuni load in C. elegans were negatively correlated, indicating that some LAB may alleviate the damage caused by infected pathogens in other ways, such as through the regulation of immune gene expression in nematodes.

In non-mammalian taxa, mice and some primates, calorie restriction is normally used to increase longevity and ease the consequences of ageing [28,29,30]. Researchers have also investigated the caloric intake of C. elegans after LAB intervention through measurements of body size and the pharyngeal pumping rate [10,11,12,13,14]. But in this article, under the intervention of LAB, there was no relationship between C. jejuni- infected C. elegans lifespan extension and its morphological or pharyngeal pumping.

With the growth and reproduction of LAB in a host’s intestine, the immune system is regulated through pivotal signalling pathways with LAB involvement [31]. The host’s immune response to pathogens may be induced by LAB [32]. In addition, LAB strains with protective efficacy, such as L. rhamnosus GG [33], L. plantarum [34] and Lactobacillus delbrueckii [35], can enhance the host’s immune defence against E. coli, S. typhimurium and Streptococcus pyogenes infection by exciting the MAPK signalling pathway and through toll-like receptors. Moreover, LAB may defend the host by adjusting gene expression through cytokine and chemokine activity [36, 37]. Another possible mechanism is the enhancement of the membrane barrier [38, 39]. Caenorhabditis elegans has two main signalling pathways: P38-MAPK and TGF-β. Its specific defence system, including antimicrobial responses, falls short of an adaptive immune system [40]. Nevertheless, the upregulation of MAPK pathway gene (tir-1, nys-1, sek-1 and pmk-1) expression can enable C. elegans to resist various microbial infections. These findings demonstrated the activation of the MAPK pathway through LAB intervention [31]. In the present study, the LAB strains offered good levels of protection for C. elegans against C. jejuni infection, and significantly enhanced the transcription of the MAPK pathway genes. As a downstream molecule regulated by the MAPK signalling pathway, the fork-head family transcription factor DAF-16 has been shown to adjust gene expression to improve dauer formation in the larval phase of nematodes, and to increase resistance and longevity when mature [41,42,43]. The results of the present study were consistent with the above findings, suggesting that the studied LAB strains prolonged the lifespan of C. elegans by upregulating DAF-16 via the MAPK signalling pathway.

In addition, it is considered that the cluster of C-type lectins can mask bacterial attachment and enhance resistance to microbial infection [44]. The increased levels of clec-60, clec-85, spp-1 and abf-2 upon LAB pre-treatment in this study also indicate the ability of some LAB to protect nematodes against C. jejuni infection. Skn-1 and bar-1 may also play critical roles in defending a host and prolonging its survival. The increased level of skn-1 in pre-treated nematodes has been found to be due to antioxidant defence followed by their improved survival [45,46,47]. Partly consistent with this claim, skn-1 transcription in the current study was significantly enhanced by the LAB strains that prolonged the lifespan of C. elegans. Although the TGF-β pathway is a major defence system in C. elegans, dbl-1 showed almost no obvious changes in the transcription level across the LAB intervention groups, indicating that this gene may not play an essential role in protecting against C. jejuni infection. Moreover, the immune genes of C. elegans without C. jejuni infection did not change significantly after intervention by the LAB strains. The studied LAB only played a significant role when the nematodes received external infection, suggesting that LAB are safe for a healthy host.

As the growth temperature of C. elegans is much lower than the temperature in the human gut, and the microbiota of C. elegans is much simpler than that of a mammal, the reliability of this lifespan model of C. elegans in testing the efficiency of LAB antagonism against C. jejuni colonisation in mammals should be confirmed. In addition, because the short cycle of acute infection in mice represents a research limitation, chickens were used to investigate the long-term effects in this study. Nevertheless, the C. jejuni-infected chicken model has been widely used in screening probiotics for feed with C. jejuni antagonism, which could be applied as antibiotic substitutes [16]. The significant correlation between the ability of LAB to remove C. jejuni in mice and chicken and their ability to upregulate immune gene transcription to protect C. elegans against C. jejuni infection indicates that the lifespan model of C. elegans infected with C. jejuni can be applied to some extent to C. jejuni-infected mammals and birds. Although the correlation between the different models was relatively good, it was undeniable that some strains had different antibacterial effects in these models. The reason for the difference may be physiological differences (such as body temperature) on different hosts or different growth characteristics of LAB in the body (the ability of colonization or production of antibacterial substances). The results indicate that C. elegans is a good model organism for screening C. jejuni-resistive LAB strains on a large scale.

In this study, the observed effects of LAB strains on the antagonistic ability of C. jejuni infection in nematodes with its genome determinants were also investigated. Taking L. plantarum N9 as an example, a glycosyltransferase-encoding gene that differed to that of other L. plantarum strains was found in COG and NR annotations. This gene regulates the synthesis of extracellular polysaccharides in bacterial strains. Exopolysaccharides have been proven to have a series of physiological health care effects, such as antioxidant properties, immune regulation and resistance to pathogenic bacteria. Therefore, the analysis results suggest that the core functional genes of LAB endow them with the ability to alleviate the infection of C. jejuni in nematodes.

Conclusion

This study established a lifespan model of C. elegans that can measure the response of this nematode to C. jejuni. Different LAB species exhibited different effects on the response of C. elegans to infection with C. jejuni. The inhibition of intestinal colonisation by C. jejuni may have been one of the mechanisms through which LAB protected C. elegans, and the protection offered by LAB may also have derived partly from their activation of the defence immune genes of C. elegans. This lifespan model of C. elegans can be used to screen for C. jejuni-antagonistic LAB on a large scale.

Methods

C. elegans, LAB and C. jejuni

C. elegans N2 Bristol wild-type strain and E. coli OP50 (Caenorhabditis Genetics Center, Minnepolis, University of Minnesota) was used in the study. 20 °C was used for nematodes maintence and cultivation. The procedures for the cultivation, maintenance and synchronization of the nematode have been reported [48, 55]. Before using E. coli OP50 on every experiment, its concentration was adjusted to 1× 108 colony-forming units (CFU)/mL in sterile phosphate-buffered saline solution (PBS).

Six LAB were purchased from the American Type Culture Collection (ATCC) or the Japan Collection of Microorganisms and 38 were isolated from human faeces from different habitats and traditional fermented food. The selected LAB were identified through 16S gene sequencing. All the isolates were deposited at the Culture Collection of Food Microorganisms at Jiangnan University (Table S1). The whole genome sequence of 12 isolates, namely N8, N9, 422, 427, 430, Z5, L103, X13, JS-SZ-1-5, JS-WX-9-1, 9–5 and H27-1 L were aligned and identified as new strains of the corresponding species reported. The other 26 isolates are assumed tentative strains, for they were isolated from samples of different origin, geographical location and span over seven years. All of the LAB were cultivated in deMan, Rogosa and Sharpe (MRS) agar at a 2% (v/v) inoculum size at 37 °C for 18 to 20 h with an atmosphere of 85% N2, 10% CO2, and 5% H2. Before using LAB on every experiment, its concentration was adjusted to 1× 109 colony-forming units (CFU)/mL in sterile phosphate-buffered saline solution (PBS).

The source and cultivation of C. jejuni NCTC 11168 have been reported [55]. Before using C. jejuni on every experiment, its concentration was adjusted to 1× 109 colony-forming units (CFU)/mL in sterile phosphate-buffered saline solution (PBS).

Life-span experimental analysis of C. elegans

The nematodes were synchronised as previously described [27]. To build a model for the experimental analysis of worm death induced by C. jejuni, 109 CFU/mL of fresh and live C. jejuni was prepared for worms every day during the whole experiment. Each group had 80–100 worms at L4 stage and three independent replications were performed for each assay. As shown in Fig. S1, after L4 stage, worms fed only with E. coli OP50 formed the negative control group (OP50 group). The worms fed only with C. jejuni was called C. jejuni group. Throughout the experiment, OP 50 + C. jejuni group was the nematodes fed by E. coli OP50 and C. jejuni at the same time after L4 stage. In an experiment undertaken to evaluate the role of LAB in protecting the worms against death caused by C. jejuni, the worms were either fed 108 CFU/mL E. coli OP50 or 109 CFU/mL LAB strains for the first 3 days. After 72 h of incubation, the worms were moved to new 6 cm plates with C. jejuni at a concentration of 109 CFU/mL. The worms that were first fed E. coli OP50 (72 h) and next C. jejuni formed the C. jejuni reference group (OP50/C. jejuni group); those treated with LAB (72 h) before C. jejuni were regarded as the LAB protection groups (LAB/C. jejuni group). A worm was considered dead when it failed to respond to gentle touch with a worm picker. The numbers of live worms were recorded, and the probability of their survival was calculated as described previously [27].

Examination of bacterial load in the intestine of C. elegans

The numbers of E. coli OP50, Lactobacillus and C. jejuni in the nematodes’ intestine were determined with some modification of the method described previously [27]. Examination of Lactobacillus was executed on LAB/C. jejuni groups. The sampling (50 worms per sample) was done every 2 days and day 0 marked the arrival of the nematodes at the L4 stage before being fed thallus. At the same time, on day 6, E.coli OP50 load in all groups was detected. C. jejuni test also followed the same method on Lactobacillus. But, day 0 marked the point at which the nematodes were first fed C. jejuni. After surface sterilization, the worms were mashed mechanically with a pellet pestle motor, re-suspended in the M9 buffer, and inoculated onto eosin-methylene blue medium (EMB medium), MRS or Columbia blood agar for counting of E. coli OP50, Lactobacillus and C. jejuni, respectively. At least three independent replications were performed for each assay.

Measurement of body size and pharynx pumping

The live worms were examined for their body size measurements every 2 days and their pharynx pumping on the eighth day after they were fed thallus at the L4 stage. Images of adult nematodes were taken with a VCT-VBIT digital microscope (Shimadzu, Kyoto, Japan) and analyzed using the ImageJ software. In this system, the area of the worm’s projection was estimated automatically and used as an index of body size. And the worm’s pharynx pumping was measured per 30s.

Evaluation of the effects on the growth of C. jejuni by co-culturing E. coli OP50

The growth of C. jejuni co-cultured with E. coli OP50 was determined by the following method as previously described [53]. The C. jejuni cells (107 CFU/mL) suspended in antibiotic-free brain heart infusion broth (BHIB) containing 5% serum were incubated under microaerophilic conditions for 48 h at 37 °C in the presence of a 10% volume of live E. coli OP50 (107 CFU/mL). Both were incubated together in liquid medium. The viability of C. jejuni was evaluated from the number of viable CFUs in C. jejuni culture as described above on C. jejuni-selective plates. At least three independent replications were performed for this assay.

RNA extraction, reverse transcription and quantitative real-time PCR analysis

The whole RNA of C. elegans and of the bacteria used in the life-span experiment was extracted. About 100 worms were prepared for the lysates. The RNA was extracted as previously described [27]. Total RNA of bacteria and C. elegans from the life span assays was extracted from lysates of the worms, using the mirVana miRNA Isolation Kit (Ambion, TX) according to manufacturer’s instructions.

The transcription of mitogen-activated protein kinase (MAPK) pathway genes (tir-1, nsy-1, sek-1 and pmk-1), antimicrobial peptide genes (spp-1, clec-85, abf-2, clec-60 and lys-7), Daf-16 pathway genes (daf-16 and age-1), a TGF-β pathway gene (dbl-1) and antioxidant genes (skn-1 and bar-1) in C. elegans was determined by quantitative polymerase chain reaction (qPCR) [49]. SuperScript first-strand synthesis system (Invitrogen, Carlsbad, CA) was used for the synthesis of cNDA. GapA, as a housekeeping gene, was used to determine the levels of mRNA transcription of the C. elegans immune genes and to normalize the input amounts of RNA. PCR primers specific to each of the genes were experimentally validated and used in the RT-qPCR assay (Table S9). One μl of each cDNA sample was included in a 24 μl reaction mixture containing 12.5 μl Master Mix, 3.75 μl each of the primers at 150 nM, and 4 μl irradiated and double autoclaved dH2O. The QPCR programs included 10 min at 95 °C and 40 cycles of 95 °C for 30 s, 56 °C for 1 min, and 72 °C for 30 s. Fluorescence wasmeasured after each annealing during the cycles. The delta Ct method was used to analyse the RT-qPCR data and to determine the relative abundance of the target genes (fold changes) (fold changes) [50, 51]. The data of OP50 group was taken as benchmark 1. All experiment was repeated three times.

Induction of acute iletis for C. jejuni infection and LAB intervention in mice

Three-week-old female C57BL/6 mice obtained from Shanghai Laboratory Animal Center (Shanghai, China) were used in the experiments. All the experimental procedures (#JIPD2017030) have been reported [55]. All the experiments conformed to the Ministry of Science and Technology of China’s Guide for the Care and Use of Laboratory Animals.

To induce ileitis, the C57BL/6 mice were infected orally with T. gondii ME49 cysts according to previous reports [52, 55]. As shown in Fig. S2A, 4 days later, mice were infected with LAB and C. jejuni NCTC 11168 by gavage at a volume of 0.3 mL every day individually. The mice were successively treated with LAB (109 CFU) and C. jejuni (109 CFU) at 1 h intervals on 2 consecutive days. The C. jejuni loads in their faeces were checked on day 9. The faeces were resuspended in sterile PBS and serially diluted. The diluted samples were spread on Columbia blood agar with a C. jejuni selective supplement and incubated at 5% oxygen concentration at 37 °C for 48 h. After incubation, the numbers of C. jejuni in the samples were counted. The mice were euthanized with CO2 after experiment. For each mice, the treatment based on the different LAB, the blood collection and execution and analysis of indicators were all done by different investigator. The first investigator was the only person aware of the treatment group allocation. All experiment was repeated three times.

C. jejuni infection and LAB intervention in chicken

White leghorn chicken eggs (Jinan Baizhun Biologic Inspection Company, Ltd., China) were maintained in an egg incubator until the chicks hatched. About 24 h after hatching, 8 chicks were randomly assigned to several groups. All the experimental procedures were approved by the Animal Care and Use Committee at Jiangsu Nannong Hi-technology company, LTD. All the experiments adhered to the Ministry of Science and Technology of China’s Guide for the Care and Use of Laboratory Animals.

As shown in Fig. S2B, all birds were administered 109 CFU of C. jejuni NCTC 11168 in a 0.3 mL suspension by oral gavage. Twenty-four hours after oral gavage, LAB (109 CFU in 0.3 mL) were orally administered daily to 11 groups (in total 88 birds) of C. jejuni-inoculated birds for two weeks. PBS was administered to the remaining one C. jejuni group of birds (8 birds). Chicks were euthanized with CO2 at 20% chamber replacement rate on 23 days post-inoculation, and the cecal contents were diluted in ice-cold PBS to 0.1 g/mL. Ten-fold serial dilutions of each sample were prepared and then plated on Columbia blood agar with a C. jejuni selective supplement and incubated at a 5% oxygen concentration at 37 °C for 48 h. For each chicken, the treatment based on the different LAB, the blood collection and execution and analysis of indicators were all done by different investigator. The first investigator was the only person aware of the treatment group allocation. All experiment was repeated three times.

Statistical analysis

Kaplan-Meier survival analysis was used to assess the survival rate of C. elegans. The significance of differences on data was determined by using Student’s t-test. Means ± standard deviations (SDs) were used as a form of data pression. Differenceswere considered significant at a P-value of < 0.01.

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Abbreviations

C. jejuni :

Campylobacter jejuni

LAB:

lactic acid bacteria

E. coli :

Escherichia coli

C. elegans :

Caenorhabditis elegans

T. gondii :

Toxoplasma gondii

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Acknowledgments

Not applicable.

Funding

This work was supported by the National Natural Science Foundation of China (No. 31671839, 31972052, 31820103010, 31601444, 31301407), the Fundamental Research Funds for the Central Universities (JUSRP22006, JUSRP51501), the national first-class discipline program of Food Science and Technology (JUFSTR20180102), the Program of Collaborative Innovation Centre of Food Safety and Quality Control in Jiangsu Province and the research supported by Hunan Provincial Natural Science Foundation of China (2019JJ50014) who approved the design of the study and collection, analysis, interpretation of data and final manuscript.

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GW and XJ conceived and designed the experiments; XJ, YH and XC performed the experiments; XJ and GW analyzed the data; YZ, YL, JZ, HZ and WC contributed reagents/materials/analysis tools; XJ and GW wrote the paper. All authors contributed to manuscript revision, read and approved the submitted version.

Corresponding author

Correspondence to Gang Wang.

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All the mice experimental procedures (#JIPD2017030) had been approved by the Animal Care and Use Committee at the Jiangsu Institute of Parasitic Diseases. All the chicken experimental procedures were approved by the Animal Care and Use Committee at Jiangsu Nannong Hi-technology company, LTD. All the experiments adhered to the Ministry of Science and Technology of China’s Guide for the Care and Use of Laboratory Animals.

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Supplementary Information

Additional file 1 Fig. S1

Flow chart of LAB interventions in C. elegans. Fig. S2 Flow chart of LAB interventions in mice and chicken. (A) Experimental design for mice. (B) Experimental design for chicken. Table S1 The information of 44 LAB strains. Table S2 LAB load in the intestine of C. elegans. Table S3 C. jejuni load in the intestine of C. elegans. Table S4 E.coli OP50 load in the intestine of C. elegans. Table S5 Effects of LAB on the body size of C. elegans infected by C. jejuni. Table S6 Effects of LAB on the pharynx pumping of C. elegans infected by C. jejuni. Table S7 Differential effects of LAB on the transcription of immune genes of C. elegans infected by C. jejuni. Table S8 Differential effects of LAB alone on the transcription of immune genes of C. elegans on day 3. Table S9 qPCR primers for nematodes defense gene.

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Jin, X., He, Y., Zhou, Y. et al. Lactic acid bacteria that activate immune gene expression in Caenorhabditis elegans can antagonise Campylobacter jejuni infection in nematodes, chickens and mice. BMC Microbiol 21, 169 (2021). https://doi.org/10.1186/s12866-021-02226-x

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Keywords

  • Lactic acid bacteria
  • Campylobacter jejuni
  • Caenorhabditis elegans
  • Life-span
  • Immune gene